Silica nanoparticle-based microfluidic immunosensor with laser-induced fluorescence detection for the quantification of immunoreactive trypsin

Article abstract of DOI:10.1016/j.ab.2014.06.016

The purpose of this study was to develop a silica nanoparticle-based immunosensor with laser-induced fluorescence (LIF) as a detection system. The proposed device was applied to quantify the immunoreactive trypsin (IRT) in cystic fibrosis (CF) newborn screening. A new ultrasonic procedure was used to extract the IRT from blood spot samples collected on filter papers. After extraction, the IRT reacted immunologically with anti-IRT monoclonal antibodies immobilized on a microfluidic glass chip modified with 3-aminopropyl functionalized silica nanoparticles (APSN-APTES-modified glass chips). The bounded IRT was quantified by horseradish peroxidase (HRP)-conjugated anti-IRT antibody (anti-IRT-Ab) using 10-acetyl-3,7-dihydroxyphenoxazine (ADHP) as enzymatic mediator. The HRP catalyzed the oxidation of nonfluorescent ADHP to highly fluorescent resorufin, which was measured by LIF detector, using excitation lambda at 561 nm and emission at 585 nm. The detection limits (LODs) calculated for LIF detection and for a commercial enzyme-linked immunosorbent assay (ELISA) test kit were 0.87 and 4.2 ng ml^-1, respectively. The within- and between-assay variation coefficients for the LIF detection procedure were below 6.5%. The blood spot samples collected on filter papers were analyzed with the proposed method, and the results were compared with those of the reference ELISA method, demonstrating a potential usefulness for the clinical assessment of IRT during the early neonatal period.

Full text of DOI:10.1016/j.ab.2014.06.016

YABIO 11781
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Analytical Biochemistry xxx (2014) xxx–xxx
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Contents lists available at ScienceDirect
Analytical Biochemistry
journal homepage: www.elsevier.com/locate/yabio
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Silica nanoparticle-based microfluidic immunosensor with laser-induced
fluorescence detection for the quantification of immunoreactive trypsin
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⇑
Marco A. Seia, Patricia W. Stege, Sirley V. Pereira, Irma E. De Vito, Julio Raba, Germán A. Messina
INQUISAL, Department of Chemistry, National University of San Luis, CONICET, D5700BWS San Luis, Argentina
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a r t i c l e i n f o
a b s t r a c t
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Article history:
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The purpose of this study was to develop a silica nanoparticle-based immunosensor with laser-induced
fluorescence (LIF) as a detection system. The proposed device was applied to quantify the immunoreac-
tive trypsin (IRT) in cystic fibrosis (CF) newborn screening. A new ultrasonic procedure was used to
extract the IRT from blood spot samples collected on filter papers. After extraction, the IRT reacted
immunologically with anti-IRT monoclonal antibodies immobilized on a microfluidic glass chip modified
with 3-aminopropyl functionalized silica nanoparticles (APSN–APTES-modified glass chips). The bounded
IRT was quantified by horseradish peroxidase (HRP)-conjugated anti-IRT antibody (anti-IRT–Ab) using
10-acetyl-3,7-dihydroxyphenoxazine (ADHP) as enzymatic mediator. The HRP catalyzed the oxidation
of nonfluorescent ADHP to highly fluorescent resorufin, which was measured by LIF detector, using exci-
tation lambda at 561 nm and emission at 585 nm. The detection limits (LODs) calculated for LIF detection
Received 28 April 2014
Received in revised form 19 June 2014
Accepted 21 June 2014
Available online xxxx
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Keywords:
Newborn screening
Immunoreactive trypsin
Microfluidic chip
Silica nanoparticles
Ultrasonic procedure
LIF
and for a commercial enzyme-linked immunosorbent assay (ELISA) test kit were 0.87 and 4.2 ng mlÀ1
,
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respectively. The within- and between-assay variation coefficients for the LIF detection procedure were
below 6.5%. The blood spot samples collected on filter papers were analyzed with the proposed method,
and the results were compared with those of the reference ELISA method, demonstrating a potential
usefulness for the clinical assessment of IRT during the early neonatal period.
Ó 2014 Elsevier Inc. All rights reserved.
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Nanomaterials are currently widely used for the construction of
biosensors [1]. Among them, silica nanoparticles (SNs)1 represent
an interesting choice to be used in sensing [2–7] and as a support
material [8–13] due to SNs’ good monodispersity, high pore volume,
and thermal and mechanical stability [14]. Furthermore, the large
surface of SNs makes them an appropriate option for the immobili-
zation of recognition biological moieties such as antibodies and
antigens, allowing their use as solid support for immunoassays.
Quantitative immunological methods have been essential to
many clinical, pharmaceutical, and scientific applications [15].
These methods can be performed in micrometer-scale analytical
devices that consist of microchannels for transporting fluids
with part or all of the necessary components of an integrated
immunoassay [16–18]. The recent trend to miniaturize assays pro-
motes the development of the microfluidic technology, which
results in a substantial reduction in the consumption of reactive
solutions [19], shorter analysis time, improved portability, and in
part better detection limits (LODs) [20–23].
The use of nanoparticles as an immunological platform incorpo-
rated into a microfluidic device results in an increase in the
effective area and the reduction of incubation times by reducing
the diffusional distances [24].
Detection systems for microfluidic devices must be capable of
providing sensitive measurements in low volumes. The most
commonly used is laser-induced fluorescence (LIF) due to its high
sensitivity and suitability for analyzing analytes in small volumes
of fluid in chip formats [25].
The goal of this work was to develop a novel method to carry
out cystic fibrosis (CF) newborn screening in a sensitive, fast, and
automated way, measuring immunoreactive trypsin (IRT) in
patients’ blood. CF is an autosomal recessive disease and is also
the most common severe genetic disease, found mainly in people
of European descent with an incidence of approximately 1 in every
3000 live births worldwide [26–28,20]. The main characteristic of
this pathology is the multi-organ involvement, including severe
respiratory disease, pancreatic insufficiency, and male infertility
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Corresponding author. Fax: +54 2652 43 0224.
E-mail address: messina@unsl.edu.ar (G.A. Messina).
Abbreviations used: SN, silica nanoparticle; LOD, detection limit; LIF, laser-induced
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fluorescence; CF, cystic fibrosis; IRT, immunoreactive trypsin; CCh, central channel;
anti-IRT–Ab, anti-IRT antibody; HRP, horseradish peroxidase; ADHP, 10-acetyl-3,
7-dihydroxyphenoxazine; PR, photoresist; HF, hydrofluoric acid; APTES, 3-aminopro-
pyl triethoxysilane; ELISA, enzyme-linked immunosorbent assay; UV, ultraviolet;
PDMS, poly(dimethylsiloxane); HPLC, high-performance liquid chromatography;
APSN, 3-aminopropyl-functionalized silica nanoparticle; PBS, phosphate-buffered
saline; RFU, relative fluorescence units; CV, coefficient of variation.
http://dx.doi.org/10.1016/j.ab.2014.06.016
0003-2697/Ó 2014 Elsevier Inc. All rights reserved.
Please cite this article in press as: M.A. Seia et al., Silica nanoparticle-based microfluidic immunosensor with laser-induced fluorescence detection for the
quantification of immunoreactive trypsin, Anal. Biochem. (2014), http://dx.doi.org/10.1016/j.ab.2014.06.016

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[21,29,23,30,31]. Depending on the country, the IRT cutoff value
currently used for CF newborn screening ranges between 65 and
70 ng ml–1 [32–34].
Because CF has no cure [35], an early and adequate diagnosis is
essential.
Absorbance was detected by a Bio-Rad Benchmark microplate
reader (Japan) and a Beckman DU 520 general UV/VIS (ultravio-
let/visible) spectrophotometer.
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Microchip fabrication
To achieve the above-mentioned goal, SNs were incorporated
into the central channel (CCh) of the microfluidic glass chip. The
surface of SNs was functionalized with anti-IRT antibody
(anti-IRT–Ab), which allowed the capture of IRT. The subsequent
detection was achieved by adding horseradish peroxidase (HRP)-
conjugated anti-IRT–Ab using 10-acetyl-3,7-dihydroxyphenox-
azine (ADHP) as enzymatic mediator. The oxidation of ADHP to
highly fluorescent resorufin was measured by LIF using excitation
at 561 nm and emission at 585 nm.
The results indicated that our method, designed for the quanti-
fication of IRT, could provide a fast, sensitive, and automated CF
diagnosis. Due to the benefits observed in the SN-based microflu-
idic immunosensor with LIF detection and the need for an early
and adequate diagnosis, we believe that it could represent an
attractive and alternative analytical strategy for CF newborn
screening.
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The procedure for the fabrication of glass microfluidic chips is
shown in Fig. 1. The construction was carried out according to
the procedure described in Ref. [38] with the following modifica-
tions. The device layout was drawn using CorelDraw software
version 11.0 (Corel) and printed on a high-resolution transparency
film in a local graphic service, which was used as a mask in the
photolithographic step. The microfluidic chip design consisted of
a T-type format. The lengths of the central and accessory channels
were 15 and 60 mm, respectively. The printed transparency mask
was placed on top of a glass wafer previously coated with a
5-lm layer of AZ4330 PR. The substrate was exposed to UV radia-
tion for 30 s and developed in AZ 400 K developer solution for
2 min. Glass channels were etched with an etching solution of
20% HF for 4 min under continuous stirring (check the HF material
safety data sheet). The etching rate was 8
1 l
m minÀ1. Following
the etching step, substrates were rinsed with deionized water and
the PR was removed with acetone. To access the microfluidic net-
work, holes were drilled on glass-etched channels with a Dremel
tool (MultiPro 395JU model, USA) using 1-mm diamond drill bits.
To achieve the final chip format, another glass plate was spin-
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Materials and methods
Reagents and solutions
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Soda lime glass wafers (26 Â 76 Â 1 mm) were purchased from
Glass Técnica (São Paulo, Brazil). Sylgard 184 and AZ4330 photore-
sist (PR) were obtained from Dow Corning (Midland, MI, USA) and
Clariant (Sommerville, NJ, USA), respectively. Hydrofluoric acid
coated with
a thin poly(dimethylsiloxane) (PDMS) layer at
3000 rpm for 10 s. PDMS was prepared with a 10:1 mixture of
Sylgard 184 elastomer and a curing agent. The thickness of this
layer was 50 lm. Before sealing, the PDMS layer was cured at
(HF),
3-aminopropyl-functionalized
silica
(nanoparticles,
100 °C for 5 min on a hot plate. Glass channels and PDMS-coated
glass substrate were placed in an oxygen plasma cleaner (Plasma
Technology PLAB SE80 plasma cleaner) and oxidized for 1 min.
The two pieces were brought into contact immediately after the
plasma treatment, obtaining a strong and irreversible sealing.
The final device format was achieved in less than 30 min. The
bonding resistance of the device was evaluated under different
pressure values by using a high-performance liquid chromatogra-
ꢀ100 nm particle size, DLS), 3-aminopropyl triethoxysilane
(APTES), and ADHP were purchased from Sigma–Aldrich. Glutaral-
dehyde (25% aqueous solution) and acetone were purchased from
Merck. The enzyme immunoassay for the quantitative determina-
tion of IRT, ImmunoChem Blood Spot Trypsin-MW ELISA (enzyme-
linked immunosorbent assay) Kit (MP Biomedicals, USA), was pur-
chased from Laboratorios Bacon (Argentina) and was used
according to the manufacturer’s instructions [36]. Anti-IRT–mouse
monoclonal Ab (10 mg ml–1) and HRP-conjugated anti-IRT–Ab
phy (HPLC) pump. The flow rate ranged from 10 to 300
l .
l minÀ1
(10 mg ml–1
) were purchased from Abcam (USA). All buffer
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Glass chip surface modification
solutions were prepared with Milli-Q water.
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Coating of central channel’s chip with APTES
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Instrumentation
The CCh was pretreated according to the following steps:
washed for 15 min with 1 M HCL, rinsed for 15 min with deionized
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The optical system was constructed using the procedure of
Ref. [37] according to the following modification. A 532-nm sin-
gle-frequency DPSS laser (Cobolt Jive, 561 nm, USA) operated at
25 mW served as the fluorescence excitation source. It was focused
on the detection channel at 45° to the surface using a lens with a
focal distance of 30 cm. The relative fluorescence signal of ADHP
was measured using excitation at 561 nm and emission at 585 nm.
The paths of the reflected beams were arranged so that they did
not strike the capillary channels elsewhere and to avoid photoble-
aching. The fluorescent radiation was detected with the optical
axis of the assembly perpendicular to the plane of the device. Light
was collected with a microscope objective (10:1, NA 0.30, working
distance 6 mm, PZO, Poland) mounted on a microscope body
(BIOLAR L, PZO). A fiber-optic collection bundle was mounted on
a sealed housing at the end of the lens of the microscope, which
was connected to a QE65000-FL scientific-grade spectrometer
(Ocean Optics, USA). The entire assembly was covered with a large
box to eliminate ambient light.
water, and rinsed for 15 min with methanol at 2 l
l minÀ1. After the
preconditioning procedure, the 2.5% (v/v) APTES in methanol
solution was introduced into the glass channel inner surface at
2 l
l minÀ1 for 30 min. It was left to stand filled with the silaniza-
tion solution for 2 h (Fig. 2). Then, the channel was rinsed with
methanol and dried under a nitrogen flow.
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Immobilization of APSNs onto APTES-modified glass chip surface
We mixed 1 mg of 3-aminopropyl-functionalized silica nano-
particles (APSNs) with 1 ml of aqueous solution of 5% (w/w)
glutaraldehyde in 0.20 M CO23À/HCOÀ3 buffer (pH 10.0) to induce
the formation of aldehyde groups at 25 °C for 2 h. Then, glutaralde-
hyde–ASNPs were pumped into the CCh of APTES-modified glass
chip at 2 l
l minÀ1 for 10 min. After that, it was left to stand filled
with the same solution for 60 min and finally rinsed with
phosphate buffer (pH 7.2) for 15 min (Fig. 2).
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Covalent binding of IRT-specific Ab onto APSN–APTES-modified glass
chip surface
The syringe pump system (Baby Bee Syringe Pump, Bioanalyti-
cal Systems) was used for pumping reagent solutions and stopping
flow.
A solution of 10
fer (pH 7.2) was injected through the CCh of the modified chip and
l
g mlÀ1 anti-IRT–Ab in 0.10 M phosphate buf-
Please cite this article in press as: M.A. Seia et al., Silica nanoparticle-based microfluidic immunosensor with laser-induced fluorescence detection for the
quantification of immunoreactive trypsin, Anal. Biochem. (2014), http://dx.doi.org/10.1016/j.ab.2014.06.016

YABIO 11781
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LIF immunosensor for IRT newborn screening / M.A. Seia et al. / Anal. Biochem. xxx (2014) xxx–xxx
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Fig.1. Microfabrication process of glass microfluidic chip with sealing procedure based on the use of a thin layer of PDMS. The process involves wet chemical etching of glass
with 20% HF for 4 min under continuous stirring, spin-coating of a PDMS and photoresist layer over flat glass surface at 3000 rpm for 15 s, and plasma-oxidized treatment for
1 min.
Fig.2. Schematic representation of the glass chip surface modification and the immunological reaction. Anti-IRT–Abs were covalently bounded onto APSNs that were
covalently attached over APTES-modified glass chip surface. The IRT present in the blood spot collected filter papers reacted immunologically with anti-IRT–Ab-immobilized
APSN–APTES-modified glass chips. The bound IRT was quantified by HRP-conjugated anti-IRT–Ab using ADHP as enzymatic mediator. The highly fluorescent resorufin (HP)
generated was measured by LIF using excitation at 561 nm and emission at 585 nm.
Please cite this article in press as: M.A. Seia et al., Silica nanoparticle-based microfluidic immunosensor with laser-induced fluorescence detection for the
quantification of immunoreactive trypsin, Anal. Biochem. (2014), http://dx.doi.org/10.1016/j.ab.2014.06.016

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left to react at 25 °C for 2 h (Fig. 2). Finally, the CCh was rinsed with
phosphate buffer (pH 7.2) to remove the unbound anti-IRT–Ab and
stored in the same buffer at 4 °C. Residual aldehyde groups of
glutaraldehyde present on the APSN surface allowed the covalent
attachment of the amino groups of anti-IRT–Ab (Fig. 2). The
antibody preparation was left stable for at least 1 month.
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Results and discussion
Optimization
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APSN–APTES amplification effect
To evaluate the signal amplification effect of silica nanoparti-
cles, we compared the signal intensity of APSN–APTES-modified
glass chips with the signal obtained from the microfluidic glass
chip modified only with APTES over the flat CCh surface using
HRP as an indicator model. For these experiments, on the one hand,
the CCh was modified according to the procedure explained in the
first and second subsections of the ‘‘Glass chip surface modifica-
tion’’ section in Materials and Methods. On the other hand, the
microfluidic glass chip was modified only with APTES according
to the procedure explained in the first subsection. After that, a
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Preparation of sample
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For the quantification of human IRT, blood samples were spot-
ted in filter paper number 903 in the center of a 1-cm circle and
allowed to diffuse outward, trying to avoid tearing or disrupting
the filter paper surface. The specimens were allowed to air dry
completely (overnight), avoiding heat, direct sunlight, and
absorbent surfaces. After drying overnight, these were stored in
an air-tight plastic envelope at less than À15 °C until assay. In this
work, we used neonatal samples and control samples provided by
the Blood Spot Trypsin-MW ELISA Kit.
To start the IRT measurement, a disk was punched from a blood
spot collected filter paper number 903. After that, it was placed
into an Eppendorf tube with 200 ll in 0.01 M phosphate-buffered
saline (PBS, pH 7.2) and exposed to a sonication procedure for
2 min. To compare IRT extraction procedures, our proposed ultra-
sonic extraction process and the Blood Spot Trypsin-MW ELISA
Kit were performed simultaneously. In both cases, the content of
all tubes was aspirated and the eluted samples were stored at
4 °C until use.
solution of 10 l
g mlÀ1 HRP in 0.10 M phosphate buffer (pH 7.2)
was injected through the CCh of both chips and left to react at
25 °C for 2 h. Finally, the CCh was rinsed with phosphate buffer
(pH 7.2) to remove the unbound HRP and stored in the same buffer
at 4 °C. The HRP toward the reduction of H2O2 catalyzed the oxida-
tion of nonfluorescent ADHP to highly fluorescent resorufin, which
was measured by LIF, using excitation at 561 nm and emission at
585 nm. According to the obtained results, APSN incorporated into
the channel generated important signal amplification, more than 4
times compared with the flat channel surface (Fig. 3).
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Effects of flow rate and incubation times
The flow rates of samples and reagents have a significant effect
on the efficiency of antigen–antibody interactions in microfluidic
immunosensors. This effect is based on the fact that in microfluidic
devices, unlike conventional immunoassays, samples and reagents
are continuously flowing through the device.
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IRT LIF determination
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The IRT determination process included several steps in which
reactive and washing solutions were pumped at a flow rate of
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For the determination of IRT, the flow rate was analyzed by
studying the relative fluorescence obtained in the IRT control
sample of 154 ng mlÀ1 at different flow rates: 1, 2, 3, 4, 5, and
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2.0 l
l minÀ1. After each reagent solution injection, the CCh of the
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l
l minÀ1 (Fig. 4). The response signal was significantly reduced
device was exposed to a washing procedure with 0.01 M PBS (pH
7.2) for 5 min in order to remove the reagent excess.
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when the flow rate exceeded 3
2
l
l minÀ1. Therefore, a flow rate of
l minÀ1 was used for samples, reagents, and washing solutions.
As a first step, a blocking treatment was performed through
injecting 1% of bovine serum albumin (BSA) in 0.01 M PBS (pH
7.2) for 5 min in order to avoid unspecific bindings. Once the
device was blocked, the eluted sample was injected into the PBS
carrier stream for 10 min. The Ab bounded on the APSNs in the
CCh wall reacted with IRT present in the eluted sample. Bound
IRT was quantified using HRP-conjugated anti-IRT–Ab (dilution of
1:1000 in 0.01 M PBS, pH 7.2) injected for 5 min.
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l
For the relative fluorescence measurement, the substrate
solution was prepared by dissolving 0.01 M ADHP stock solution
in dimethyl sulfoxide (DMSO) and stored at À20 °C. The ADHP
solution previously obtained and the H2O2 solution were diluted
to 0.001 M with 0.1 M phosphate–citrate buffer (pH 5.05) before
being used. The substrate solution was injected into the carrier
stream for 1 min, and the enzymatic product was measured by
LIF. The HRP in the presence of H2O2 catalyzed the oxidation of
nonfluorescent ADHP to highly fluorescent resorufin, which was
measured using excitation at 561 nm and emission at 585 nm.
After each sample measurement, the device was exposed to a
flow of desorption buffer (0.1 M glycine–HCl, pH 2.0) at a flow rate
of 2.0 l
l minÀ1 for 5 min and then washed with PBS (pH 7.2). With
this treatment, bound immunocomplexes were desorbed, allowing
us to start with the next determination. The storage of the device
was done in 0.01 M PBS (pH 7.2) at 4 °C. The proposed device could
be used with no significant loss of sensitivity for 15 days, whereas
its useful lifetime was 1 month with a sensitivity decrease of 10%.
The storage of the device was done in 0.01 M PBS (pH 7.2) at 4 °C.
Table A in the online supplementary material summarizes the
complete analytical procedure required for the IRT immunoassay.
Fig.3. Evaluation of signal amplification effect of APSNs. A comparison between the
signal intensity of the proposed method (APSN–APTES-modified glass chip) with
the signal obtained from the microfluidic glass chip modified only with APTES over
the flat CCh surface using HRP as an indicator model was carried out. For this study,
0.1 M phosphate–citrate buffer (pH 5.05) containing different concentrations of
H2O2 and 0.001 M ADHP was injected into the carrier stream at a flow rate of
2 l
l minÀ1, and the enzymatic product was measured by LIF using an excitation
wavelength of 561 nm and an emission wavelength of 580 nm. RFU, relative
fluorescence units.
Please cite this article in press as: M.A. Seia et al., Silica nanoparticle-based microfluidic immunosensor with laser-induced fluorescence detection for the
quantification of immunoreactive trypsin, Anal. Biochem. (2014), http://dx.doi.org/10.1016/j.ab.2014.06.016

YABIO 11781
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No. of Pages 7, Model 5G
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Fig.4. Effect of flow rate analyzing a 154-ng mlÀ1 IRT control sample at different
flow rates. Here, 0.1 M phosphate–citrate buffer (pH 5.05) containing 0.001 M H2O2
and 0.001 M ADHP was injected into the carrier stream at different flow rates. The
enzymatic product was measured by LIF using an excitation wavelength of 561 nm
and an emission wavelength of 580 nm.
Fig.5. Fluorescence intensity as
a
function of reaction time for 53, 154, and
l minÀ1
580 ng mlÀ1 of IRT control sample concentrations. The flow rate was 2
l
.
Here, 0.1 M phosphate–citrate buffer (pH 5.05) containing 0.001 M H2O2 and
0.001 M ADHP was injected into the carrier stream at different flow rates. The
enzymatic product was measured by LIF using an excitation wavelength of 561 nm
and an emission wavelength of 580 nm.
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Regarding incubation time, the minimum time required for IRT
binding is also a critical assay factor, especially when the use of a
minimum total analysis time is desired. Fig. 5 shows the measured
fluorescence for 53, 154, 282, and 580 ng mlÀ1 IRT control sample
concentrations. The fluorescence intensity increased when the IRT
concentration grew. As expected, the intensity of the fluorescence
increased with the reaction time. The intensity of the fluorescence,
however, did not increase considerably until 10 min had passed,
which was likely due to saturation of the specific antibody sites
in the APSN–APTES-modified glass chips. Therefore, the optimal
reaction time was 10 min.
A study of the rates of enzymatic response under flow condi-
tions was performed in the pH range of 3.0 to 8.0 and showed a
maximum value of activity at pH 5.05 (data not shown). Besides,
the enzyme exhibits its best activity over the temperature range
of 20 to 25 °C; higher temperatures would be harmful to its activity
[39].
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Blood Spot Trypsin-MW ELISA Kit. As shown in Table 1, we
obtained similar IRT concentrations employing both procedures
for all neonatal samples and control samples analyzed, indicating
a good correspondence between them. The acquired results prove
that APSN–APTES-modified glass chips have excellent selectivity
and sensitivity for the specific detection of IRT antigens.
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Quantitative test for detection of IRT in APSN–APTES-modified glass
chips
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The IRT calibration plot was obtained by plotting relative fluo-
rescence versus IRT concentration in the range of 0 to 580 ng mlÀ1
.
A
linear relation, relative fluorescence units (RFU) = 0.281 +
0.179 Â CIRT, was obtained. The correlation coefficient (r) for this
plot was 0.998. The coefficient of variation (CV) for determination
of the IRT control sample of 154 ng mlÀ1 was below 5% (six repli-
cates). The LOD is the concentration that gives a signal 3 times
the standard deviation of the blank. For LIF detection procedures
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Ultrasonic procedure for IRT extraction
We compared our ultrasonic procedure for the IRT extraction
and the Trypsin-MW ELISA Kit procedure as has already been
explained. For this experiment, two high-level control samples,
two high-level neonatal samples, two low-level control samples,
and two low-level neonatal samples were compared using both
extraction procedures for the IRT blood spot elution. After that,
all of these samples were analyzed by our LIF immunosensor and
and the ELISA test kit, the LODs were 0.87 and 4.20 ng mlÀ1
,
respectively. This indicates that the proposed method exhibits a
wide measurable concentration range and a low LOD.
The precision of the proposed method was checked with IRT
control samples at 53-, 154-, and 580-ng mlÀ1 concentrations. This
Table 1
Comparison between ultrasonic and Trypsin-MW ELISA Kit procedures for the IRT blood spot extraction, analyzed by the proposed method and the Trypsin-MW ELISA Kit (five
measurements in the same run for each control sample).
IRT Ultrasonic procedure extraction
eIRT ELISA Kit procedure extraction
Sample number (IRT ng mlÀ1
)
Immunosensor (IRT ng mlÀ1
)
ELISA kita (IRT ng mlÀ1
)
Immunosensor (IRT ng mlÀ1
)
ELISA kita (IRT ng mlÀ1
)
HLCS1
HLCS2
HLNS1
HLNS2
LLCS1
LLCS2
LLNS1
LLNS2
96.3 3.3
223.2 8.4
310.3 12.2
256.5 10.7
54.3 1.4
26.2 0.8
33.4 1.2
22.1 0.7
97.9 5.2
226.7 7.5
317.9 16.8
252.4 8.9
56.4 2.2
26.8 1.2
32.9 1.8
22.4 0.9
96.6 2.6
222.9 9.1
313.4 11.9
253.7 9.5
55.4 2.6
27.3 1.1
32.6 1.1
22.7 0.4
96.4 4.6
219.8 8.7
311.3 13.9
259.2 11.3
55.3 2.9
26.8 1.6
31.7 1.3
21.9 1.0
Q7
Note. HLCS, high-level control samples; HLNS, high-level neonatal samples; LLCS, low-level control samples; LLNS, low-level neonatal samples.
a
Trypsin-MW ELISA Kit.
Please cite this article in press as: M.A. Seia et al., Silica nanoparticle-based microfluidic immunosensor with laser-induced fluorescence detection for the
quantification of immunoreactive trypsin, Anal. Biochem. (2014), http://dx.doi.org/10.1016/j.ab.2014.06.016

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Table 2
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on dissociative fluorescence enhancement applying the principle
of cofluorescence, and on time-resolved fluorometry [40]. Second,
Lindau-Shepard and Pass developed a multiplex immunoassay
using two different Luminex bead sets for IRT isoform detection
[41]. It is relevant to emphasize that the proposed method is based
on microfluidic technology, coupled to LIF detection, with an
APSN–APTES biorecognition platform that allowed the successful
immobilization of anti-IRT–Ab as a strategy to provide specificity
to the device. In addition, the achieved LOD was lower than that
obtained by the above-mentioned articles [40,41]. Considering
the CF neonatal screening cutoff value, all obtained LODs were rea-
sonably good. Thus, we came to the conclusion that our device has
inherent benefits such as miniaturization, integration, portability,
and the possibility to perform on-site analysis.
Within-assay precision (five measurements in the same run for each IRT control
sample) and between-assay precision (five measurements for each IRT control
sample, repeated for 3 consecutive days).
Control samplea (ng mlÀ1
)
Within assay
Mean
Between assay
CV%
Mean
CV%
53
154
580
52.64
154.72
581.77
4.26
4.94
2.59
52.13
157.53
586.82
4.81
6.42
5.26
a
ng mlÀ1 IRT control sample.
389
Conclusions
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391
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393
394
395
396
397
398
399
400
401
402
403
404
405
406
407
408
409
410
411
412
413
In this research, we successfully performed the microfabrication
of a glass microfluidic chip sealed by a thin layer of PDMS together
with the modification of the glass channel surface by anti-IRT–Ab
covalently bounded onto APSN–APTES-modified glass chip. This
modified microfluidic chip was applied to the CF newborn screen-
ing through the selective and sensitive quantification of IRT in neo-
natal blood samples. In addition, we proposed and evaluated a new
ultrasonic procedure for the IRT blood spot extraction collected on
filter papers. This allowed us to obtain an important reduction of
the elution time compared with the extraction process proposed
by the conventional ELISA analysis. The total assay time for IRT
determination by the proposed method was less than 40 min con-
sidering the sample elution step, whereas the conventional batch-
well ELISA requires approximately 10 h of time consumption
including the sample elution step. Our developed system combines
the high sensitivity of LIF detection and the inherent properties of
optical fibers (e.g., chemical inertness of the surface, high transmis-
sion, flexibility, low cost) with microfluidic technology features,
which translates into more rapid manipulation, lower power
requirements, and increased portability of the device. To conclude,
the APSN–APTES-modified glass chip described above enables a
fast, accurate, and selective IRT analysis during the early neonatal
period, demonstrating its potential usefulness for CF newborn
screening.
Fig.6. Correlation between proposed method and ELISA. PM, proposed method.
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series of analyses was repeated for 3 consecutive days in order to
estimate the between-assay precision. The IRT assay showed CV
within-assay values that were below 5%, and the CV between-assay
values were below 6.5% (Table 2).
The accuracy of the LIF immunosensor was tested with a dilu-
tion test performed using an IRT control sample of 580 ng mlÀ1
that was serially diluted in 0.01 M PBS (pH 7.2). The linear
regression equation was RFU = 0.491 + 107.01 Â CIRT, with a linear
regression coefficient r = 0.997 (see supplementary material).
In addition, the proposed method was compared with a com-
mercial ELISA procedure for the quantification of IRT in neonatal
samples. The slopes obtained were reasonably close to 1, indicating
a good correspondence between the two methods (Fig. 6).
In this work, we analyzed three IRT high-level neonatal sam-
ples, three IRT high-level control samples (provided with the
Trypsin-MW ELISA Kit), and 10 low-level neonatal samples. All
samples had been previously confirmed for IRT levels using the
commercial Trypsin-MW ELISA Kit. High-level samples and con-
trols were later analyzed by our proposed quantitative method,
which revealed high concentrations of IRT in all of them. The
analysis of the low-level samples also showed low concentrations
of IRT.
The total assay time required for the determination of IRT con-
centration using the proposed method was 37 min without the
ultrasonic sample elution preparation and less than 40 min includ-
ing it. The conventional batch-well ELISA employs an assay time of
2 h without the sample elution procedure and approximately 10 h
including the sample elution procedure provided by the Blood Spot
Trypsin-MW ELISA Kit.
According to an intensive search, few previously reported arti-
cles were found on IRT detection. First, Xu and coworkers proposed
an assay based on immunoreagents labeled with lanthanide ions,
414
Acknowledgments
415
Q45 416
417
The authors are thankful for financial support from the Univers-
idad Nacional de San Luis, the Agencia Nacional de Promoción
Científica y Tecnológica, and the Consejo Nacional de Investigaci-
ones Científicas y Técnicas (CONICET).
418
419
Appendix A. Supplementary data
420
421
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.ab.2014.06.016.
422
References
[1] T. Asefa, C.T. Duncan, K.K. Sharma, Recent advances in nanostructured Q6 423
424
425
426
427
428
429
430
431
432
chemosensors and biosensors, Analyst 134 (2009) 1980–1990.
[2] Q. Wei, R. Li, B. Du, D. Wu, Y.Y. Han, Y.Y. Cai, Y.F. Zhao, X.D. Xin, H. Li, M.H.
Yang, Multifunctional mesoporous silica nanoparticles as sensitive labels for
immunoassay of human chorionic gonadotropin, Sens. Actuator, B 153 (2011)
256–260.
[3] Q. Wei, X.D. Xin, B. Du, D. Wu, Y.Y. Han, Y.F. Zhao, Y.Y. Cai, R. Li, M.H. Yang, H.
Li, Electrochemical immunosensor for norethisterone based on signal
amplification strategy of graphene sheets and multienzyme functionalized
mesoporous silica nanoparticles, Biosens. Bioelectron. 26 (2010) 723–729.
Please cite this article in press as: M.A. Seia et al., Silica nanoparticle-based microfluidic immunosensor with laser-induced fluorescence detection for the
quantification of immunoreactive trypsin, Anal. Biochem. (2014), http://dx.doi.org/10.1016/j.ab.2014.06.016

YABIO 11781
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7
433
434
435
436
437
438
439
440
441
442
443
444
445
446
447
448
449
450
451
452
453
454
455
456
457
458
459
460
461
462
463
464
465
466
467
468
469
470
471
472
473
474
475
476
477
478
479
480
481
482
483
484
485
486
487
488
489
490
491
492
493
494
495
496
497
498
499
500
501
502
503
504
505
506
507
508
509
510
511
512
513
514
515
516
517
518
519
520
521
522
523
524
525
526
527
528
529
530
531
532
533
534
535
536
537
538
539
540
541
542
543
544
545
546
547
548
549
[4] D. Wu, R. Li, H.X. Wang, S.Q. Liu, H. Wang, Q. Wei, B. Du, Hollow mesoporous
silica microspheres as sensitive labels for immunoassay of prostate-specific
antigen, Analyst 137 (2012) 608–613.
[5] J.H. Lin, Z.J. Wei, C.M. Mao, A label-free immunosensor based on modified
mesoporous silica for simultaneous determination of tumor markers, Biosens.
Bioelectron. 29 (2011) 40–45.
[6] Y.Y. Cai, H. Li, B. Du, M.H. Yang, Y. Li, D. Wu, Y.F. Zhao, Y.X. Dai, Q. Wei,
Ultrasensitive electrochemical immunoassay for BRCA1 using BMIMÁBF4-
coated SBA-15 as labels and functionalized graphene as enhancer,
Biomaterials 32 (2011) 2117–2123.
[7] Y. Wu, C. Chen, S. Liu, Enzyme-functionalized silica nanoparticles as sensitive
labels in biosensing, Anal. Chem. 81 (2009) 1600–1607.
[8] H. Wang, Y. Zhang, H.Q. Yu, D. Wu, H.M. Ma, H. Li, B. Du, Q. Wei, Label-free
electrochemical immunosensor for prostate-specific antigen based on silver
hybridized mesoporous silica nanoparticles, Anal. Biochem. 434 (2013) 123–
127.
[9] S. Liu, Q. Lin, X.M. Zhang, X.R. He, X.R. Xing, W.J. Lian, J. Li, M. Cui, J.D. Huang,
Electrochemical immunosensor based on mesoporous nanocomposites and
HRP-functionalized nanoparticles bioconjugates for sensitivity enhanced
detection of diethylstilbestrol, Sens. Actuators, B 562 (2012) 166–167.
[10] J.H. Lin, Z.J. Wei, H.H. Zhang, M.J. Shao, Sensitive immunosensor for the label-
free determination of tumor marker based on carbon nanotubes/mesoporous
silica and graphene modified electrode, Biosens. Bioelectron. 41 (2013) 342–
347.
[11] B.Q. Liu, B. Zhang, Y.L. Cui, H.F. Chen, Z.Q. Gao, D.P. Tang, Multifunctional gold–
silica nanostructures for ultrasensitive electrochemical immunoassay of
streptomycin residues, ACS Appl. Mater. Interfaces 3 (2011) 4668–4676.
[12] D. Tang, B. Su, J. Tang, J. Ren, G. Chen, Nanoparticle-based sandwich
electrochemical immunoassay for carbohydrate antigen 125 with signal
enhancement using enzyme-coated nanometer-sized enzyme-doped silica
beads, Anal. Chem. 82 (2010) 1527–1534.
[24] J.W. Choi, K.W. Oh, J.H. Thomas, W.R. Heineman, H.B. Halsall, J.H. Nevin, A.J.
Helmicki, H.T. Henderson, C.H. Ahn, An integrated microfluidic biochemical
detection system for protein analysis with magnetic bead-based sampling
capabilities, Lab Chip 2 (2002) 27–30.
[25] M.E. Johnson, J.P. Landers, Fundamentals and practice for ultrasensitive laser-
induced fluorescence detection in microanalytical systems, Electrophoresis 25
(2004) 3513–3527.
[26] H. Corvol, J. Beucher, P. Boëlle, P. Busson, C. Muselet-Charlier, A. Clement, F.
Ratjen, H. Grasemann, J. Laki, C.N.A. Palmer, J.S. Elborn, A. Mehta, Ancestral
haplotype 8.1 and lung disease severity in European cystic fibrosis patients, J.
Cyst. Fibros. 11 (2012) 63–67.
[27] D. Debray, D. Kelly, R. Houwen, B. Strandvik, C. Colombo, Best practice
guidance for the diagnosis and management of cystic fibrosis-associated liver
disease, J. Cyst. Fibros. 10 (2011) 29–36.
[28] A. Leonard, P. Lebecque, J. Dingemanse, T. Leal,
A randomized placebo-
controlled trial of miglustat in cystic fibrosis based on nasal potential
difference, J. Cyst. Fibros. 11 (2012) 231–236.
[29] M.R. Knowles, P.R. Durie, In vivo nasal potential difference: techniques and
protocols for assessing efficacy of gene transfer in cystic fibrosis, N. Engl. J.
Med. 347 (2002) 439–442.
[30] B.J. Rosenstein, G.R. Cutting, The diagnosis of cystic fibrosis: a consensus
statement, J. Pediatr. 132 (1998) 589–595.
[31] C.E. Wainwright, A.L. Quittner, D.E. Geller, C. Nakamura, J.L. Wooldridge, R.L.
Gibson, S. Lewis, A.B. Montgomery, Aztreonam for inhalation solution (AZLI) in
patients with cystic fibrosis, mild lung impairment, and P. aeruginosa, J. Cyst.
Fibros. 10 (2011) 234–242.
[32] R. Weber, M. Pavan, A. Canto de Souza, S. Martins de Castro, An evaluation of
IRT neonatal analytical performance in AutoDELFIA, J. Bras. Patol. Med. Lab. 49
(2013) 388–390.
[33] O. Sommerburg, V. Krulisova, J. Hammermann, M. Lindner, N.M. Stahl, M.
Muckenthaler, D. Kohlmueller, M. Happich, A.E. Kulozik, F. Votava, M.
Balascakova, V. Skalicka, M. Stopsack, M. Gahr, M. Macek Jr., M.A. Mall, G.F.
Hoffmann, Comparison of different IRT–PAP protocols to screen newborns for
cystic fibrosis in three central European populations, J. Cyst. Fibros. 13 (2014)
15–23.
[34] J. Sarles, R. Giorgi, P. Berthézène, A. Munck, D. Cheillan, J.C. Dagorn, M. Roussey,
Neonatal screening for cystic fibrosis: comparing the performances of IRT/DNA
and IRT/PAP, J. Cyst. Fibros. 13 (2014) 384–390.
[35] C. Minasian, A. McCullagh, A. Bush, Cystic fibrosis in neonates and infants,
Early Hum. Dev. 81 (2005) 997–1004.
[36] MP Biomedicals, ImmunoChem Blood Spot Trypsin-MW ELISA Kit, MP
Biomedicals, 2011.
[37] K. Seiler, D.J. Harrison, A. Manz, Planar glass chips for capillary electrophoresis:
repetitive sample injection, quantitation, and separation efficiency, Anal.
Chem. 65 (1993) 1481–1488.
[38] T.P. Segato, W.K. Coltro, A.L. Almeida, M.H. Piazetta, A.L. Gobbi, L.H. Mazo, E.
Carrilho, A rapid and reliable bonding process for microchip electrophoresis
fabricated in glass substrates, Electrophoresis 31 (2010) 2526–2533.
[39] G.D. Liu, J.T. Yan, G.L. Shen, R.Q. Yu, Renewable amperometric immunosensor
for complement 3 (C3) assay in human serum, Sens. Actuators, B 80 (2001) 95–
100.
[13] J. Wang, G. Liu, M.H. Engelhard, Y. Lin, Sensitive immunoassay of a biomarker
tumor necrosis factor-
a based on poly(guanine)-functionalized silica
nanoparticle label, Anal. Chem. 78 (2006) 6974–6979.
[14] L.L. Chen, Z.J. Zhang, P. Zhang, X.M. Zhang, A.H. Fu, An ultra-sensitive
chemiluminescence immunosensor of carcinoembryonic antigen using HRP-
functionalized mesoporous silica nanoparticles as labels, Sens. Actuators, B
155 (2011) 557–561.
[15] C.H. Self, D.B. Cook, Advances in immunoassay technology, Curr. Opin.
Biotechnol. 7 (1996) 60–65.
[16] H. Becker, L.E. Locascio, Polymer microfluidic devices, Talanta 56 (2002) 267–
287.
[17] S.K.
Sia,
G.M.
Whitesides,
Microfluidic
devices
fabricated
in
poly(dimethylsiloxane) for biological studies, Electrophoresis 24 (2003)
3563–3576.
[18] D. Erickson, D. Li, Integrated microfluidic devices, Anal. Chim. Acta 507 (2004)
11–26.
[19] J. Wang, Portable electrochemical systems, Trends Anal. Chem. 21 (2002) 226–
232.
[20] C. Farra, R. Menassa, J. Awwad, Y. Morel, P. Salameh, N. Yazbeck, M. Majdalani,
R. Wakim, K. Yunis, S. Mroueh, F. Cabet, Mutational spectrum of cystic fibrosis
in the Lebanese population, J. Cyst. Fibros. 9 (2010) 406–410.
[21] C. Colombo, P.M. Battezzati, A. Crosignani, A. Morabito, D. Costantini, R.
Padoan, A. Giunta, Liver disease in cystic fibrosis: a prospective study on
incidence, risk factors, and outcome, Hepatology 36 (2002) 1374–1382.
[22] M.R. Knowles, P.R. Durie, What is cystic fibrosis?, N Engl. J. Med. 347 (2002)
439–442.
[40] Y.Y. Xu, K. Pettersson, K. Blomberg, I. Hemmilä, H. Mikola, T. Lövgren,
Simultaneous quadruple-label fluorometric immunoassay of thyroid-
stimulating hormone, 17a-hydroxyprogesterone, immunoreactive trypsin,
and creatine kinase MM isoenzyme in dried blood spots, Clin. Chem. 38
(1992) 2038–2043.
[41] B.A. Lindau-Shepard, K.A. Pass, Newborn screening for cystic fibrosis by use of
a multiplex immunoassay, Clin. Chem. 56 (2010) 445–450.
[23] A. Lindblad, H. Glaumann, B. Strandvik, Natural history of liver disease in cystic
fibrosis, Hepatology 30 (1999) 1151–1158.
550
Please cite this article in press as: M.A. Seia et al., Silica nanoparticle-based microfluidic immunosensor with laser-induced fluorescence detection for the
quantification of immunoreactive trypsin, Anal. Biochem. (2014), http://dx.doi.org/10.1016/j.ab.2014.06.016