micromachines
Article
Paper-Based Analytical Device for Zinc Ion
Quantification in Water Samples with Power-Free
Analyte Concentration
Hiroko Kudo, Kentaro Yamada, Daiki Watanabe, Koji Suzuki and Daniel Citterio *
Department of Applied Chemistry, Keio University, 3-14-1 Hiyoshi, Kohoku-ku, Yokohama 223-8522, Kanagawa,
Japan; [email protected] (H.K.); [email protected] (K.Y.); [email protected] (D.W.);
[email protected] (K.S.)
* Correspondence: [email protected]; Tel.: +81-45-566-1568
Academic Editors: Frank A. Gomez and Carlos D. Garcia
Received: 25 March 2017; Accepted: 13 April 2017; Published: 18 April 2017
Abstract: Insufficient sensitivity is a general issue of colorimetric paper-based analytical devices
(PADs) for trace analyte detection, such as metal ions, in environmental water. This paper
demonstrates the colorimetric detection of zinc ions (Zn2+ ) on a paper-based analytical device with an
integrated analyte concentration system. Concentration of Zn2+ ions from an enlarged sample volume
(1 mL) has been achieved with the aid of a colorimetric Zn2+ indicator (Zincon) electrostatically
immobilized onto a filter paper substrate in combination with highly water-absorbent materials.
Analyte concentration as well as sample pretreatment, including pH adjustment and interferent
masking, has been elaborated. The resulting device enables colorimetric quantification of Zn2+ in
environmental water samples (tap water, river water) from a single sample application. The achieved
detection limit of 0.53 µM is a significant improvement over that of a commercial colorimetric Zn2+
test paper (9.7 µM), demonstrating the efficiency of the developed analyte concentration system not
requiring any equipment.
Keywords: colorimetry; analyte enrichment; PAD; inkjet printing; 3D-printing
1. Introduction
Zinc ions (Zn2+ ) are a major metal contaminant in environmental water originating for example
from mine drainage, industrial waste water, and galvanized steel pipes, among others. Environmental
standards defining maximum allowable concentrations of Zn2+ are provided by various organizations.
For instance, the WHO guideline for drinking water defines the standard value as 3 mg·L−1 (46 µM) [1].
In Japan, the maximum permissive concentration in drinking water of 1.0 mg·L−1 (15 µM) is set by the
Ministry of Health, Labor, and Welfare [2]. In addition, the recommended water quality criteria for
aquatic life provided by the EPA is even more strict (120 µg·L−1 ; 1.8 µM of Zn2+ ) [3]. Conventional
analytical techniques such as inductively coupled plasma-mass spectrometry (ICP-MS) [4] and atomic
absorption spectrometry (AAS) [5,6] enable selective detection of trace amounts of Zn2+ . However,
the necessity of sophisticated analytical instruments operated by trained specialists at relatively high
running costs hampers their field use in routine environmental monitoring.
In 2007, the Whitesides group has introduced microfluidic paper-based analytical devices (µPADs)
as a novel chemical analysis platform [7]. Aside from advantages such as low-cost, light-weight,
and safe disposability by incineration, the availability of capillary action allowing spontaneous sample
transportation in paper channels has made µPADs simple, yet highly-functional analytical tools.
From the original purpose of medical diagnosis [7], the application of µPADs has been expanded to a
variety of fields [8–14], including environmental monitoring [15]. Examples of µPAD analysis of trace
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metal contaminants in environmental water include electrochemical lead detection [16], colorimetric
iron detection [17], and a lateral flow immunoassay for cadmium detection [18]. Despite its importance,
the demonstration of Zn2+ quantification on µPADs remains scarce. Although not being applied to
environmental monitoring, electrochemical measurement of Zn2+ on a µPAD has been reported with
a limit of detection of 1.0 µg·L−1 (15 nM) [19]. In an example of a colorimetric detection approach,
Feng and co-workers have achieved the detection of 50 µM (3.3 mg·L−1 ) of Zn2+ with the aid of
hierarchical cluster analysis (HCA) [20].
The current work demonstrates the colorimetric detection of Zn2+ on a paper-based analytical
device equipped with a target analyte concentration system to achieve sufficient detection sensitivity for
environmental monitoring. Although colorimetric methods offer an easily detectable signal observable
by the naked eye, their limited detection sensitivity is a general issue for the quantification of low
analyte concentrations [21]. Several reports describe target analyte concentration approaches on PADs,
for example by using heating [22], ion concentration polarization [23], or a pumping system [24].
Most notably, Satarpai et al. have demonstrated the colorimetric detection of low levels of Pb2+ in
environmental water by using a peristaltic pumping system [24]. In the current work, concentration of
Zn2+ from a comparably large amount of sample has been achieved by simple stacking of multiple
layers of cellulosic porous substrates and a filter paper layer for colorimetric detection. Compared to
conventional µPADs, the overlaid highly liquid absorbent cellulose pads allow wicking of an enlarged
sample volume (1 mL), enabling external power-free concentration of Zn2+ from the sample into the
colorimetric detection area. In addition, the requirement for sample pretreatment has been overcome
by integrating the necessary reagents into the sample application area. Most importantly, the influence
of Cu2+ , a major interfering metal ion of the used Zincon colorimetric indicator, has been eliminated
up to a concentration of 50 µM. The developed PAD has been applied to Zn2+ quantification in tap
water and river water samples.
2. Materials and Methods
2.1. Reagents and Equipment
All reagents were used as received without further purification. ZnCl2 , CaCl2 , MgCl2 ,
MnCl2 , CuCl2 , NiCl2 , FeCl3 , and standard solutions of Pb(NO3 )2 , Cd(NO3 )2 , and HgCl2
were purchased from Wako Pure Chemical Industries (Osaka, Japan). Tetramethylammonium
hydroxide·5H2 O (TMAOH), N-Tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid (TAPS) and
poly(diallyldimethylammonium chloride) (PDDA) were purchased from Sigma-Aldrich (St. Louis,
MO, USA). Salicylaldoxime was obtained from TCI (Tokyo, Japan). 1-(2-Hydroxycarbonylphenyl)-5(2-hydroxy-5-sulfophenyl)-3-phenylformazan, sodium salt (Zincon) was obtained from Dojindo
(Kumamoto, Japan). Whatman No. 1 filter paper (460 mm × 570 mm) and CF7 absorbent pads
(22 mm × 50 m) were purchased from GE Healthcare Life Sciences (Buckinghamshire, UK). Glass fiber
(GFDX203000) and cellulose pads (GFCP103000) were purchased from Merck Milllipore (Darmstadt,
Germany). Baking paper was purchased from Toyo Aluminium Ekco Products Co., Ltd. (Osaka, Japan).
Ultrapure water ( >18 MΩ·cm) was obtained from a PURELAB flex water purification system (ELGA,
Veolia Water, Marlow, UK) and used for the preparation of all solutions.
A Xerox ColorQube 8570 wax printer (Xerox, Norwalk, CT, USA) was used to print hydrophobic
barriers designed in Adobe Illustrator CC software. An iP2700 inkjet printer (Canon, Tokyo, Japan) was
used to deposit the colorimetric assay reagent and the polymeric additive (Zincon and PDDA). For this
purpose, standard Canon ink cartridges have been cut open and the sponges inside were removed,
followed by washing with copious amounts of ultrapure water. A Silhouette Cameo electronic knife
blade cutting device (Silhouette, Lehi, UT, USA) in double cutting mode was used to cut glass fiber
disks into the desired sizes. Hot lamination was performed on a QHE325 laminator (Meiko Shokai,
Tokyo, Japan) set for plain copy paper and a total thickness of 100 µm. A 9000F MARK II color scanner
(Canon, Tokyo, Japan) was used to acquire images for quantitative evaluation of colorimetric response
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and numerical color intensity values were measured by the Image J color analysis software (NIH,
Bethesda, MD, USA). An Objet30 Prime (Stratasys, Eden Prairie, MN, USA) 3D-printer was used
to fabricate the device holder designed on 123D design software (Autodesk, San Rafael, CA, USA).
A potentiometric Ca2+ monitoring device (LAQUA twin) was purchased from Horiba (Kyoto, Japan).
Zinc test paper was purchased from Kyoritsu Chemical-Check Lab., Corp. (Tokyo, Japan).
2.2. Investigation of Reaction Time of Cu2+ -Salicylaldoxime Chelation
150 µL of aqueous CuCl2 solution (50 µM) and 150 µL of salicylaldoxime solution (7.15 mM) in
TAPS/TMAOH buffer (pH 8.5, 400 mM) were mixed in a micro centrifuge tube. 200 µL of the mixed
solution was added to a microtiter plate well containing 10 µL of Zincon solution (1.6 mM) immediately
(for a reaction time of 0 min) or 1 min after mixing (for a reaction time of 1 min). Absorption spectra
were acquired with a VarioskanTM Flash multi spectra microplate reader (Thermo Fisher Scientific,
Waltham, MA, USA).
2.3. Device Fabrication
As shown in Figure 1a,b, the device for the detection of low Zn2+ concentrations is composed of
four sections: Glass fiber layers, colorimetric detection area, absorbent pad layers, and a device holder.
The glass fiber layers were prepared by stacking seven glass fiber disks with different diameters (5, 6,
8, 11, 14, 17, and 20 mm from the bottom to top). Before stacking, salicylaldoxime solution (14.3 mM),
and aqueous CuCl2 solution (100 µM) were pipetted onto the glass fiber disks (detailed conditions are
summarized in Table 1), followed by drying at 40 ◦ C for 1 h.
Table 1. Summary of reagents added onto the glass fiber layers.
Glass Fiber Disk Diameter
5 mm
6 mm
8 mm
11 mm
14 mm
17 mm
20 mm
Added Reagent
14.3 mM of salicylaldoxime in
buffer solution (TAPS/TMAOH,
pH 8.5, 400 mM)
100 µM of aqueous CuCl2 solution
Pipetting Amount
10.0 µL
14.4 µL
25.6 µL
50.0 µL
80.0 µL
116.0 µL
160.0 µL
The detection area was prepared by using the wax printing technique [25,26]. Whatman No.
1 filter paper was cut into A4 size and fed into the ColorQube 8570 printer to print black wax on
both sides of the filter paper in greyscale printing mode. A single detection area consists of an
unmodified hydrophilic circular paper region (4 mm diameter) surrounded by a 20 × 20 mm2 solid
black square-shaped hydrophobic wax region. Seventy detection areas were printed on a single A4
sheet. In order to achieve penetration of the printed wax throughout the paper thickness, the filter
paper sandwiched by baking paper was passed through a hot laminator [27]. Finally, aqueous solutions
of PDDA (1 wt %) and Zincon (1.6 mM) were inkjet-printed onto the hydrophilic circular area in 10 print
cycles from the black and magenta ink reservoirs of the Canon printer, respectively.
Absorbent pad layers were prepared by using two types of cellulose pads (CFSP223000 and CF7).
One sheet of CFSP223000 (cut into 30 × 30 mm2 ) and two sheets of CF7 (cut into 22 × 30 mm2 ) were
manually piled up.
Finally, all porous substrate materials were assembled with the aid of a 3D-printed device holder
composed of two parts (Figure 1c). The glass fiber pads were sequentially put onto the funnel-shaped
pocket prepared on the top side of the holder. Patterned filter paper, CFSP223000, and CF7 were
manually stacked and sandwiched by the screwed 3D-printed parts.
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Figure 1. Design of the 3D-PAD for the detection of Zn2+ with integrated pretreatment and
Figure
1. Design
of the(a)3D-PAD
forillustration;
the detection
of Zn2+
with integrated
pretreatment
and
concentration
systems:
schematic
(b) actual
photograph;
(c) schematic
illustration
concentration
systems:
(a)
schematic
illustration;
(b)
actual
photograph;
(c)
schematic
illustration
of
of disassembled device holder fabricated by 3D-printing.
disassembled device holder fabricated by 3D-printing.
2.4. Detection and Quantification Method
2.4. Detection and Quantification Method
Colorimetric detection was carried out by depositing 1 mL of sample onto the sample application
Colorimetric detection was carried out by depositing 1 mL of sample onto the sample application
area. After the entire liquid volume was absorbed by the absorbent pad layers (approximately 3 min
area. After the entire liquid volume was absorbed by the absorbent pad layers (approximately 3 min
after sample introduction), the detection filter paper was removed from the assembled device for
after sample introduction), the detection filter paper was removed from the assembled device for
color observation. For quantitative evaluation of colorimetric response, the detection filter paper was
color observation. For quantitative evaluation of colorimetric response, the detection filter paper was
attached to a sheet of copy paper with double-sided tape and dried for 15 min at room temperature,
attached to a sheet of copy paper with double-sided tape and dried for 15 min at room temperature,
followed by scanning at 600 dpi resolution and software-based digital color analysis of the photograph.
followed by scanning at 600 dpi resolution and software-based digital color analysis of the
photograph.
3. Results and Discussion
3.1.
Roles ofand
theDiscussion
3D-PAD Components
3.
Results
In order to achieve a low detection limit for Zn2+ with a simple colorimetric indicator on paper,
3.1. Roles of the 3D-PAD Components
a sample volume as large as 1 mL has been handled throughout this research, which is significantly
2+ is
Inthan
orderintocommon
achieve µPAD
a low detection
limit for Zn
with
a simple
indicator on
paper,
larger
analyses (typically
up2+ to
several
tens colorimetric
of µL). Concentration
of Zn
aachieved
sample volume
as large
as 1 mL
beensample
handled
throughout
which
is significantly
by the vertical
passage
ofhas
a large
volume
throughthis
theresearch,
filter paper
detection
area with
larger
than in
common μPAD
analyses
(typically
up to several
tens of μL).detection
Concentration
Zn2+ an
is
the Zincon
colorimetric
indicator.
For ease
of operation,
the colorimetric
of Zn2+ofwith
achieved
the vertical passage
of a largeonly
sample
volume
through
the filter
detectioninarea
integratedbyconcentration
system requiring
a single
sample
application
haspaper
been targeted
this
with
Zincon
colorimetric
indicator.
ease
of operation,
the colorimetric
detection
of additional
Zn2+ with
work.the
The
elaborated
PAD consists
of aFor
filter
paper-based
colorimetric
detection
area and
an
integrated
concentration
systemabsorbent
requiringpad
onlylayers,
a single
has been targeted in
functional
parts:
glass fiber layers,
andsample
deviceapplication
holder.
this work. The elaborated PAD consists of a filter paper-based colorimetric detection area and
3.1.1. Filterfunctional
Paper Detection
Areafiber layers, absorbent pad layers, and device holder.
additional
parts: glass
The colorimetric Zn2+ assay was carried out by using the Zincon indicator. For this purpose,
3.1.1.
Filter
Detection
Area of an inkjet printer onto a circular detection region (3.85 ± 0.03 mm
Zincon
wasPaper
deposited
by means
diameter
after wax perfusion;
n =was
5) prepared
on filter
paper.
an additional
immobilizing
The colorimetric
Zn2+ assay
carried out
by using
theHowever,
Zincon indicator.
For this
purpose,
agent
was
necessary,
because
of
the
water-solubility
of
Zincon.
With
the
current
device
design
Zincon was deposited by means of an inkjet printer onto a circular detection region (3.85 ± 0.03
mm
where upafter
to 1 wax
mL of
sample liquid
is passed on
through
the colorimetric
sensing
layer,immobilizing
it is essential
diameter
perfusion;
n = 5) prepared
filter paper.
However, an
additional
to immobilize
the indicator
itswater-solubility
Zn2+ complex formed
during
Indevice
our previous
study,
agent
was necessary,
becauseand
of the
of Zincon.
Withthe
theassay.
current
design where
a
cationically-charged
nanoparticle
has
been
employed
for
anchoring
sulfonated
colorimetric
indicators
up to 1 mL of sample liquid is passed through the colorimetric sensing layer, it is essential to
onto a filter paper
substrateand
[28].
use
of a piezoelectrically-actuated
inkjetInprinter
was inevitable
immobilize
the indicator
itsThe
Zn2+
complex
formed during the assay.
our previous
study,for
a
dispensing
of
the
nanoparticle
ink.
In
the
present
study,
a
water-soluble
cationic
polymer
(PDDA)
[29]
cationically-charged nanoparticle has been employed for anchoring sulfonated colorimetric
was employed
placepaper
of thesubstrate
cationically-charged
nanoparticle.
Since aqueous solutions
PDDAwas
are
indicators
onto in
a filter
[28]. The use
of a piezoelectrically-actuated
inkjetof
printer
inevitable for dispensing of the nanoparticle ink. In the present study, a water-soluble cationic
polymer (PDDA) [29] was employed in place of the cationically-charged nanoparticle. Since aqueous
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solutions of PDDA are compatible with the thermally-actuated printer used, this allows for the
compatible with
the thermally-actuated
printer
used,
this
allows for
colorimetric
indicator
and As
the
colorimetric
indicator
and the immobilizing
agent
to be
deposited
bythe
using
a single inkjet
printer.
immobilizing
agent
to
be
deposited
by
using
a
single
inkjet
printer.
As
compared
to
the
state
before
compared to the state before sample liquid introduction, Zincon was completely washed away after
sample liquid
Zincon
completely
washed
away
of 1 mL
water
introduction
ofintroduction,
1 mL of water
in thewas
absence
of PDDA
(Figure
2a).after
Withintroduction
10 printing cycles
ofof
PDDA
in
the
absence
of
PDDA
(Figure
2a).
With
10
printing
cycles
of
PDDA
solution
(1
wt
%),
the
pink
color
solution (1 wt %), the pink color of Zincon was maintained after exposure to 1 mL of water (Figure
of
Zincon
was
maintained
after
exposure
to
1
mL
of
water
(Figure
2b).
It
is
postulated
that
the
positive
2b). It is postulated that the positive charge of the quaternary amino groups of PDDA allows
charge of theattractive
quaternary
amino groups
PDDA
allows electrostatic
with[30]
bothand
the
electrostatic
interaction
with of
both
the negatively
charged attractive
cellulosicinteraction
paper surface
negatively
charged
cellulosic
paper
surface
[30]
and
the
sulfonate
groups
of
Zincon.
The
importance
the sulfonate groups of Zincon. The importance of the PDDA immobilizing agent has been
of the PDDA immobilizing
agent 2c).
has been
quantitatively
evaluated
(Figure 2c). In contrast
to the
Zn2+
2+ concentration-dependent
quantitatively
evaluated (Figure
In contrast
to the Zn
color
change
concentration-dependent
change of
with
PDDA
(red markers),
the absence
of PDDA
resulted
in no
with
PDDA (red markers),color
the absence
PDDA
resulted
in no colorimetric
response
(blue
markers).
colorimetric
response
(blue
markers).
The
increased
red
color
intensity
reflects
the
whiteness
of
The increased red color intensity reflects the whiteness of the detection area due to washing out the
of
detection
due to washing
out of the Zincon colorimetric indicator.
the
Zinconarea
colorimetric
indicator.
Thereproducibility
reproducibility of
of inkjet-deposition
inkjet-deposition of
in
The
of the
theZincon
Zinconcolorimetric
colorimetricindicator
indicatorhas
hasbeen
beenexamined
examined
a
quantitative
manner.
For
this
purpose,
digital
color
analysis
of
an
inkjet
printed
PDDA-Zincon
spot
in a quantitative manner. For this purpose, digital color analysis of an inkjet printed PDDA-Zincon
before
exposure
to a sample
was was
performed.
The The
measured
red intensity
was was
found
to beto208
1.47
spot
before
exposure
to a sample
performed.
measured
red intensity
found
be ±
208
±
(n
=
34).
The
small
standard
deviation
clearly
indicates
the
reproducibility
of
reagent
deposition
by
1.47 (n = 34). The small standard deviation clearly indicates the reproducibility of reagent deposition
inkjet
printing.
by
inkjet
printing.
Figure 2. Immobilization of Zincon by poly(diallyldimethylammonium chloride) (PDDA). Scanned
Figure 2. Immobilization of Zincon by poly(diallyldimethylammonium chloride) (PDDA). Scanned
images of the detection areas before and after introduction of 1 mL water (a) in the absence and
images of the detection areas before and after introduction of 1 mL water (a) in the absence and (b)
(b) presence of 10 cycles of inkjet deposited PDDA (scale bars: 4 mm); (c) quantitative evaluation of
presence of 10 cycles of inkjet deposited
PDDA (scale bars: 4 mm); (c) quantitative evaluation of the
the colorimetric response to Zn2+ in the presence (red markers) and absence (blue markers) of PDDA
2+ in the presence (red markers) and absence (blue markers) of PDDA (in
colorimetric
response
to
Zn
(in this experiment, the glass fiber layers contain salicylaldoxime, buffer component, and CuCl2 ).
this experiment, the glass fiber layers contain salicylaldoxime, buffer component, and CuCl2).
3.1.2. Glass Fiber Layers
3.1.2. Glass Fiber Layers
The glass fiber layers on the top of the PAD serve as an inlet with integrated sample pretreatment
The glass fiber layers on the top of the PAD serve as an inlet with integrated sample pretreatment
function. Among various possible interferences in a Zincon-based Zn2+2+assay, colorimetric response to
function. Among2+various possible interferences in a Zincon-based Zn assay, colorimetric response
copper ions (Cu )2+is most cumbersome owing to the overlapping working pH range (8.5−9.5 for Zn2+ ;
to copper ions (Cu
) is most cumbersome owing to the overlapping working pH range (8.5−9.5 for
5.52+−9.5 for Cu2+ )[31]
and similar maximum absorption wavelength of the colored metal-indicator
2+
Zn ; 5.5−9.5 for Cu )[31] and
similar maximum absorption wavelength of the colored metal-indicator
complexes (620 nm for Zn2+2+ , 600 nm for Cu2+2+ )[31]. Salycilaldoxime, known as an effective chelating
complexes (6202+nm for Zn , 600 nm for Cu )[31]. Salycilaldoxime, known as an effective chelating
agent for Cu [32–34] as well as effective masking agent for other metal ions interfering the
agent for Cu2+[32–34] as well as
effective masking agent2+for other metal 2+
ions interfering the ZinconZincon-based colorimetric
Zn2+ detection including
Fe [35–37]2+and Ni [35,37], has been deposited
2+
2+
based colorimetric Zn detection including Fe [35–37] and Ni [35,37], has been deposited on the
on the glass fiber layers together with the buffer components (TAPS/TMAOH, pH = 8.5). Additionally,
glass
fiber
layers
together
with
the
buffer
components
(TAPS/TMAOH,
CuCl has been placed on the topmost glass fiber layer for further suppressing interference from Cu2+
pH = 2 8.5). Additionally, CuCl2 has been placed on the topmost glass fiber layer for further
(details are discussed in Section 3.2). Glass fiber has been selected as the substrate material of choice
suppressing interference from Cu2+ (details are discussed in Section 3.2). Glass fiber has been selected
for the sample pretreatment region, due to its ability of smooth reagent release.
as the substrate material of choice for the sample pretreatment region, due to its ability of smooth
reagent
release. Pad Layers
3.1.3. Absorbent
Cellulose pad materials (CFSP223000 and CF7) placed at the bottom of the PAD beneath the
detection layer work as a “wicking pad” as seen in lateral flow immunoassay devices. Two sheets
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3.1.3. Absorbent Pad Layers
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Cellulose pad materials (CFSP223000 and CF7) placed at the bottom of the PAD beneath the
detection layer work as a “wicking pad” as seen in lateral flow immunoassay devices. Two sheets of
of CF7
cellulose
provide
absorption
capacity
of waste
sample.
The CFSP223000
cellulose
CF7
cellulose
padpad
provide
highhigh
absorption
capacity
of waste
sample.
The CFSP223000
cellulose
pad,
pad,
placed
between
the
detection
filter
paper
layer
and
the
CF7
pads,
results
in
moderate
sample
placed between the detection filter paper layer and the CF7 pads, results in moderate sample
transportationrate,
rate,ensuring
ensuringsufficient
sufficientreaction
reactiontime
timefor
forZn
Zn2+2+and
andZincon.
Zincon.
transportation
3.1.4.Device
DeviceHolder
Holder
3.1.4.
Thereexist
existseveral
severalapproaches
approachestotofabricate
fabricateμPADs
µPADscomposed
composedof
ofstacked
stackedmultiple
multiplepaper
paperlayers
layers
There
(3D-µPADs). InIn2008,
2008,Martinez
Martinezetetal.
al.first
firstreported
reporteda a3D-μPAD
3D-µPADassembled
assembledby
byusing
using double-side
double-side
(3D-μPADs).
adhesive
tape
and
cellulose
powder
[38].
In
2011,
the
Crooks
group
utilized
an
aluminum
housingtoto
adhesive tape and cellulose powder [38]. In 2011, the Crooks group utilized an aluminum housing
supportan
anorigami
origami3D-μPAD[39].
3D-µPAD [39].InIn2012
2012and
and2013,
2013,sprayed
sprayedadhesive
adhesive[40]
[40]and
andtoner
toner[41]
[41]have
havebeen
been
support
employed
to
attach
multiple
layers
of
paper
substrates.
In
the
current
study,
a
3D-printed
device
holder
employed to attach multiple layers of paper substrates. In the current study, a 3D-printed device
has been
to assemble
the 3D-PAD,
since nosince
modification
of substrate
surfaces surfaces
is involved.
holder
hasprepared
been prepared
to assemble
the 3D-PAD,
no modification
of substrate
is
The
3D-printed
holder
consists
of
two
(top
and
bottom)
parts
fastened
by
screws
(Figure
1c).
The
top
involved. The 3D-printed holder consists of two (top and bottom) parts fastened by screws (Figure
partThe
bears
a funnel-shaped
pocket to accommodate
seven layers ofthe
glass
fiberlayers
substrates
serving
as
1c).
top
part bears a funnel-shaped
pocket totheaccommodate
seven
of glass
fiber
an
inlet
and
sample
pretreatment
region.
substrates serving as an inlet and sample pretreatment region.
3.2. Suppression of Cu2+ Interference
3.2. Suppression of Cu2+ Interference
At first, the effect of Cu2+ masking by salicylaldoxime was confirmed by comparing the
At first, the effect of Cu2+ masking by salicylaldoxime was confirmed by comparing the
colorimetric response obtained for devices with and without the masking agent impregnated glass fiber
colorimetric response obtained for devices with and without the masking agent impregnated glass
layers. As compared to the pink color observed after application of a pure water sample (Figure 3a),
fiber layers. As compared to
the pink color observed after application of a pure water sample (Figure
the presence of 50 µM Cu2+ resulted in a color change of the detection area to blue (Figure 3b) in the
3a), the presence of 50 μM Cu2+ resulted in a color change of the detection area to blue (Figure 3b) in
absence of the glass fiber layers. On the other hand, the color change was significantly suppressed in
the absence of the glass fiber layers. On the other hand, the color change was significantly suppressed
the presence of the glass fiber layers impregnated with salicylaldoxime and the buffer components
in the presence of the glass fiber layers
impregnated with salicylaldoxime and the buffer components
(Figure 3c), indicating successful Cu2+ masking.
(Figure 3c), indicating successful Cu2+ masking.
2+ masking
2+ masking
Figure
ofof
CuCu
by salicylaldoxime:
scanned
images
of theoffilter
paperpaper
detection
area
Figure3.3.Effect
Effect
by salicylaldoxime:
scanned
images
the filter
detection
after
of 1 mL
(a) pure
device
with reagent
impregnated
glass fiber
layers,
50
areaaddition
after addition
of of
1 mL
of (a)water
pure on
water
on device
with reagent
impregnated
glass
fiber (b)
layers,
(b) aqueous
50 µM aqueous
CuCl2 solution
onwithout
device without
glass
fiber(c)layers,
50 µM CuCl
aqueous
CuCl2
on device
glass fiber
layers,
50 μM(c)
aqueous
2 solution
μM
CuCl2 solution
solution
onwith
a device
with impregnated
fiber
layers
(scale
bars: 4 mm).
on
a device
impregnated
glass fiberglass
layers
(scale
bars:
4 mm).
However,
However, deposition
depositionofofsalicylaldoxime
salicylaldoximeand
andbuffer
buffercomponents
componentswas
wasnot
notsufficient
sufficienttotoallow
allow
2+
2+
2+ still differed
accurate
Zn
quantification
in
the
presence
of
Cu
.
Typical
calibration
curves
for
Zn
2+
2+
2+
accurate Zn quantification in the presence of Cu . Typical calibration curves for Zn still differed in
inthethe
absence
(0 μM)
or presence
a Cu2+ background
in the
sample
4a).
absence
(0 µM)
or presence
(30, 50(30,
µM)50
of μM)
a Cu2+ofbackground
in the sample
(Figure
4a). (Figure
Interestingly,
2+. Based on
Interestingly,
no
differences
were
observed
between
the
presence
of
30
and
50
μM
of
Cu
2+
no differences were observed between the presence of 30 and 50 µM of Cu . Based on the observed
the
observedshift
downward
shift of the
of the
calibration
curve with
the
downward
of the y-intercept
ofy-intercept
the calibration
curve
with identical
slopeidentical
upon theslope
initialupon
addition
2+ only and no further shifts at
2+ background concentration, it was
initial
addition
of
Cu
increased
Cu
2+
2+
of Cu only and no further shifts at increased Cu background concentration, it was postulated that
2+ reaches the detection area despite the presence
postulated
a constant
amountCu
of2+unmasked
Cudetection
a constant that
amount
of unmasked
reaches the
area despite the presence of the masking
ofagent
the in
masking
agent
in
the
glass
fiber
areas.
To
evaluate
the this
possible
cause behind
this
the glass fiber areas. To evaluate the possible cause behind
experimentally
observed
experimentally
observed
behavior,
a
simple
solution-based
experiment
was
performed.
Although
the
behavior, a simple solution-based experiment was performed. Although the reported complex
2+
reported
complex
formation
higher for Cu -salicyladoxime
(log
K = 7.94) to
[42]
2+ -Zincon
formation
constant
is higherconstant
for Cu2+is-salicyladoxime
(log K = 7.94) [42]
compared
Cucompared
2+-Zincon (log K = 7.5) [43,44], there is a difference in binding kinetics. As shown in Figure 4b, a
to
Cu
(log K = 7.5) [43,44], there is a difference in binding kinetics. As shown in Figure 4b, a significant
significant
at 600 nm,the
representing
Cu2+-Zincon
complex,
is observed
absorbanceabsorbance
peak at 600 peak
nm, representing
Cu2+ -Zinconthe
complex,
is observed
immediately
after
immediately
after
mixing
of
reagents,
while
this
peak
is
completely
suppressed
and
the
resulting
mixing of reagents, while this peak is completely suppressed and the resulting spectra overlapping
spectra
overlapping
the metal
free absorption
Zincon
(480 nm)
when
Cu2+ andto
the metal
free absorption
spectrum
of Zinconspectrum
(480 nm) of
when
allowing
Cu2+
andallowing
salicylaldoxime
salicylaldoxime
to interact
for 1 mintobefore
addition
to the Zincon
indicator
solution.
Thisinteraction
indicates
interact for 1 min
before addition
the Zincon
indicator
solution.
This indicates
a fast
of Cu2+ ions with Zincon, followed by the slower displacement with the thermodynamically more
Micromachines
Micromachines 2017,
2017, 8,
8, 127
127
Micromachines 2017, 8, 127
77 of
of 12
12
7 of 12
2+ ions with Zincon, followed by the slower displacement with the
a fast interaction of Cu2+
2+ present
thermodynamically more stable salicyladoxime complex. The fact
that the chelation of Cu2+
stable salicyladoxime complex. The fact that the chelation of Cu2+ present in the sample solution by
2+
in the sample solution by salicylaldoxime is not instantaneous results in a 2+
certain amount of free Cu2+
salicylaldoxime is not instantaneous results in a certain amount of free Cu reaching the colorimetric
reaching the colorimetric detection area of the device, inducing a colorimetric signal. To reduce the
detection area of the device, inducing a colorimetric signal. To reduce the shift in the calibration curve
shift in the calibration curve caused by this phenomenon, it was attempted to pre-deposit CuCl22 as a
caused by this phenomenon, it was 2+
attempted to pre-deposit CuCl as a sample-independent source of
sample-independent
source of Cu 2+ on the topmost glass fiber 2layer. Calibration curves obtained
Cu2+ on the topmost glass fiber layer. Calibration
curves obtained using devices with pre-deposited
2+ (Figure 4c) demonstrate that, through this strategy, the
using
devices with pre-deposited Cu2+
2+
Cu (Figure 4c) demonstrate
that, through this strategy, the influence of different Cu2+ background
2+ background concentrations in samples (0, 30, and 50 μM) were finally
influence of different Cu2+
concentrations in samples (0, 30, and 50 µM) were finally significantly reduced.
significantly reduced.
2+ in the presence of 0, 30, and 50 µM of Cu2+ background
Figure 4. (a)
(a) Calibration curves
curves for Zn
Zn2+
2+ background
Figure
in
Figure 4.
4. (a) Calibration
Calibration curves for
for Zn2+
in the
the presence
presence of
of 0,
0, 30,
30, and
and 50
50 μM
μM of
of Cu
Cu2+
background
obtained
on
devices
with
glass
fiber
layers
impregnated
with
salicylaldoxime
and
buffer
components.
obtained
obtained on
on devices
devices with
with glass
glass fiber
fiber layers
layers impregnated
impregnated with
with salicylaldoxime
salicylaldoxime and
and buffer
buffer components.
components.
2+ masking by salicylaldoxime
(b) Absorbance spectra
spectra in solution
solution showing the
the delayed kinetics
of Cu
2+ masking
(b)
masking by
by salicylaldoxime
salicylaldoxime
(b) Absorbance
Absorbance spectra in
in solution showing
showing the delayed
delayed kinetics
kinetics of
of Cu
Cu2+
2+ in the
(detailed
experimental
conditions
available
in
Section
2.2).
(c)
Calibration
curves
for Zn
2+ in
(detailed
available
in the
the
(detailed experimental
experimental conditions
conditions 2+
available in
in Section
Section 2.2).
2.2). (c)
(c) Calibration
Calibration curves
curves for
for Zn
Zn2+
presence
of
0,
30,
and
50
µM
of
Cu
background
in
the
sample
obtained
on
devices
with
glass
fiber
2+
presence
background in
in the
the sample
sample obtained
obtained on
on devices
devices with
with glass
glass fiber
fiber
presence of
of 0,
0, 30,
30, and
and 50
50 μM
μM of
of Cu
Cu2+ background
layers treated with
with salicylaldoxime, buffer
buffer components, and
and CuCl22..
layers
layers treated
treated with salicylaldoxime,
salicylaldoxime, buffer components,
components, and CuCl
CuCl2.
3.3. Selectivity
Selectivity Study
Study
3.3.
3.3.1. Primary
Primary Heavy
Heavy Metal
Metal Contaminants
Contaminants
3.3.1.
2+ detecting 3D-PAD has been evaluated using heavy metal ions known as
The selectivity
selectivity of
2+ detecting 3D-PAD has been evaluated using heavy metal ions known
The
of the
the Zn
Zn2+
2+ , Hg2+ , Ni2+ , Fe3+ , Cu2+ , and Mn2+ ).
primary
contaminants
in
environmental
water
samples
(Pb2+
, Cd
2+
2+, Cd2+
2+, Hg2+
2+, Ni2+
2+, Fe3+
3+, Cu2+
2+, and Mn2+
2+).
as primary contaminants in environmental
water
samples
(Pb
2+ was tested at 7.5 µM
The
metal
ion
samples
have
been
prepared
in
water
at
10
µM,
whereas
Zn
2+ was tested at 7.5 μM
The metal ion samples have been prepared in water at 10 μM, whereas Zn2+
2+
(Figure 5).
the
presence
of Mn
significantly lowered red
2+
2+ exhibited significantly lowered
(Figure
5). Among
Among the
thetested
testedheavy
heavymetal
metalions,
ions,
the
presence
of Mnexhibited
2+
color
intensity.
This
result
is
attributed
to
the
inability
of
Mn
masking
due
to
its
poor
binding
2+
to its
poor property
binding
red color intensity. This result is attributed to the inability of Mn 2+ masking due
2+ complexes compared to that
with
salicylaldoxime
[35],
and
the
weaker
absorbance
of
the
Zincon-Mn
2+ complexes
property with salicylaldoxime [35], and the weaker absorbance of the Zincon-Mn2+
of free Zincon
[45].
compared
to that
of free Zincon [45].
Figure 5. Selectivity of the Zn2+ detection device against other primary heavy metal contaminants.
2+ detection
Figure
5.
of
device
primary
The
Figure
5. Selectivity
Selectivity
of the
the Zn
Zn2+
detectionto
device
against
other
primary
heavy
metal
contaminants.
The
The graph
shows colorimetric
response
singleagainst
heavyother
metals
(Zn2+ : heavy
7.5
µM,metal
othercontaminants.
metals: 10 µM).
2+
2+
graph
7.5 μM,
μM, other
other metals:
metals: 10
10 μM).
μM).
graph shows
shows colorimetric
colorimetric response
response to
to single
single heavy
heavy metals
metals (Zn
(Zn :: 7.5
Micromachines 2017, 8, 127
Micromachines 2017, 8, 1272+
3.3.2.
Influence of Ca
8 of 12
8 of 12
2+
It Influence
is well known
Ca2+ ions undergo significant interaction with Zincon and that the chelation
3.3.2.
of Cathat
2+
of Ca induces UV/VIS spectral
changes [31,45]. Nevertheless, this does not pose a problem for
It is well known
that Ca2+ ions undergo significant interaction with Zincon and that the chelation
2+
Zincon-based
ZnUV/VIS
assaysspectral
in solution,
since
selectivity
can be readily
achieved
by athe
selection
of Ca2+ induces
changes
[31,45].
Nevertheless,
this does
not pose
problem
forof a
2+ and Ca2+ complexes with the indicator show distinguishable
suitable
measurement
wavelength.
Zn
2+
Zincon-based Zn assays in solution, since selectivity can be readily achieved by the selection of a
2+ assay however, a specific wavelength selection
spectral
In the
case of a paper-based
suitablebehavior.
measurement
wavelength.
Zn2+ and Ca2+Zn
complexes
with the indicator show distinguishable
isspectral
not achievable
inspectionZn
or2+ simple
colorimetric
datawavelength
analysis and
therefore,
behavior. through
In the casevisual
of a paper-based
assay however,
a specific
selection
is
2+ interferes with Zn2+ detection. Considering the inability of selective Ca2+
the
presence
of
Ca
not achievable through visual inspection or simple colorimetric data analysis and therefore, the
masking
its2+abundance
in environmental
samples,
effectofofselective
the presence
of Ca2+ on
presenceand
of Ca
interferes with
Zn2+ detection. water
Considering
the the
inability
Ca2+ masking
2+
2+
quantitative
Zn detection
with the
current
paper-based
was investigated.
On the basis
and its abundance
in environmental
water
samples,
the effect device
of the presence
of Ca on quantitative
2+ detection
the for
current
deviceofwas
investigated.
On the basis
of the Japanese
ofZn
the
Japanesewith
criteria
totalpaper-based
water hardness
< 300
mg·L−1 , [46]
investigated
maximum
2+
−
1
−1, [46] the investigated
2+ concentration 2+
criteria
for
total
water
hardness
of
<
300
mg·L
maximum
Ca
Ca concentration has been selected as 7.5 mM (300 mg·L ). As expected, the presence of Ca
−1). As expected,
has been aselected
as 7.5influence
mM (300 mg·L
presence(Figure
of Ca2+ 6a).
exhibited
a significant
exhibited
significant
on colorimetric
Zn2+the
detection
On the
other hand,
2+
2+ showed no significant −1
2+
influence
on colorimetric
Zninterference
detection (Figure
6a). On the
other hand, Mg
Mg
showed
no significant
when present
at concentration
up
to 12.3 mM (300 mg·L )
−1) (Figure 6b), reflecting the
interference
when present
at concentration
12.3 mMspectral
(300 mg·L
(Figure
6b), reflecting
the reported
absenceupoftoUV/VIS
response
of Zincon towards that
reported
absence
UV/VIScalibration
spectral response
Zincon towards
that cation
[31,45].
different
2+ are The
cation
[31,45].
Theof
different
curves of
recorded
in the presence
of Ca
attributed
to its
2+ are attributed to its hypochromic spectral change
calibration
curves
recorded
in
the
presence
of
Ca
hypochromic spectral change [31], where Zincon-Ca2+ complexes exhibit decreased absorbance and
[31], where Zincon-Ca2+ complexes exhibit decreased absorbance and thus show a weaker color
thus show a weaker color intensity than metal-free Zincon. It was postulated that the decrease of
intensity than metal-free Zincon. It was2+postulated that the decrease of the pink color due to the 2+
the pink color due
to the presence of Ca -bound Zincon leads to an increased sensitivity of the Zn
presence of Ca2+-bound Zincon leads to an increased sensitivity of the Zn2+ concentration-dependent
concentration-dependent calibration curve. On the other hand, the experimental results shown in
calibration curve. On the other2+hand, the experimental results shown in Figure 6a demonstrate that
Figure
6a demonstrate that Ca background concentrations of 500 µM and 7.5 mM result in identical
Ca2+ background concentrations
of 500 μM and 7.5 mM result in identical response curves for Zn2+.
2+ . Therefore, Ca2+ concentration-independent colorimetric Zn2+ measurements
response
curves
for
Zn
2+
Therefore, Ca concentration-independent colorimetric Zn2+ measurements with the paper-based
with
the are
paper-based
are achievable
in water
samples
with
a presence
of Ca
5002+ µM
to 7.5 mMisCa2+
device
achievabledevice
in water
samples with
a presence
of 500
μM
to 7.5 mM
if calibration
ifperformed
calibrationaccordingly.
is performedWhile
accordingly.
While
might
not allow the
application
of the in
present
device
this might
not this
allow
the application
of the
present device
all types
inofall
types
of water
samples,
it can
to be useful
in common
environmental
water
samples,
it can
reasonably
bereasonably
assumed tobe
be assumed
useful in common
environmental
water
samples
water
samples
including
tap water, asin
demonstrated
the following section.
including
tap water,
as demonstrated
the followingin
section.
Figure 6. Zn2+
concentration-dependent response curves recorded in the presence of (a) Ca2+ and
Figure2+6. Zn2+ concentration-dependent response curves recorded in the presence of (a) Ca2+ and (b)
(b) Mg
ion background of various concentrations.
Mg2+ ion background of various concentrations.
3.4.
Water Sample
SampleMatrix
Matrix
3.4.Application
Applicationin
in Environmental
Environmental Water
2+ concentration measurements with the paper-based device developed in this
Quantitative
Quantitative Zn
Zn2+ concentration
measurements with the paper-based device developed in this
2+ into tap water
2+ into
study
matricesby
byspiking
spikingofof5.0
5.0and
and
10.0
µM
studywere
wereperformed
performed in
in real sample
sample matrices
10.0
μM
of of
ZnZn
tap water
and
calculated based
based on
andriver
riverwater
water(Yagami
(YagamiRiver,
River,Yokohama,
Yokohama,Japan)
Japan)samples.
samples. Recovery
Recovery values were calculated
2+2+ solutions
2+ 2+
a calibration
curve
obtained
from
aqueousZn
Zn
solutions with
background
(red(red
lineline
a on
calibration
curve
obtained
from
aqueous
withaa500
500μM
µMCa
Ca
background
Figure7)7)under
underthe
the assumption
assumption that
in in
thethe
real
sample
matrix
is within
the the
ininFigure
that the
the Ca
Ca2+2+concentration
concentration
real
sample
matrix
is within
range
of
500
μM
and
7.5
mM.
This
assumption
was
experimentally
confirmed
by
direct
range of 500 µM and 7.5 mM. This assumption was experimentally confirmed by direct measurements
of Ca2+ in
the collected
using a potentiometric
Ca2+device.
monitoring
device. The
ofmeasurements
Ca2+ in the collected
samples
using asamples
potentiometric
Ca2+ monitoring
The recovery
values
Micromachines 2017, 8, 127
9 of 12
Micromachines 2017, 8, 127
9 of 12
summarized
in Table
2 suggestinthat
reasonable
Zn2+
quantification
regardless
varying
recovery
values
summarized
Table
2 suggest
that
reasonable was
Zn2+possible
quantification
wasofpossible
2+
Ca
concentrations
(0.825
mM
for
tap
water,
1.65
mM
for
river
water).
regardless of varying Ca2+ concentrations (0.825 mM for tap water, 1.65 mM for river water).
Finally, to
to demonstrate
demonstrate the
the superior
superior sensitivity
sensitivity and
and lower
lower detection
detection limit
limit of
of the
the current
current device
device
Finally,
2+ test paper, a calibration curve in the presence of Ca2+ was also recorded
compared
to
a
common
Zn
compared to a common Zn2+ test paper, a calibration curve in the presence of Ca2+ was also recorded
(green line
line in
in Figure
Figure 7)
7) with
with aa commercially
commercially available
available test
test strip.
strip. In
Incomparison
comparisonwith
withthe
theconventional
conventional
(green
2+
Zn2+ test
testpaper
paperbased
basedon
onthe
the same
same Zincon
Zincon indicator,
indicator, the
the current
current device
device exhibited
exhibited aa significantly
significantly
Zn
improved
sensitivity
(slope
of
the
calibration
curves:
−
4.8
for
the
current
device;
−
fortest
the
improved sensitivity (slope of the calibration curves: −4.8 for the current device; −0.95 0.95
for the
2+
test paper).
achieved
detection
of 0.53
allows
thedetection
detectionofofZn
Zn2+ even
evenbelow
below the
the
paper).
The The
achieved
detection
limitlimit
of 0.53
μMµM
allows
the
environmental
standard
provided
by
the
EPA
(1.8
µM),
in
contrast
to
the
much
higher
detection
environmental standard provided by the EPA (1.8 μM), in contrast to the much higher detection limit
2+ test paper currently on the market (9.7 µM). The material cost has been
limit obtained
a Zn
obtained
with with
a Zn2+
test paper
currently on the market (9.7 μM). The material cost has been
calculated
as
$0.398
for
a
single
3D-PAD
andand
$14.0
for the
devicedevice
holderholder
(calculation
details
calculated as $0.398 for a single 3D-PAD
$14.0
for 3D-printed
the 3D-printed
(calculation
on
3D-PAD
and
3D-printed
device
holder
are
available
in
Tables
S1
and
S2
of
the
Supplementary
details on 3D-PAD and 3D-printed device holder are available in Tables S1 and S2 of the
Materials). Considering
their
relatively high-cost,
re-use ofhigh-cost,
the 3D-printed
is desired
in
Supplementary
Materials).
Considering
their relatively
re-usedevice
of theholders
3D-printed
device
practicalisuse.
holders
desired in practical use.
Figure 7.
7. Comparison
Comparison of
of Zn
Zn2+2+detection
detectionwith
withthe
thepaper-based
paper-baseddevice
devicedeveloped
developedin
inthis
thisstudy
studyand
and aa
Figure
2+
2+ in a background of 500 µM Ca2+ targeting
commercial
Zn
test
strip.
(a)
Calibration
curves
for
Zn
commercial Zn2+ test strip. (a) Calibration curves for Zn2+ in a background of 500 μM Ca2+ targeting
real sample
sample analysis
analysis recorded
recorded with
with the
the paper-based
paper-based device
devicedeveloped
developedin
inthis
thisstudy
study(red
(red line)
line) and
and aa
real
2+
2+
commercial Zn
Zn2+ test
teststrip
strip(green
(greenline).
line).(b)
(b)Scanned
Scannedimages
imagesof
of the
the Zn
Zn2+ testing
testingarea
areaof
ofthe
the developed
developed
commercial
2+ concentrations in
device
(top
row)
and
a
commercial
test
strip
(bottom
row)
at
the
corresponding
Zn
2+
device (top row) and a commercial test strip (bottom row) at the corresponding Zn concentrations
2+ .
a background
of of
500
µM
CaCa
2+.
in
a background
500
μM
2+ in spiked tap water and river water samples (n = 3).
Table 2.
Quantification of
of Zn
Zn2+
Table
2. Quantification
in spiked tap water and river water samples (n = 3).
Sample
Sample
water
Tap Tap
water
River water
River water
AddedZn/
Zn/µM
Added
μM
FoundZn/
Zn/µM
Found
μM
Recovery/%
Recovery/%
00
55
10
100
05
510
10
Not
Notdetectable
detectable
5.76±±1.04
1.04
5.76
8.93 ± 1.08
8.93detectable
± 1.08
Not
± 0.72
Not6.48
detectable
10.6 ± 0.47
6.48 ± 0.72
10.6 ± 0.47
-115
115
89
-89
129106
129
106
Micromachines 2017, 8, 127
10 of 12
4. Conclusions
This work has demonstrated concentration and colorimetric detection of Zn2+ on a paper-based
analytical device. Integration of multiple layers of porous substrates, including glass fiber for sample
pre-treatment and cellulose pads for liquid absorption, enables power-free concentration of the
analyte from a single sample application. Importantly, the influence of Cu2+ possessing an identical
working pH range and colorimetric response with Zn2+ has been significantly suppressed with the
aid of the salicylaldoxime masking agent. Although successful application to Zn2+ determination in
environmental water matrix has been achieved, challenges remain in real sample analysis under more
extreme conditions, such as high heavy metal ion contamination or extremely low concentration of
Ca2+ . However, it is believed that the current analyte concentration approach requiring no electrical
power source is compatible with the inherent simplicity of paper-based analytical devices. We hope
that the current system helps to overcome the inherently limited sensitivity of simple colorimetric
devices and contributes to the expanded applicability of paper-based analytical devices for rapid and
simple colorimetric detection of trace metals.
Supplementary Materials: The following are available online at www.mdpi.com/2072-666X/8/4/127/s1;
Table S1: Material cost estimation for the developed 3D-PAD. Table S2: Material cost estimation for the 3D-printed
device holder.
Acknowledgments: K.Y. kindly acknowledges the funding from a Research Fellowship of the Japan Society for
the Promotion of Science (JSPS) for Young Scientists.
Author Contributions: K.Y. and D.C. conceived and designed the experiments; H.K. performed the experiments;
H.K. and K.Y. analyzed the data; K.S. attended the discussions and edited the final version of the paper;
D.W. designed the 3D-printed holder and created the corresponding data file for 3D-printing; H.K., K.Y., and D.C.
wrote the paper; D.C. is the principal investigator of the study.
Conflicts of Interest: The authors declare no conflict of interest.
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