Analytical and Bioanalytical Chemistry (2018) 410:2665–2669

https://doi.org/10.1007/s00216-018-0965-2

COMMUNICATION

Sensitive paper-based analytical device for fast colorimetric

detection of nitrite with smartphone
Xiu-Xiu Zhang 1 & Yi-Zhen Song 1 & Fang Fang 1 & Zhi-Yong Wu 1

Received: 8 December 2017 / Revised: 5 February 2018 / Accepted: 14 February 2018 / Published online: 19 March 2018
# Springer-Verlag GmbH Germany, part of Springer Nature 2018

Abstract
On-site rapid monitoring of nitrite as an assessment indicator of the environment, food, and physiological systems has drawn
extensive attention. Here, electrokinetic stacking (ES) was combined with colorimetric reaction on a paper-based device (PAD) to
achieve colorless nitrite detection with smartphone. In this paper, nitrite was stacked on the paper fluidic channel as a narrow band
by electrokinetic stacking. Then, Griess reagent was introduced to visualize the stacking band. Under optimal conditions, the
sensitivity of nitrite was 160-fold increased within 5 min. A linear response in the range of 0.075 to 1.0 μg mL−1 (R2 = 0.99) and a
limit of detection (LOD) of 73 ng mL−1 (0.86 μM) were obtained. The LOD was 10 times lower than the reported PAD, and close
to that achieved by a desktop spectrophotometer. The applicability was demonstrated by nitrite detection from saliva and water
with good selectivity, adding 100 times more concentrated co-ions. High recovery (91.0~108.7%) and reasonable intra-day and
inter-day reproducibility (RSD < 9%) were obtained. This work shows that the sensitivity of colorless analyte detection-based
colorimetric reaction can be effectively enhanced by integration of ES on a PAD.

Keywords Paper-based analytical device . Nitrite . Colorimetry . Electrokinetic stacking . Smartphone

Introduction Nitrite can be detected by a variety of laboratory methods

Nitrite is an important potential pathogenic chemical, which
exists widely in physiological, food, environmental, and in-
dustrial systems. Improper use of nitrogen fertilizers can result
in increase of nitrite in soil and water system [1]. Excessive
intake of nitrate and nitrite leads to serious health problems,
for example, stomach cancer, since nitrite is susceptible to the
formation of carcinogenic N-nitrosamine in the human diges-
tive system [2]. Detection of nitrite is also used as an indicator
in the diagnosis of sepsis, infectious gastroenteritis, and men-
ingitis [3]. Bhakta et al. [4] have indicated that nitrite content
from saliva can be used as diagnostic indicator of certain oral
diseases, such as periodontitis.
Electronic supplementary material The online version of this article
(https://doi.org/10.1007/s00216-018-0965-2) contains supplementary
material, which is available to authorized users.
* Zhi-Yong Wu
zywu@mail.neu.edu.cn
1
Research Center for Analytical Sciences, Chemistry Department
College of Sciences, Northeastern University,
Shenyang, Liaoning 110819, China
based on analytical instruments, such as chromatographic,
chemiluminescence, flow injection analysis, capillary electro-
phoresis, spectrophotometry, and electrochemical analysis [1,
5–7]. In recent years, various graphene nanoparticle compos-
ites have also been applied to nitrite detection [8]. However,
these analytical methods generally require expensive equip-
ment, specialized personnel, and are not suitable for fast on-
site analysis.
Paper-based analytical device (PAD) as a new detection plat-
form was introduced by Whitesides in 2007 [9, 10]. PAD is
advantageous in low cost, portability, easy to use, and low
sample and reagent consumption, which is particularly suitable
for on-site rapid analysis and point-of-care tests (POCT)
[11–13]. Numerous PAD methods for determination of nitrite
have been reported, and the major detection methods include
colorimetric, fluorescent, and electrochemical [14, 15].
Colorimetric analysis of nitrite on PAD with Griess reagent is
the simplest and most commonly used [16]. Nitrite reacts with
sulfanilamide in the presence of citric acid, and a diazonium ion
bond with N-1-napthylethylenediamine dihydrochloride
(NED) is produced, which forms a magenta azo compound
(see Electronic Supplementary Material (ESM) Fig. S1) [17,
18]. Colorimetric analysis of nitrite can be achieved based on
2666
image analysis, and the most relevant reported results are sum-
marized in Table S1 in the ESM [4, 16–22]. As can be seen
from the table, nitrite reacts with Griess reagent on the paper
fluidic channel of various shapes to form an azo dye on detec-
tion zone. The lowest limit of detection (LOD) of 1.0 μM was
achieved by flatbed scanner, and LOD of 11.3 μM was
achieved with smartphone. A smartphone is more superior to
a bulky scanner in the light of on-site analysis. In addition to
Griess reagent, tetrazine-based chemistry and paper indicators
coated single walled carbon nanotubes dispersions, poly (4-
aminosytrene) aqueous solutions, and 2-naphthol single walled
carbon nanotubes dispersions were also used for indication of
nitrite [23, 24].
Although image-based colorimetric detection on PAD is
simple and intuitive, the sensitivity is still lower than desktop
spectrophotometer-based methods. Several methods have
been proposed to enhance sensitivity of PAD, such as surface
modification, evaporation concentration, isotachophoresis
(ITP), ion concentration polarization (ICP), and field-
amplified sample stacking (FASS) have been reported [25].
In this work, electrokinetic stacking (ES) mechanism was
successfully integrated with Griess reaction for real-time anal-
ysis of colorless nitrite on a simple PAD with smartphone. The
LOD was 10 times lower than the reported, and the detect
ability became comparable to desktop spectrophotometer
methods. Fast detection of nitrite from saliva and water sam-
ples were successfully demonstrated by the proposed PAD
method with good selectivity and satisfactory recovery rate.
Experiment
Reagents and instruments
Tris(hydroxymethyl)aminomethane (Tris), hydroxyethyl cel-
lulose (HEC), potassium nitrite (KNO2), sulfanilamide, citric
acid, and N-1-napthylethylenediamine dihydrochloride
(NED) were purchased from Aladdin Reagent Co., Ltd.
(Shanghai, China). Hydrochloric acid (HCl), sodium nitrate
(NaNO3), sodium phosphate (Na3PO4·12H2O), sodium chlo-
ride (NaCl), sodium pyrophosphate (Na4P2O7·10H2O), anhy-
drous sodium sulfite (Na2SO3), anhydrous sodium carbonate
(Na2CO3), and sodium fluoride (NaF) were obtained from
Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China).
The working solutions of nitrite were freshly prepared by de-
ionized water-dilution from its stock solution (0.02 g mL−1).
A small booster converter (GRB12300D, ESSO Latham
Electronic Technology Co., Ltd., Shenzhen, China) connected
with a portable battery (PH 50, Romoss Technology Co., Ltd.,
Shenzhen, China) was used as the power supply, which can
output a constant voltage of 300 V. The smartphone (M1 Note,
Meizu, China) was fixed on the stereomicroscope (SZM45-
B1, RuiHoge, Nanjing, China) or 12.5 × macro lens (Micro
Zhang X.-X. et al.
Technology Co., Ltd., Shengzhen, China) to acquire the image
of the color band. Multimeter (MT-1232, ProsKit, Taiwan)
was used to monitor the current. A centrifuge (TGL-16G,
Fitjar Analytical Instruments Co., Ltd., Shanghai, China)
was used for sample treatment. The control experiment was
conducted by a desktop spectrophotometer with a cell
(V56000, Shanghai Metash Instruments Co., Ltd., Shanghai,
China). Glass fiber paper purchased from Kejia
Environmental Products Co., Ltd. (Dezhou, China) was cut
into paper fluidic channel with horizontal channel (32 mm ×
3 mm) and vertical channel (5 mm × 2 mm). Platinum wire
electrodes of 0.5 mm were used to apply the voltage.
Measurement protocol
A smartphone was fixed on stereomicroscope or macro lens by
an attaching clamp. Glass fiber strip was placed between two
reservoirs, and 5 mm platinum wire was immersed into the
reservoir. 200 μL 0.4 M Tris-HCl with electrical conductivity
19.51 mS/cm (pH = 8.0, containing 0.2% HEC) was loaded in
the anode reservoir, and the horizontal channel of glass fiber
strip was wetted by capillary action. Two hundred microliters
of sample solution was added to the cathode reservoir. With the
application of a voltage, the target component from the sample
reservoir was stacked on the horizontal channel. The circuit
was disconnected when the stacking was finished, and 5 μL
Griess reagent (30 mM sulfanilamide, 330 mM citric acid, and
10 mM N-1-napthylethylenediamine dihydrochloride) was in-
troduced to the end of the side channel with a pipette to visu-
alize the stacking band. The stacking band was imaged by the
camera Fv5 software of smartphone in dark conditions, which
was stored in JPG format. Then, the JPEG images were proc-
essed with ImageJ by adjusting some parameters such as
BImage,^ BAdjust,^ and BColor Threshold^. The colorimetric
process was shown in Fig. 1b.
Sample collection and treatment
Saliva samples from three volunteers were collected directly
into a 10-mL centrifuge tube in the morning 30 min after the
oral hygiene. All subjects received informed consent forms
before inclusion in the study and voluntarily agreed to partic-
ipate. The samples were filtered through a 0.22-μm syringe
filter and centrifuged at 2600 rpm for 15 min by a desktop
centrifuge. Saliva supernatant marked as 1, 2, and 3 were kept
at − 20 °C when not in use and defreezed at room temperature
before the detection, following the procedures reported [15].
The samples were diluted 50, 50, and 100 times, respectively,
to fit the detection range of the PAD method.
The water samples were obtained from the local river water
and supermarket, respectively, and the sample preparation was
the same as described for the saliva samples preparation.
Sensitive paper-based analytical device for fast colorimetric detection of nitrite with smartphone 2667

Fig. 1 a Illustration and

schematic of electrokinetic
stacking process. b Colorimetric
procedure illustration. c Display
of typical visualization results.
The anode background
electrolyte: 0.4 M Tris-HCl
(contain 0.2% HEC); the cathode
solution: nitrite standard solution;
voltage 300 V; the experimental
devices are systematically
protected from light

Results and discussion
It has been visually demonstrated that an electric field gradient
can be established on a paper fluidic channel based on titration
reaction between background electrolyte (BGE) and the OH−/
H+ ions from water, which has been used for fluorescent de-
tection [24, 25]. Charged analytes can be electrokinetically
stacked as a narrow and stable band on a paper fluidic channel
taking advantage of a field-amplification effect. Nitrite, as a
negatively charged component, should be stacked following
the same mechanism as shown in Fig. 1a. Given that nitrite
itself is colorless, a side channel was used to introduce Griess
reagent to visualize the stacking band (see ESM Fig. S2). In
this way, the visualized band image could be acquired in real
time by a smartphone for colorimetric detection of nitrite.
Figure 1c shows the image of a typical visualized stacking
band as a result of Griess reagent introduction after ES.
6 mm diameter and the images of disks were acquired by the
same smartphone and processed following the same proce-
dure as that for the PAD with ES. Figure 3 is the calibration
result. The calibration curve of PAD is also provided in the
same figure for comparison purposes. The same intensity val-
ue of 56.72 (horizontal dotted line) corresponds to
119.4 μg mL−1 (point a) and 0.75 μg mL−1 (point b), respec-
tively, for the two calibration curves, indicating an estimated
enhancement factor of 160-fold. This means that with ES, the
sensitivity was over two orders of magnitude improved.
Selectivity to co-ions
PAD ES can be performed in two modes, positive and nega-
tive [24]. With negative working ES mode as used in this
work, nitrite and all other negatively charged analytes can be
stacked into the same band. Co-ion interference could be an
issue for detection of nitrite. In order to evaluate the selectivity

Calibration

The calibration of nitrite (0.075~2 μg mL−1) was conducted

under the optimized conditions (see ESM Figs. S3 and S4 for
condition optimization), and the results are shown in Fig. 2. A
linear response was obtained in the range of 0.075~1.0 μg mL−1
(R2 = 0.99), and the relative standard deviation (RSD) was less
than 7.3%. The estimated limit of detection was 0.073 μg mL−1
(0.86 μM) based on 3-fold of standard deviation of blank sam-
ples and the slope of linear equation (LOD =3σ/k. σ = 0.85 was
the standard deviation of the blank sample signal, and k = 34.8
was the slope of the standard curve). This LOD was closed to
detection capability that obtained by a desktop spectrophotom-
eter based on the same color reaction (see ESM Fig. S5).
To quantitatively evaluate the contribution of ES to the
sensitivity, a comparative assay was conducted. Ten microli- Fig. 2 Electrokinetic stacking analytical curve. Anode reservoir: 0.4 M
Tris-HCl (contain 0.2% HEC); cathode reservoir: nitrite standard solution
ters of nitrite standard solution (5.00~200 μg mL−1) was (0.075~2.5 μg mL−1); 5 μL Griess reagent for color development; what
mixed with excessive Griess reagents to ensure the full forma- inset is the standard curve in range of 0.075~1.0 μg mL−1. Each intensity
tion of pink color and loaded directly onto the paper disks of value is averaged from four measurements
2668 Zhang X.-X. et al.

To compensate the poor focusing ability of a smartphone

for short working distance (~ 5 cm), a stereomicroscope was
used to improve the sharpness of the acquired image.
However, the microscope was a bit bulky for on-site analysis.
To maintain the portability of the PAD system, a small com-
mercial available micro lens clip as a substitute of stereomi-
croscope was adapted to the smartphone camera, and similar
results were obtained as shown in Table 1. In addition, this
PAD method is suitable for on-site analysis of real samples,
since sample pretreatment can be very simple. Laboratory
operations like centrifugation and filtration treatment could
be eliminated without major difference of the results (see
ESM Fig. S8).

Fig. 3 Calibration curves of nitrite with and without ES. The red curve
represents the electrokinetic enrichment curve. The blue curve represents
the non-electrokinetic enrichment curve. Each intensity value is averaged
from four measurements
of this PAD detection method, we investigated the nitrite sig-
nal intensity variations due to addition of 100 times more
concentrated co-ions of PO43−, Cl−, P2O74−, SO32−, CO32−,
F−, NO3−, respectively. As can be seen from ESM Fig. S6,
no obvious changes were noticed due to addition of these ions.
Application for saliva and water samples
The applicability of this PAD was demonstrated by detection
of nitrite from three saliva samples and two water samples,
and the results were shown in Table 1. Nitrite in saliva was
relatively high, so the samples were diluted before PAD de-
tection to satisfy the linear range. The reproducibility was
demonstrated by intra-day and inter-day measurements of sa-
liva samples, and less than 9% RSD were obtained (see ESM
Fig. S7). The recovery rate ranges from 91.0 to 108.7% (see
ESM Table S2). The concentration of nitrite from a river water
sample was 0.60 ± 0.03 and 0.57 ± 0.01 μg mL−1 by the en-
hanced PAD and the desktop spectrophotometer, respectively.
The two results had no obvious difference according to t test
under confident level of 95%.
Table 1 Determination of nitrite form saliva and water samples
Conclusions
In this work, a smartphone-based PAD combined with ES and
post color reaction for fast and sensitive real-time detection
was successfully demonstrated. Through system optimization,
the sensitivity was improved by 160-fold and a LOD
(73 ng mL−1) comparable to that of a desktop spectrophotom-
eter was achieved. Fast detection of nitrite from saliva and
water samples with minimum sample pretreatment were dem-
onstrated with high recoveries and good intra-day and inter-
day RSD. This PAD method is particularly applicable for fast
on-site analysis or POCT. PAD colorimetric detection of other
colorless charged target analytes could also be developed fol-
lowing this method.
Acknowledgements Financial supports from the National Natural
Science Foundation of China (21575019) and Educational Department
of Liaoning Province (LZ2015036) are acknowledged.
Compliance with ethical standards
The saliva samples involved in this work were obtained from voluntary
participants with informed consent. This study was carried out in accor-
dance with the ethical principles for medical research involving human
subjects as described by the World Medical Association.
Conflict of interest The authors declare that they have no conflict of
interest.

No. Steromicroscope RSD Micro lens RSD

(μg mL−1) (%) (μg mL−1) (%) References
Saliva 1 12.7 8.2 12.4 7.0
1. Wu J, Wang X, Lin Y, Zheng Y, Lin J-M. Peroxynitrous-acid-
Saliva 2 20.6 2.7 21.5 6.3 induced chemiluminescence detection of nitrite based on
Saliva 3 26.5 3.3 27.3 8.9 microfluidic chip. Talanta. 2016;154:73–9.
Drinking – – – – 2. Zhu J, Li C, Liu S, Liu Z, Yang J, Tian J, et al. A non-diazotization-
water coupling reaction-based colorimetric determination of nitrite in tap
River water 0.60 5.0 0.61 8.4 water and milk. Eur Food Res Technol. 2014;238(5):889–94.
3. Moorcroft MJ, Davis J, Compton RG. Detection and determination
Note: Each intensity value is averaged from four measurements of nitrate and nitrite: a review. Talanta. 2001;54(5):785–803.
Sensitive paper-based analytical device for fast colorimetric detection of nitrite with smartphone
4. Bhakta SA, Borba R, Taba M, Garcia CD, Carrilho E.
Determination of nitrite in saliva using microfluidic paper-based
analytical devices. Anal Chim Acta. 2014;809:117–22.
5. Romitelli F, Santini SA, Chierici E, Pitocco D, Tavazzi B, Amorini
AM, et al. Comparison of nitrite/nitrate concentration in human
plasma and serum samples measured by the enzymatic batch
Griess assay, ion-pairing HPLC and ion-trap GC-MS: the impor-
tance of a correct removal of proteins in the Griess assay. J
Chromatogr B Analyt Technol Biomed Life Sci. 2007;851(1–2):
257–67.
6. Wang X, Masschelein E, Hespel P, Adams E, Van Schepdael A.
Simultaneous determination of nitrite and nitrate in human plasma
by on-capillary preconcentration with field-amplified sample stack-
ing. Electrophoresis. 2012;33(2):402–5.
7. Helaleh MI, Korenaga T. Ion chromatographic method for simulta-
neous determination of nitrate and nitrite in human saliva. J
Chromatogr B Biomed Sci Appl. 2000;744(2):433–7.
8. Yang J-H, Yang H, Liu S, Mao L. Microwave-assisted synthesis
graphite-supported Pd nanoparticles for detection of nitrite. Sensor
Actuat B Chem. 2015;220:652–8.
9. Martinez AW, Phillips ST, Butte MJ, Whitesides GM. Patterned
paper as a platform for inexpensive, low-volume, portable bioas-
says. Angew Chem Int Ed. 2007;46(8):1318–20.
10. Ellerbee AK, Phillips ST, Siegel AC, Mirica KA, Martinez AW,
Striehl P, et al. Quantifying colorimetric assays in paper-based
microfluidic devices by measuring the transmission of light through
paper[J]. Anal Chem. 2009;81(20):8447–52.
11. Li B, Fu L, Zhang W, Feng W, Chen L. Portable paper-based device
for quantitative colorimetric assays relying on light reflectance
principle[J]. Electrophoresis. 2014;35(8):1152–9.
12. Wang H, Li YJ, Wei JF, Xu JR, Wang YH, Zheng GX. Paper-based
three-dimensional microfluidic device for monitoring of heavy
metals with a camera cell phone[J]. Anal Bioanal Chem.
2014;406(12):2799–807.
13. Li B, Yu L, Qi J, Fu L, Zhang P, Chen L. Controlling capillary-
driven fluid transport in paper-based microfluidic devices using a
movable valve[J]. Anal Chem. 2017;89(11):5707–12.
14. Wang P, Wang M, Zhou F, Yang G, Qu L, Miao X. Development
of a paper-based, inexpensive, and disposable electrochemical
sensing platform for nitrite detection. Electrochem Commun.
2017;81:74–8.
2669
15. Zheng X-J, Liang R-P, Li Z-J, Zhang L, Qiu J-D. One-step,
stabilizer-free and green synthesis of Cu nanoclusters as fluorescent
probes for sensitive and selective detection of nitrite ions. Sensor
Actuat B Chem. 2016;230:314–9.
16. Shariati-Rad M, Irandoust M, Mohammadi S. Multivariate analysis
of digital images of a paper sensor by partial least squares for de-
termination of nitrite. Chemom Intell Lab Syst. 2016;158:48–53.
17. Giustarini D, Rossi R, Milzani A, Dalle-Donne I. Nitrite and nitrate
measurement by Griess reagent in human plasma: evaluation of
interferences and standardization. Methods Enzymol. 2008;440:
361–80.
18. Zurcher DM, Adhia YJ, Romero JD, McNeil AJ. Modifying a
known gelator scaffold for nitrite detection. Chem Commun.
2014;50(58):7813–6.
19. Klasner SA, Price AK, Hoeman KW, Wilson RS, Bell KJ,
Culbertson CT. Paper-based microfluidic devices for analysis of
clinically relevant analytes present in urine and saliva. Anal
Bioanal Chem. 2010;397(5):1821–9.
20. Jayawardane BM, Wei S, McKelvie ID, Kolev SD. Microfluidic
paper-based analytical device for the determination of nitrite and
nitrate. Anal Chem. 2014;86(15):7274–9.
21. Cardoso TMG, Garcia PT, Coltro WKT. Colorimetric determina-
tion of nitrite in clinical, food and environmental samples using
microfluidic devices stamped in paper platforms. Anal Methods.
2015;7(17):7311–7.
22. Wang Z, Wang J, Xiao Z, Xia J, Zhang P, Liu T, et al. A paper
indicator for triple-modality sensing of nitrite based on colorimetric
assay, Raman spectroscopy, and electron paramagnetic resonance
spectroscopy. Analyst. 2013;138(24):7303–7.
23. Ortiz-Gomez I, Ortega-Muñoz M, Salinas-Castillo A, Álvarez-
Bermejo JA, Ariza-Avidad M, de Orbe-Payá I, et al. Tetrazine-
based chemistry for nitrite determination in a paper microfluidic
device. Talanta. 2016;160:721–8.
24. Ma B, Xie S-F, Liu L, Fang F, Wu Z-Y. Two orders of magnitude
electrokinetic stacking of proteins in one minute on a simple paper
fluidic channel. Anal Methods. 2017;9(18):2703–9.
25. Song YZ, Zhang XX, Ma B, Wu ZY, Zhang ZQ. Performance of
electrokinetic stacking enhanced paper-based analytical device with
smartphone for fast detection of fluorescent whitening agent. Anal
Chim Acta. 2017;995:85–90.