biosensors

Article
Development of Lateral Flow Test-System for the Immunoassay
of Dibutyl Phthalate in Natural Waters
Anna N. Berlina * , Maria Y. Ragozina, Nadezhda S. Komova, Kseniya V. Serebrennikova, Anatoly V. Zherdev
and Boris B. Dzantiev

A. N. Bach Institute of Biochemistry, Research Center of Biotechnology of the Russian Academy of Sciences,
119071 Moscow, Russia
* Correspondence: anberlina@yandex.ru

Abstract: The use of a large amount of toxic synthetic materials leads to an increase in the pollution
of environmental objects. Phthalates are compounds structurally related to esters of phthalic acid that
are widely used in the manufacturing of synthetic packaging materials as plasticizers. Their danger
is conditioned by leaching into the environment and penetrating into living organisms with negative
consequences and effects on various organs and tissues. This work presents the first development of
lateral flow immunoassay to detect dibutyl phthalate, one of the most common representatives of
the phthalates group. To form a test zone, a hapten–protein conjugate was synthesized, and gold
nanoparticles conjugated with antibodies to dibutyl phthalate were used as a detecting conjugate.
The work includes the preparation of immunoreagents, selectivity investigation, and the study of the
characteristics of the medium providing a reliable optical signal. Under the selected conditions for the
analysis, the detection limit was 33.4 ng/mL, and the working range of the determined concentrations
was from 42.4 to 1500 ng/mL. Time of the assay—15 min. The developed technique was successfully
applied to detect dibutyl phthalate in natural waters with recovery rates from 75 to 115%.

Keywords: phthalates; dibutyl phthalate; environment; contaminant; lateral flow immunoassay;

Citation: Berlina, A.N.; Ragozina, natural water; spring water
M.Y.; Komova, N.S.; Serebrennikova,
K.V.; Zherdev, A.V.; Dzantiev, B.B.
Development of Lateral Flow
Test-System for the Immunoassay of 1. Introduction
Dibutyl Phthalate in Natural Waters.
Disubstituted phthalates (phthalic acid esters) are widely used as plasticizers in con-
Biosensors 2022, 12, 1002. https://
doi.org/10.3390/bios12111002
sumer products–food packaging, bottles for drinking water, containers for storing food
products, and raw materials for their manufacturing. A feature of their inclusion to end
Received: 7 October 2022 products is the absence of covalent chemical bonds; therefore phthalates can migrate into
Accepted: 8 November 2022 the environment–water and food from packages of non-decomposable waste [1]. The
Published: 10 November 2022
biodegradation of disubstituted phthalates leads to the formation of the more toxic effects
Publisher’s Note: MDPI stays neutral of phthalates [2–4]. Esters of phthalic acid demonstrate embryotoxic action, damaging
with regard to jurisdictional claims in effects on DNA, toxic effects on nervous and immune systems, and other negative health
published maps and institutional affil- effects [5,6]. Their destructive influence on the reproductive system is associated with the
iations. chemical similarity with estrogens that causes binding with their receptors [7–9], as well
as with other pathological pathways, including apoptosis [10]. According to the USEPA,
priority phthalates for monitoring are dimethyl phthalate (DMP), diethyl phthalate (DEP),
di-n-butyl phthalate (DBP), di-n-octyl phthalate (DnOP), butyl benzyl phthalate (BBP), and
Copyright: © 2022 by the authors.
di-ethyl-hexyl phthalate (DEHP) [11].
Licensee MDPI, Basel, Switzerland.
Dibutyl phthalate and other phthalates are released from microplastics, which are
This article is an open access article
recognized as significant environmental pollutants along with chemical wastes [12]. An
distributed under the terms and
important feature is the more intense release of DBP in the combined presence of salts
conditions of the Creative Commons
and fulvic acids–natural components of soil [12]. In addition to these factors, Ye et al. [13]
Attribution (CC BY) license (https://
showed that light emission, the small particle size of the plastic, and elevated temperature
creativecommons.org/licenses/by/
4.0/).
are also factors contributing to the release of DBP from plastic. With the growth in the

Biosensors 2022, 12, 1002. https://doi.org/10.3390/bios12111002 https://www.mdpi.com/journal/biosensors

Biosensors 2022, 12, 1002 2 of 16

manufacturing and the use of plastic products, the need to control DBP content in water
and food is increasing.
The values of the reference dose (RfD) for DBP in rats are 0.1 mg/kg-day (no side
effects), 125 mg/kg-day (observed side effects), and 600 mg/kg-day (mortality) [7]. The
officially controlled maximum allowable concentrations for DBP in drinking water vary
by country and range from 0.2 mg/L [14] to 0.45 mg/L [15,16]. The main sources of DBP
entry into the body are drinks, grains, and cereal products [17]. At the same time, some
products accumulate phthalates from the package, while others accumulate freely circulat-
ing phthalates from the environment. Song et al. [18] showed that DBP is the dominant
pollutant of natural river waters among all used phthalate esters, and its concentrations
exceed the permissible levels of phthalates in water.
Among the methods used for detecting DBP and other phthalic acid esters, instrumen-
tal methods such as gas and liquid chromatography dominate [19–21]. These methods have
undeniable advantages in terms of sensitivity, selectivity, and reproducibility, but require
long sample preparation, as well as a trained specialist to work with the special expensive
equipment. However, routine monitoring of pollution requires rapid and efficient analytical
methods with sufficient sensitivity and reliability. Most often, immunoassay methods are
proposed for this purpose, as they are widely used in clinical practice and environmental
monitoring [22]. The advantages of immunochemical analytical techniques are specificity,
accuracy, the ability to analyze multiple samples simultaneously, and the possibility of their
implementation in most laboratories. Such immunochemical techniques as enzyme im-
munoassay [23,24], immunofluorescence [25], polarization fluorescent immunoassay [26],
and electrochemical immunoassay [27] have been developed and applied to detect dibutyl
phthalate in natural waters (rivers), tap water, drinking water, and leachate from plastic
bottles in drinking water [28,29].
Due to the absence of lateral flow immunoassays for phthalates in earlier develop-
ments, the aim of this work was to develop and characterize this simple and rapid analytical
technique. Widely presented dibutyl phthalate was the target analyte. The study integrated
the estimation of factors influencing the parameters of the lateral flow assay and finding
solutions to ensure sensitive and reproducible measurements. To carry out this work,
the necessary reagents have been synthesized, and factors affecting the interaction in the
immunochromatographic system were studied.

2. Materials and Methods

2.1. Materials and Components
Di-n-butyl phthalate (DBP), monobutyl phthalate (MBP), diethyl phthalate (DEP),
mono- n-octyl phthalate (MnOP), mono-2-octyl phthalate (M2OP), di- n-octyl phthalate
(DnOP), mono benzyl phthalate (MBzP), mono cyclohexyl phthalate (McHP), mono methyl
phthalate (MMP), Dimethyl phthalate (DMP), Butyl benzyl phthalate (BBzP), diheptyl
phthalate (DHP), monobutyl phthalate (MBP), diethylhexyl phthalate (DEHP), diphenyl
phthalate (DPhP), were from Sigma-Aldrich (St. Louis, MO, USA). 4-amino-DBP was
synthesized and provided by Prof. Suqing Zhao (Guangdong University of Technology,
Guangzhou, China) in lyophilized form. Sodium tetraborate was from Sigma-Aldrich (St.
Louis, MO, USA).
Monoclonal antibody to DBP was from Fapon (Songshan Lake, Dongguan, China)
and had a concentration of 2.4 mg/mL (in 0.1 M PBS, pH 7.2–7.4). Goat anti-mouse IgG
polyclonal antibodies were from IMTEK (1 mg/mL in 0.05 M PBS, pH 7.4, Moscow, Russia).
Albumin from bovine serum was from Sigma-Aldrich (St. Louis, MO, USA). Gold (III)
chloride and sodium citrate were from Fluka (St. Louis, MO, USA). Dimethylformamide,
sodium citrate, potassium carbonate, sodium borate, boric acid, Tris, and Tween-20 were
from Sigma-Aldrich (St. Louis, MO, USA). Potassium dihydrogen phosphate, sodium
chloride, potassium carbonate, potassium hydroxide, and hydrochloric acid were from
Chimmed (Moscow, Russia). All salts, powders, and liquids in their pure form were of
Biosensors 2022, 12, 1002 3 of 16

analytical grade. The preparation of solutions was carried out in accordance with the rules
of work in an analytical laboratory.
Milli-Q water with a resistance of no more than 18.6 MΩ·cm at 25 ◦ C was obtained
with the use of the Simplicity Water Purification System (Millipore, Bedford, MA, USA).
Both water and water solutions were filtered by 0.22 µm filters. Nitrocellulose membranes
(CNPC type) for lateral flow assays were from Advanced Microdevices (MDI, Ambala
Cantt, India) and from Millipore (Millipore 180 type, Bedford, MA, USA). Absorption pad
CFSP223000 and macroporous CFCP203000 glass-fiber membrane were also from Millipore
(Bedford, MA, USA).

2.2. Methods
2.2.1. Synthesis of Hapten-Protein Conjugates
The procedure was based on the azo coupling reaction, when the primary amino
group of the hapten reacted with the carboxyl group of the protein as a carrier [30]. For this
purpose, 12.5 µL of 31% hydrochloric acid, 375 µL of H2 O, and 15 µL of Tween-20 were
added at 0 ◦ C to 5.8 mg of amino-dibutyl phthalate in 25 µL dimethylsulfoxide (DMSO),
with the followed addition of 500 µL of water containing 1.375 mg of NaNO2 .
Next, 10 mg of soybean trypsin inhibitor (STI, carrier protein) was dissolved in 5 mL
0.1 M Na-borate buffer, pH 8.3, and added to the mixture. The color of the reaction mixture
changed to terracotta. Then, after incubation for 2 h at room temperature, dialysis was
performed against 10 mM phosphate buffer, pH 7.4. The concentration of preparation was
calculated from the carrier protein, based on the data of the material balance. The resulting
conjugate STI-DBP was aliquoted into small volumes of 10–40 µL and stored at −20 ◦ C
until use. In this way, each aliquot was defrosted once before use. The molar ratio STI: DBP
under the synthesis was 1:40.

2.2.2. Synthesis of Gold Nanoparticles and Their Conjugation with Antibody

Gold nanoparticles (GNPs) were synthesized via protocol from [31]. First, 200 µL of
5% HAuCl4 and 93 mL of Milli-Q water were mixed in the glass vessel and maintained at
boiling point. Then, 6 mL of 1% sodium citrate solution in water was added to the solution,
and the boiling was continued for the next 5 min. After this, 200 µL of 5% HAuCl4 was
dropped and kept boiling for 15 min. The freshly synthesized nanoparticles were cooled in
the same vessel and used for the conjugation with the antibody. Excess of nanoparticles
was stored at 4–6 ◦ C.
Mouse IgG antibody against DBP (mAb) was dialyzed against 10 mM Tris-HCl buffer
(pH 8.6). Then, freshly prepared GNPs were pre-adjusted to a pH of 8.6 by 0.2 M K2 CO3 .
To prepare the conjugate, 2 mL of GNPs were dropped into the glass vessel containing
300 µL of diluted mAb with followed stirring (at Shaker IntelliMixer (ELMI, Riga, Latvia))
for incubation within 45 min at room temperature. Then, 30 µL of 10% solution of BSA in
Milli-Q water was added to the mixture, and additional incubation followed under stirring
for 15 min at room temperature and 8 h overnight at +4 ◦ C. The obtained conjugate was
separated from the unbound antibody and excess amount of BSA by centrifugation at
13,000 g at +4 ◦ C for 15 min with decanting of the supernatant. Then, 200 µL of 10 mM
Tris-HCl buffer with 2 µL of 10% BSA water solution was added. An optical density of
the obtained conjugate was established using a Shimadzu UV-2450 spectrophotometer
(Shimadzu, Japan).

2.2.3. Characterization of the Obtained Conjugates

UV-Vis Spectroscopy
Absorption spectra of gold nanoparticles-antibody conjugate, as well as hapten–
protein conjugate, were registered by Shimadzu UV-2450 spectrophotometer (Shimadzu,
Japan). The optical density of the synthesized conjugate of gold nanoparticles at 527 nm
(OD527 nm ) with the antibody was 11.9.
Fourier transform infrared (FTIR)
Biosensors 2022, 12, 1002 4 of 16

The STI-DBP conjugate was lyophilized using a freeze dryer (ALPHA 1–2 LDplus,
Martin Christ GmbH, Germany) and stored at 4 ◦ C. The characteristic bands in the spectrum
of STI-DBP were measured using Fourier-transform infrared spectroscopy (FT-IR) in the
4000–400 cm–1 frequency range was conducted using FT/IR-6700 FT-IR Spectrometer
(JASCO Corporation, Japan).
Transmission Electron Microscopy (TEM)
First, 7 uL of native GNPs or the mAb−GNPs conjugate diluted to OD = 1.0 was
dropped onto the surface of 300-mesh grids (Pelco International, Redding, CA, USA) with
preliminarily formed polyform films. JEM CX-100 electron microscope (JEOL, Tokyo, Japan)
was used to obtain images of the nanoparticles, CanoScan 9000F Mark 2 (Canon, Tokyo,
Japan)–for scanning microphotographs at 1200 dpi resolution, and Image Tool software
(San Antonio, TX, USA)–for data handling.

2.2.4. Characterization of Antibody by ELISA Technique

ELISA was conducted using 96-well transparent polystyrene microplates (Costar
9018, Corning Costar, NY, USA). For this purpose, 100 µL of STI−DBP solution with a
concentration 2 µg/mL in 50 mM phosphate buffer saline, pH 7.4, were dropped into
wells of microplate and incubated at +4 ◦ C overnight. Then, the microplate was washed
three times with 50 mM phosphate buffer, pH 7.4, with 0.05% Tween-20 (PBST). A series
of antibody dilutions were added to interact with adsorbed STI−DBP conjugate and
incubated for 1 h at +37 ◦ C. Then, after the washing step, anti-species antibody (goat
anti-mouse IgG) conjugated with horseradish peroxidase (anti-species conjugate, Jackson,
USA, working dilution 1:3000) was added and incubated for 1 h at +37 ◦ C. Next, 100 µL
of the substrate solution (commercial TMB + H2 O2 solution, Immunotech, Russia) was
added, and after 15 min reaction was stopped by the addition of 50 µL 0.1 M H2 SO4 . The
optical density was measured at 450 nm and plotted using Origin 9.0 software (OriginLab
Corporation, MA, USA). The curve was obtained by plotting the relationship between
antibody concentration and the optical density at 450 nm. The concentration of antibody
corresponding to OD450 = 1.0 was chosen for competitive ELISA.
For the competitive assay and the study of selectivity, the same initial stages were
provided. After the incubation of STI−DBP and the washing step, different concentrations
of DBP or other structure analogues were added in the volume of 50 µL to the wells. After
that, 50 µL of antibody solution in PBST was added, and this mixture was incubated for
1 h at +37 ◦ C. Further stages were carried out as described above, namely adding the
anti-species conjugate, developing the resulting complexes with a substrate solution, and
measuring the optical density. The curve was obtained by plotting the relationship between
the optical density at 450 nm and DBP concentration. The dependences of OD450 from the
antigen concentration were approximated by a four-parametric sigmoidal equation.
Cross-reactivity values were calculated according to the following equation:

CR(%) = IC50(DBP)/IC50(analogue) × 100%

2.2.5. Composition of Lateral Flow Tests

Assembling of Lateral Flow Tests
The lateral flow strip was prepared using a working membrane, absorbent membrane
(AP 110), and membrane for sample adsorption. STI−DBP and goat-anti-mouse antibodies
were immobilized on the working membrane to form the test line and the control line,
respectively (Scheme 1). The loading regime used in this work was 0.1 µL per 1 mm.
The reagents were dispensed with the use of Isoflow dispenser (Imagene Technology,
Inc., Hanover, NH, USA). Then, the multimembrane composite was assembled, dried
at 37 ◦ C for 2 h and overnight at 21 ± 1◦ C, and cut onto the test strips with a width of
3.5 mm with the use of a guillotine cutter (IndexCutter, Port Washington, NY, USA). In the
laboratory room used for the manufacturing of test strips, a constant humidity of 27 ± 2%
was supported. All dimensional characteristics of the test strip are shown in Scheme 1.
 or 10 mM Tris-HCl buffer solution (pH 8.8) containing 1% Tween-20 with different DBP
concentrations were dropped into the microplate wells. After this, 1.0 µL of the conjugate
of GNPs with antibody was added to the solution for 10 s. Then, the test strip was inserted
vertically by a sample pad for 8 min to provide fluid current over the entire surface of the
Biosensors 2022, 12, 1002
working membrane. The depth of immersion of the test strip was 5 mm. After that,5 of the
16
test strip was removed and placed horizontally to dry for 5 min.

Scheme
Scheme 1.1. The
The composition
compositionofof the
the test
test strip
strip used
used in
in this
this investigation.
investigation. Membrane
Membrane dimensions
dimensionsand
and
distances:
distances: a—60 mm (total length), b—21 mm (sample pad), c—2 mm (overlap), d—12mm
a–60 mm (total length), b–21 mm (sample pad), с–2 mm (overlap), d–12 mm(distance
(distance
from the beginning of working membrane to test line), e–8 mm (distance between test and control
from the beginning of working membrane to test line), e—8 mm (distance between test and control
zone), f–16 mm (adsorption pad).
zone), f—16 mm (adsorption pad).

Collection
Lateral Flow andAssay
Processing
of DBPData of Lateral Flow Assays
The
The assay was carriedthe
color intensity in test
out at zone
roomafter the assay(21
temperature was ±assessed by processing
1 ◦ C). First, scanned
100 µL of the probe
digital images. The test strips were loaded onto the working surface
or 10 mM Tris-HCl buffer solution (pH 8.8) containing 1% Tween-20 with different DBP of the CanoScan
9000F Mark II (Canon,
concentrations Japan) scanner,
were dropped into the and the images
microplate were
wells. saved
After this,and processed.
1.0 µL For this
of the conjugate
purpose, TotalLab
of GNPs with software
antibody (Nonlinear
was added to theDynamics,
solution forNewcastle
10 s. Then,upon Tyne,
the test stripUK)
waswith 1D
inserted
regimen was used. When processing the images of the test strips, the image
vertically by a sample pad for 8 min to provide fluid current over the entire surface of the was automat-
ically
workingconverted
membrane. to grayscale,
The depththen the test zones
of immersion of thewere selected
test strip wasin accordance
5 mm. with
After that, thetheir
test
location, and the intensity of staining was calculated.
strip was removed and placed horizontally to dry for 5 min. Calibration curves were obtained by
plotting the relationship between color intensity
Collection and Processing Data of Lateral Flow Assays in the test zone and concentration of DBP
in theThe
solution
color by using Origin
intensity 9.0 zone
in the test software
after(OriginLab)
the assay was with the useby
assessed ofprocessing
the four-paramet-
scanned
ric sigmoidal
digital images.equation.
The test strips were loaded onto the working surface of the CanoScan 9000F
Mark II (Canon, Japan) scanner, and the images were saved and processed. For this purpose,
2.2.6. Collection
TotalLab software and Preparation
(Nonlinear of WaterNewcastle
Dynamics, Samples upon Tyne, UK) with 1D regimen was
used.The
When processing
probes of springthewater
images
wereof the test strips,
collected thefrom
(n = 10) image was to
April automatically
July 2022 fromconverted
points
to Russia
in grayscale,
wherethen the testhave
springs zones were for
existed selected
morein accordance
than 20 years.withAbouttheir location,
50–100 and
mL of the
each
intensity of staining was calculated. Calibration curves were obtained by plotting the
relationship between color intensity in the test zone and concentration of DBP in the
solution by using Origin 9.0 software (OriginLab) with the use of the four-parametric
sigmoidal equation.

2.2.6. Collection and Preparation of Water Samples

The probes of spring water were collected (n = 10) from April to July 2022 from points
in Russia where springs have existed for more than 20 years. About 50–100 mL of each
probe was gathered into the glass vial and sent for storage in the laboratory. For the
storage at −20 ◦ C, the probes were aliquoted and frozen. In most cases, water was used
without additional pretreatment. If it was needed, spring waters were filtered through a
0.22 µm syringe filter (Millipore, Bedford, MA, USA) to remove insoluble impurities. All
the samples were confirmed by gas chromatography as pure from dibutyl phthalate and
used for further experiments.

Biosensors 2022, 12, 1002
probe was gathered into the glass vial and sent for storage in the laboratory. For the stor-
age at −20 °C, the probes were aliquoted and frozen. In most cases, water was used with-
out additional pretreatment. If it was needed, spring waters were filtered through a 0.22
µm syringe filter (Millipore, Bedford, MA, USA) to remove insoluble impurities. All the
samples were confirmed by gas chromatography as pure from dibutyl phthalate and used
for further experiments. 6 of 16

3. Results and Discussion

3. 3.1.
Results and Discussion
Synthesis and Characterization of DBP–Protein Conjugate
3.1. Synthesis and Characterization
To form a test of DBP–Protein
zone of the lateral Conjugate
flow test, the hapten–protein conjugate was obtained
fromTo an
form a testderivative
amino zone of theoflateral
DBP and flowatest, the hapten–protein
soybean trypsin inhibitor conjugate
(STI). was obtained
As native DBP
from
doesannot
amino derivative
contain a reactiveof DBP and a soybean
(carboxyl, hydroxyl,trypsin inhibitor
or amino) group, (STI). As native
its amino DBP
derivative
does not contain
substituted a reactive
in position 4 was(carboxyl,
used forhydroxyl,
the synthesis. or amino) group, itswas
The conjugation amino derivative
based on an azo
substituted in position 4 was used for the synthesis. The
coupling reaction and the use of succinic anhydride [30]. The resulting conjugateconjugation was based on an was
azo coupling reaction
characterized by FT-IR andandthe UV-vis
use of succinic anhydride [30]. The resulting conjugate was
spectrophotometry.
characterized
The FT-IRby FT-IR
spectraandof UV-vis spectrophotometry.
the STI−DBP conjugate and the initial STI are shown in Figure
The FT-IR spectra of the STI − DBP
1. In the vibrational spectra of proteins, the amide conjugate and the initial
group STI are showncan
of polypeptides in Figure 1.
be distin-
Inguished
the vibrational spectra of proteins, the amide group of polypeptides
by the characteristic frequencies in the regions of 1700–1600 cm , 1575–1480 cm−1, can be
−1 distinguished
byand
the1300–1230
characteristic −1 , 1575–1480 cm−1 , and
cm−1frequencies
[32]. The peak in the regions
at 1645 cm−1 of(Figure
1700–1600 cmdue
1a) is to stretching vibrations
1300–1230 − 1
cmgroups [32].[33].
TheThepeak at 1645 − 1
cm cm(Figure 1a) is to
due
of the C=O band at 1549 −1 is denoted thetoC–N
stretching vibrations
stretching of
vibrations,
the C=O groups [33]. The band at 1549 cm −1 is denoted to the C–N stretching vibrations,
as well as to the bending of NH in the peak plane [34]. The peak at 1248 cm−1 indicates
asbending
well as to the bending
vibrations of theof N–H
NH in the peak
bond. plane [34].ofThe
The similarity thepeak cm−1isindicates
at 1248peaks
absorption shown in
bending vibrations
both FT-IR spectraofofthepure N–HSTIbond.
(a, black Theline)
similarity
and theofsynthesized
the absorption peaks(b,
STI−DBP is red
shownline).
inSpecifically,
both FT-IR the spectra of pure STI
band at 1066 cm refers−1(a, blackto line)
the and
C=O the synthesized
vibrations. It was STI − DBP
recorded (b, in
both red the
line). Specifically, the band at 1066 cm−1 refers to the C=O vibrations. It was recorded
spectrum of pure DBP and in the protein molecule (ether bond) with other abovemen-
both in the spectrum of pure DBP and in the protein molecule (ether bond) with other
tioned bands. However, after conjugation, the IR spectra revealed the resulting peaks at
abovementioned bands. However, after conjugation, the IR spectra revealed the resulting
1725 cm−1 (ester-C=O stretching vibrations) and 996 cm−1 (C–O–C stretching vibrations)
peaks at 1725 cm−1 (ester-C=O stretching vibrations) and 996 cm−1 (C–O–C stretching
that are obviously characteristics of dibutyl phthalate, which corresponds to the data on
vibrations) that are obviously characteristics of dibutyl phthalate, which corresponds to the
dibutyl phthalate described earlier in the literature [35,36]. The FT-IR spectrum showed
data on dibutyl phthalate described earlier in the literature [35,36]. The FT-IR spectrum
the characteristic absorption frequency at 1725 cm−1, which is associated with the vibration
showed the characteristic absorption frequency at 1725 cm−1 , which is associated with the
of the stretching of the carbonyl group of the ester (Figure 1, red line), as well as the ab-
vibration of the stretching of the carbonyl group of the ester (Figure 1, red line), as well as
sorption peaks at 2920 and 2867 cm−1 representing the saturated C–H symmetric and
the absorption peaks at 2920 and 2867 cm−1 representing the saturated C–H symmetric
asymmetric stretching vibrations of alkyl groups. The FT-IR spectrum showed the absorp-
and asymmetric stretching vibrations of alkyl groups. The FT-IR spectrum showed the
tion peaks at 2920 and 2867 cm−1 representing the saturated C–H symmetric and asym-
absorption peaks at 2920 and 2867 cm−1 representing the saturated C–H symmetric and
metric stretching
asymmetric stretching vibrations
vibrationsof alkyl
of alkylgroups
groupsof DBP. As aAs
of DBP. whole, the obtained
a whole, FT-IR
the obtained spec-
FT-IR
tra confirmed that the conjugate STI−DBP was successfully
spectra confirmed that the conjugate STI−DBP was successfully synthesized. synthesized.

(a) (b)

Figure 1. Characterization of STI−DBP by FT-IR and UV−vis spectrophotometric techniques. (a) The
FT-IR spectra of STI (black line, A) and synthesized STI−DBP (red line, B); (b) UV−vis absorption
spectra of STI−DBP conjugate, STI, and DBP.

The UV-vis absorption spectra of STI, DBP, and the synthesized STI−DBP conjugate
are given in Figure 1b. DBP has a strong absorbance peak at 295 nm. The carrier protein
STI has a peak at 280 nm. The conjugate shows a peak at 281 nm, which is characteristic
 spectra of STI−DBP conjugate, STI, and DBP.

The UV-vis absorption spectra of STI, DBP, and the synthesized STI−DBP conjugate
are given in Figure 1b. DBP has a strong absorbance peak at 295 nm. The carrier protein
STI has a peak at 280 nm. The conjugate shows a peak at 281 nm, which is characteristic
Biosensors 2022, 12, 1002 7 of 16
of the carrier protein, as well as its own unique absorption in the range of 320–430 nm
corresponding to its yellow coloration.

of the carrier protein,

In addition, as well
storage as its own
stability unique absorption
was studied in the
up to 1.5 years range
after of 320–430
synthesis. nmthe
Since
corresponding
most labile part to its
of yellow coloration.
the conjugate is the carrier protein, frequent freezing and thawing can
In addition,
adversely affectstorage stability
its stability [37].was studiedwhen
However, up tostored
1.5 years after synthesis.
in aliquots of up toSince
50 µL,thethe
most labile part of the conjugate is the carrier protein, frequent freezing
necessity of repeated defrosting–freezing cycles was excluded, and the reached retaining and thawing can
adversely affect
activity was its stability
confirmed [37]. However,
by stable whenand
values of LOD stored in aliquots
working range offorup to 50 µL, the
ELISA.
necessity of repeated defrosting–freezing cycles was excluded, and
The antibodies were characterized by enzyme immunoassay (Figure 2). Since the reached retaining
the ox-
activity
idizedwasTMB confirmed
absorbs at by450
stable
nm values of LOD
after adding and working
a stop range
reagent (1M H2for
SO4ELISA.
), all measurements
TheELISA
in the antibodies were characterized
on characterization of theby enzyme were
antibodies immunoassay
carried out(Figure
with this 2). wavelength.
Since the
oxidized TMB absorbs at 450 nm after adding a stop reagent (1M H 2 SO
The antibody concentration required to provide the competitive ELISA was determined 4 ), all measurements
in in
thenon-competitive
ELISA on characterization of the antibodies
ELISA. By replacing DBP with were carried
other out with
phthalates, the this wavelength.
selectivity of an-
The antibody concentration required to provide the competitive ELISA
tibodies was analyzed. As seen from Figure 2a, the concentration of antibodies corre- was determined in
non-competitive ELISA. By replacing DBP with other phthalates, the selectivity of antibod-
sponding to an optical density of about 1.0 was 16 ng/mL (Figure 2a), and this concentra-
ies was analyzed. As seen from Figure 2a, the concentration of antibodies corresponding to
tion was chosen for competitive ELISA. Figure 2b shows the competitive ELISA curve
an optical density of about 1.0 was 16 ng/mL (Figure 2a), and this concentration was chosen
obtained for DBP. The detection limit was 0.9 ng/mL and the working range was from 2.5
for competitive ELISA. Figure 2b shows the competitive ELISA curve obtained for DBP. The
to 83 ng/mL. The antibodies testing showed their suitability for the development of a lat-
detection limit was 0.9 ng/mL and the working range was from 2.5 to 83 ng/mL. The anti-
eral flow immunoassay due to the competition at low concentrations of DBP (Figure 2b).
bodies testing showed their suitability for the development of a lateral flow immunoassay
due to the competition at low concentrations of DBP (Figure 2b).

(a) (b)
Figure 2. The ELISA curves representing the dependence of registered signal (optical density at 450
Figure 2. The ELISA curves representing the dependence of registered signal (optical density at
nm) from (a) antibody concentration, (b) DBP concentration (inhibition curve), n = 3.
450 nm) from (a) antibody concentration, (b) DBP concentration (inhibition curve), n = 3.

TheThe specificity
specificity of the
of the antibody
antibody waswas assessed
assessed by ELISA
by ELISA testing
testing of 15 of 15 derivatives
derivatives of ph-of
phthalic acid esters whose chemical formulas are presented in Figure 3. The
thalic acid esters whose chemical formulas are presented in Figure 3. The compounds were compounds
were to
chosen chosen to take
take into into not
account account not priority
only the only theofpriority of contamination
contamination and production
and production volumes
volumes among disubstituted phthalates but also their possible degradation
among disubstituted phthalates but also their possible degradation to monoderivatives to monode-
rivatives (Figure 3). The cross-reactivity of the antibody with other structurally
(Figure 3). The cross-reactivity of the antibody with other structurally related compounds related
compounds was
was less than 0.1%. less than 0.1%.

3.2. Synthesis and Characterization of Gold Nanoparticles Conjugated with the

Anti-DBP Antibody
Gold nanoparticles are widely used as markers in immunoassays [38,39]. Usually,
preparations of GNPs with a diameter of about 30–40 nm are considered as preferable
for LFIA [40]. However, the advantages of smaller GNPs for competitive schemes were
sometimes noted–13 nm [41,42], 15 nm [43], and 20 nm [44]. The use of larger GNPs in LFIA
could be associated with unproductive competition, as described in [45]. For our work,
preparations of GNPs with a diameter of about 12 nm were obtained. Characterization
of GNPs and their conjugate with antibodies (mAb−GNPs) was provided separately in
comparison to each other.
Biosensors 2022, 12, x FOR PEER REVIEW 8 of 16
Biosensors 2022, 12, 1002 8 of 16

Figure 3. Structural
Figure formulas
3. Structural formulasof
of compounds
compounds ofof the
the phthalates
phthalates group
group used used in selectivity
in selectivity testing. testing.

The mAb–GNPs
3.2. Synthesis conjugate has
and Characterization ofbeen
Goldsynthesized by physical
Nanoparticles adsorption.
Conjugated The
with the quantity
Anti-DBP
of the antibodies used for the conjugation was 14 µg per 1 mL of the GNPs’ sol. This
Antibody
amount was calculated to reach complete stabilization of the GNPs and exclude less stable
Gold nanoparticles
multilayer adsorption. As arewell
widely
as theused as markers
commonly used 50in mM immunoassays
phosphate buffer, [38,39]. Usually,
pH 7.4,
with 0.1 M of
preparations NaCl
GNPs[46] was
withfound to be unacceptable
a diameter to exclude
of about 30–40 nm aggregation,
are considered it wasasreplaced
preferable for
LFIA with 10 However,
[40]. mM Tris-HCl, thepH 8.8. The used
advantages ofstabilizing agent was
smaller GNPs for BSA, because its
competitive effectiveness
schemes were some-
has been shown in a number of works [47,48]. In this media, the mAb−GNPs conjugate
times noted–13 nm [41,42], 15 nm [43], and 20 nm [44]. The use of larger GNPs in LFIA
was stored for 10 months without changes in the properties of its colloid solution.
could beAccording
associated with unproductive
to spectrophotometry, thecompetition,
conjugate hadasandescribed
absorptionin [45].
peak at For our work,
527 nm
preparations
(Figure 4a),of GNPs
which waswith
closeatodiameter of aboutfor12the
the peak location nm were
initial obtained.
GNPs (525 nm).Characterization
The optical of
GNPs and their
density of the conjugate with antibodies
prepared conjugate at the peak(mAb−GNPs)
wavelength waswas 11.9.provided
Based on theseparately
registered in com-
UV-Vis
parison absorption
to each other.spectrum, the location of their maximums and OD values at these
wavelengths
The mAb–GNPs were determined.
conjugateThe hasgiven
beenparameters
synthesized werebyused to control
physical the stabilityThe
adsorption. of quan-
colloidal solutions of mAb−GNP conjugates [49,50].
tity of the antibodies used for the conjugation was 14 µg per 1 mL of the GNPs’ sol. This
A histogram of the distribution of the conjugated particles by size was obtained using
amount
TEMwasdatacalculated to reach
(Figure 4b,c). complete
The average stabilization
diameter was 11.8of ±the1.3 GNPs
nm (n = and exclude
105, minimum less stable
multilayer adsorption.
value–8.76 nm, maximum As well as thenm),
value–14.86 commonly usedof50ellipticity
with a degree mM phosphate buffer,
of 1.17 ± 0.11. The pH 7.4,
withabsence
0.1 M NaCl [46] was
of aggregates in found to be unacceptable
the preparation to exclude
and small deviations fromaggregation, it was replaced
the average diameter
indicates the receipt of a high-quality preparation.
with 10 mM Tris-HCl, pH 8.8. The used stabilizing agent was BSA, because its effective-
ness has been shown in a number of works [47,48]. In this media, the mAb−GNPs conju-
gate was stored for 10 months without changes in the properties of its colloid solution.
According to spectrophotometry, the conjugate had an absorption peak at 527 nm
(Figure 4a), which was close to the peak location for the initial GNPs (525 nm). The optical
density of the prepared conjugate at the peak wavelength was 11.9. Based on the regis-
tered UV-Vis absorption spectrum, the location of their maximums and OD values at these
wavelengths were determined. The given parameters were used to control the stability of
colloidal solutions of mAb−GNP conjugates [49,50].
Biosensors2022,
Biosensors 12,x 1002
2022,12, FOR PEER REVIEW 9 of 16 9 of 16

(a) (b) (c)

Figure 4. Characterization of the GNP−antibody conjugate. (a) Absorption spectrum; (b) Fragment
Figure 4. Characterization of the GNP−antibody conjugate. (a) Absorption spectrum; (b) Fragment
of the TEM micrograph; (c) histogram showing diameter distribution of the conjugated particles.
of the TEM micrograph; (c) histogram showing diameter distribution of the conjugated particles.

3.3.AThe
histogram of the distribution of the conjugated particles by size was obtained using
LFIA Format
TEM data (Figure 4b,c). The average diameter was 11.8 ± 1.3 nm (n = 105, minimum value–
The mAb–GNPs conjugate was incubated with DBP-containing samples for about 10 s
8.76 nm, maximum value–14.86 nm), with a degree of ellipticity of 1.17 ± 0.11. The absence
for mixing. Then, the test strips were dipped by the sample pad to absorb the mixture. The
of aggregates in the preparation and small deviations from the average diameter indicates
conjugate moved along the membrane with the liquid flow, and in the absence of DBP in the
the receipt of a high-quality preparation.
sample it was bound by free antibody binding sites in the test zone with the formation of a
colored
3.3. band.
The LFIA As the concentration of DBP in the sample increased, the coloration intensity
Format
decreased and then disappeared. The sample was completely absorbed and passed the
The mAb–GNPs conjugate was incubated with DBP-containing samples for about 10
entire distance to the end of the working membrane in less than 8 min, around 2.5 min. We
s for mixing. Then, the test strips were dipped by the sample pad to absorb the mixture.
chose a time of 8 min for sufficient accumulation of the analytical signal.
The conjugate moved along the membrane with the liquid flow, and in the absence of DBP
Similarly
in the sample to the
it was boundorganic hydrophobic
by free dyes studied
antibody binding in our
sites in the test previous
zone withwork [46], the
the for-
competition
mation was absent
of a colored band. Aswhen the conjugateof
the concentration was loaded
DBP in theonto
samplethe increased,
conjugate the
pad. This effect
color-
probably is caused by the different speeds of analyte and conjugate
ation intensity decreased and then disappeared. The sample was completely absorbed and movement limiting
passed the entire distance to the end of the working membrane in less than 8 min, around LFIA
possibilities of their interaction during the lateral flow. Therefore, the pre-incubated
2.5format
min. Wewas applied
chose a timeinofthe work.
8 min The obtained
for sufficient test strips
accumulation were
of the processed
analytical as described
signal.
in Similarly
Section 2.2.5
to the organic hydrophobic dyes studied in our previous work [46],to
with image conversion to grayscale, but there are other ways theprocess
data [51,52].
competition was absent when the conjugate was loaded onto the conjugate pad. This effect
probably is caused by the different speeds of analyte and conjugate movement limiting
3.4. The Choice of STI−DBP Immobilization Medium
possibilities of their interaction during the lateral flow. Therefore, the pre-incubated LFIA
Biosensors 2022, 12, x FOR PEER REVIEW 10 of 16
format Inwastheapplied
primary in experiment,
the work. Thetheobtained
mediumtest forstrips
immobilization of theasSTI
were processed −DBP conjugate
described in
was 10
Section mMwith
2.2.5 PBS,image
pH 7.4. However,
conversion to in this case,but
grayscale, thethere
binding of theways
are other mAbto −process
GNPs conjugate
data in
the test zone was weak (Figure 5, columns 1 and 2). Similarly, weak binding was observed
[51,52].
water (Table
after the 1, strip
addition 4). However,
of 0.05% Tween-20 if to
spring
the PBSwater is diluted
(Figure with 3the
5, columns and104);mM
thus,Tris-HCl
we have
buffer,
3.4. pH
The Choice
replaced 8.8, the signal
theofimmobilizationdrops (Table
STI−DBP Immobilization
medium Medium 1, strip 3).
with Milli-Q. Test strips prepared in this way showed
a much
In themore
primaryintense bindingthe
experiment, (Figure
medium 5, columns 5–7).
for immobilization of the STI−DBP conjugate
was 10 mM PBS, pH 7.4. However, in this case, the binding of the mAb−GNPs conjugate
in the test zone was weak (Figure 5, columns 1,2). Similarly, weak binding was observed
after the addition of 0.05% Tween-20 to the PBS (Figure 5, columns 3,4); thus, we have
replaced the immobilization medium with Milli-Q. Test strips prepared in this way
showed a much more intense binding (Figure 5, columns 5–7).
Changing the medium for the interaction of DBP-containing sample and anti-
body−GNP conjugate to 10 mM Tris-HCl, pH 8.8, with 1% Tween-20 demonstrated a log-
ical increase in coloration intensity in the test zone (Table 1). Other media were also
tested–10 mM borate buffer, pH 9.0, and 10 mM carbonate buffer, pH 10.0, with 1%
Tween-20 for better conjugate mobility. The comparison of test zone coloration made it
possible to choose the optimal medium–10 mM borate buffer, pH 9.0, see Table 1, strip 2.
A similar coloration of the test zone was obtained for measurements in natural spring
Figure 5. Histogram showing the intensity of signals in the test zone depending on media used for
Figure 5. Histogram showing
the hapten–protein immobilization (STI−of
the intensity signals
DBP) andin the test
sample zone depending
dilution. OD527nm onmAb −GNPs
media used=for
0.5.
the hapten–protein
Concentration andimmobilization (STI−DBP)
medium composition and sample
are shown on thedilution. OD
right part themAb−GNPs
of527nm picture. = 0.5. Con-
centration and medium composition are shown on the right part of the picture.

Table 1. The appearance of the test strips obtained as a result of the selection of running buffer.

The Appearance of Test Strips

No. Running Buffer
Biosensors 2022, 12, 1002 10 of 16

Changing the medium for the interaction of DBP-containing sample and antibody−GNP
conjugate to 10 mM Tris-HCl, pH 8.8, with 1% Tween-20 demonstrated a logical increase in
coloration intensity in the test zone (Table 1). Other media were also tested–10 mM borate
buffer, pH 9.0, and 10 mM carbonate buffer, pH 10.0, with 1% Tween-20 for better conjugate
mobility. The comparison of test zone coloration made it possible to choose the optimal
medium–10 mM borate buffer, pH 9.0, see Table 1, strip 2. A similar coloration of the test
Figure 5. Histogram showing the intensity of signals in the test zone depending on media used for
zone was obtained for measurements in natural spring water (Table 1, strip 4). However, if
the hapten–protein immobilization
spring water (STI−DBP)
is diluted with the 10 and
mMsample dilution.
Tris-HCl buffer, OD527nm mAb−GNPs = 0.5. Con-
pH527nm
8.8, the signal drops (Table 1,
centration and
stripmedium
3). composition are shown on the right part of the picture.

Table 1. The appearance

Table of the testofstrips
1. The appearance obtained
the test as a result
strips obtained of theofselection
as a result of running
the selection buffer.
of running buffer.

The The
Appearance ofTest
Appearance of Test Strips
Strips
No. No. RunningRunning
BufferBuffer
Control Line
Control Line Test Line
Test Line
1 1 10 mM Tris-HCl (pH 8.8)
10 mM Tris-HCl (pH 8.8)
2 2 10 mM borate10 mMbuffer (pH (pH
borate buffer 9.0)9.0)
3 spring
3 water/10
springmM Tris-HCl
water/10 (pH(pH
mM Tris-HCl 8.8) 1:1
8.8) 1:1

4 4 spring water
spring water

5 10 mM carbonate bufferbuffer
5 10 mM carbonate (pH(pH9.0)9.0)

3.5.ofSelection
3.5. Selection of the Working
the Working Membrane Membrane
The choiceThe of choice of the working
the working membrane membrane
in LFIA in depends
LFIA dependson the onmedium
the medium viscosity
viscosity and and
the size of the conjugate particles. The CNPC15 membrane is often used for analysis in
the size of the conjugate particles. The CNPC15 membrane is often used for analysis in
various media. Previously, we applied this membrane for LFIA of low molecular weight
various media. Previously, we applied this membrane for LFIA of low molecular weight
analytes–antibiotic chloramphenicol and Sudan I dye [53,54], and heavy metals such as
analytes–antibiotic chloramphenicol
lead [55]. However, in the caseand Sudan
of DBP, a weakI dye [53,54],
coloration was and heavy
formed metals
during such as
the interaction
lead [55]. However, in the case of DBP, a weak coloration was formed during
in LFIA and the band in the test zone looked pale (Figure 5, columns 1 and 2). A similar the interac-
tion in LFIA and the
situation was band in thewhen
observed test zone
workinglookedwithpale (Figure 5, columns
two hydrophobic 1,2). A
compounds, similar
Sudan I [46]
situation was observedB1when
and aflatoxin working
[56]. Due to this,with two changed
we have hydrophobic compounds,
the working membrane Sudan I [46]
to Millipore
180 (capillary
and aflatoxin flowto
B1 [56]. Due rate 180we
this, s/4have
cm) which
changed allowed working with
the working water samples
membrane (Table 1)
to Millipore
unlike the CNPC15 membrane (capillary flow rate is close
180 (capillary flow rate 180 s/4 cm) which allowed working with water samples (Table 1)to 240 s/4 cm).
The choice of membrane for sample absorption also plays an important role. Two
unlike the CNPC15 membrane (capillary flow rate is close to 240 s/4 cm).
sample pads were tested–glass fiber from Millipore non-treated by detergents and pre-
The choice of membrane for sample absorption also plays an important role. Two
treated cellulose PTR7 (MDI). As seen from Table 2, the use of a pre-treated membrane
sample pads weresignal
reduces tested–glass
intensity in fiber from
the test Millipore
zone. non-treated
Strip 3 showed only 68% by coloration
detergents and pre-
intensity in the
treated cellulose PTR7 (MDI). As seen from Table 2, the use of a pre-treated
test zone as compared with strip 1, and strip 4 showed 47% of the value for strip 2. Therefore, membrane
reduces signal
a glassintensity in the test
fiber membrane waszone.
chosenStripfor3the
showed
sample. only 68% case,
In each coloration intensity
the choice dependedin
the test zone as compared
on the with stripThe
test zone coloration. 1, main
and strip 4 showed
conditions 47% of the
for choosing the concentrations
value for stripof2.the
Therefore,gold conjugate
a glass fiber were the presence
membrane was of an intense
chosen signal
for the (at leastIn
sample. 20,000
eachAU) in the
case, theabsence
choice of
DBP and its decrease with increasing analyte concentration
depended on the test zone coloration. The main conditions for choosing the concentrations (effective competition). The
optimization of the analysis conditions is summarized in Table 3.
of the gold conjugate were the presence of an intense signal (at least 20,000 AU) in the
absence of3.6.
DBP and its Characteristics
Analytical decrease withinincreasing analyte concentration (effective competi-
Optimized Conditions
tion). The optimization
Under optimized conditions (Table 3), is
of the analysis conditions summarized
a calibration curvein has
Table
been3. obtained, shown
in Figure 6. Two detection options are possible–visual (by the disappearance of a band
in the test zone in the presence of DBP) and instrumental, based on registered coloration
intensities. The cut-off level for visual detection was 1500 ng/mL when the band disap-
peared; the negative result is when the intensity of staining was less than 2700 arbitrary
units. This level has been established as a relative cut-off of visual detection when the test
line is not seen by the naked eye. For instrumental registration of the assay results, the
calculated limit of detection (LOD) was 33.4 ng/mL, the limit of quantification (LOQ) was
42.4 ng/mL, and the working range was 42.4—1500 ng/mL with linear approximation in
semi-logarithmic coordinates. The coefficient of variation was less than 12%. The value
 Biosensors 2022, 12, 1002 11 of 16
ensors 2022, 12, x FOR PEER REVIEW 11 of 16
ensors 2022, 12, x FOR PEER REVIEW 11 of 16
ensors 2022, 12, x FOR PEER REVIEW 11 of 16
ensors 2022, 12, x FOR PEER REVIEW 11 of 16
of the coefficient of variation in experiments during the day did not exceed 11.5%, and in
Table 2. The appearance
day-to-day of the test
experiments strips
it was noobtained as a12.8%.
more than result of the selection of sample pads.
Table 2. The appearance of the test strips obtained as a result of the selection of sample pads.
Table 2. The appearance of the test strips obtained as a result of the selection of sample pads.
Table 2. The appearance of the test strips obtained as a resultThe of the selection of sample
Appearance of Testpads.
Strip
No. Table 2. The appearance of
Conditions, the test strips
Sample Pad obtained as a result
TheofAppearance
the selection of of
sample
Testpads.
Strip
No. Conditions, Sample Pad The Appearance
Control Line ofTest
TestLine
Strip
No. Conditions, Sample Pad The The
Appearance
Control Line
Appearance
ofTest
of
Test
Test Strip
Line
Strip
No. Conditions,
No.10 mM borate
Sample
buffer
Conditions, (pHPad
Sample 9.0),
Pad Control Line Test Line
1 10 mM borate buffer (pH 9.0), Control Line
Control Line Test
Test Line
Line
1 (OD527 10nmmM mAbborate
−GNPsbuffer (pH 9.0),
= 0.06), glass-fiber
1 (OD527 10 mM
nm mAb borate buffer
GNPs
10−mM (pH
= buffer
borate 0.06),(pH9.0),
glass-fiber
9.0),
1 (OD527
1 nm mAb −GNPs = 0.06), glass-fiber
2 (OD52710 nmmM
(OD527
mAb borate
nm
− mAb
GNPsbuffer
−GNPs
=
10 mM borate buffer (pH 9.0)
(pH 9.0)glass-fiber
= 0.06),
0.06), glass-fiber
2 OD527 10nmmM mAb borateGNPs
10−mM buffer (pH(pH
= 0.12,
borate buffer 9.0)
glass-fiber
9.0)
2 10
OD527 nm
2 mM mAbborate
−GNPs buffer
−
(pH
= 0.12, 9.0)
glass-fiber
2 OD527 nm
10nmmM
OD527
mAb nm
−
borate
mAb
GNPs
buffer
GNPs
= =
0.12, 0.12, glass-fiber
glass-fiber
(pHglass-fiber
9.0),
3 OD527 10 mM mAb −GNPs
borate
10 mMbuffer
= 0.12,
(pH(pH
borate buffer 9.0),
9.0),
3 3 10 ODmM borate
527 = 0.06, buffer
R7 sample (pH 9.0),
pad
3 10ODmM borate
527 =
OD buffer
=
0.06,527R7 0.06, R7
sample (pHpad
sample9.0),
pad
3 10ODmM527 = 0.06,
borate R7 sample
buffer (pHpad 9.0),
4 4 10 ODmM527 = 0.06,
10 mMR7
borate sample
borate
buffer buffer
(pHR7 pad
(pH 9.0),
9.0),
4 OD527 10 Ab
mM borate= buffer
OD527
GNPs Ab − GNPs
0.12, = (pH
0.12,
R7 9.0),
samplesample pad
4 OD527 10 Ab
mM − borate= buffer
−GNPs 0.12, R7 (pH 9.0), pad
sample pad
4 OD527 Ab−GNPs = 0.12, R7 sample pad
OD527 Ab−GNPs = 0.12, R7 sample pad
Table 3. Conditions for test strips preparation and LFIA of DBP chosen by sequential optimization.
Table 3. Conditions for test strips preparation and LFIA of DBP chosen by sequential optimization.
Table 3. Conditions for test strips preparation and LFIA of DBP chosen by sequential optimization.
Table 3. Conditions for test strips preparation
Parameter
and LFIA of DBP
Variationsand LFIASignal
chosen AU
Intensity,
by sequential optimization.
Chosenoptimization.
Conditions
Table 3. Conditions for test strips preparation of DBP chosen
Signalby sequential
Chosen Con-
Parameter Variations
OD527 = 0.06 10,025
Signal Chosen Con-
Parameter Variations Signal AU
Intensity, Chosen
ditions Con-
Parameter Variations Signal
Intensity, AU Chosen
ditions Con-
Parameter
Concentration of Variations
OD
OD527 = 0.12
527= 0.06 Intensity,
25,16310,025 AU ditions
OD527 = 0.06 Intensity,
10,025 AU OD527ditions = 0.12
GNPs−mAb ODOD527527= =0.06
0.24 42,61810,025
Concentration of OD
OD 527 = 0.12 25,163
Concentration of OD527 527 == 0.06
0.12 10,025
25,163 OD527 = 0.12
Concentration
GNPs−mAb of OD
OD OD527527=
527 = =0.12
0.5
0.24 90,24725,163
42,618 OD527 = 0.12
Concentration
GNPs−mAb of OD
OD527 527 == 0.12
0.24 25,163
42,618 OD527 = 0.12
GNPs−mAb OD0.25527
OD ==0.24
mg/mL
527 0.5 13,24742,618
90,247 OD527 = 0.12
GNPs−mAb OD
OD527 527==0.24
0.5 42,618
90,247
Concentration of OD0.5 527
0.25 = 0.5
mg/mL
mg/mL 25,16390,247
13,247
OD
0.25 = 0.5
mg/mL
527 90,247
13,247 0.5 mg/mL
Concentration of STI − DBP 0.25
0.5 mg/mL
1.0mg/mL
mg/mL 46,31913,247
25,163
Concentration of 0.25 mg/mL
0.5 mg/mL 13,247
25,163 0.5 mg/mL
Concentration
STI−DBP of 0.5
2.0mg/mL
1.0 mg/mL
mg/mL 94,56725,163
46,319 0.5 mg/mL
Concentration
STI−DBP of 0.5 mg/mL
1.0 mg/mL 25,163
46,319 0.5 mg/mL
STI−DBP 10 1.0
2.0
mM mg/mL
mg/mL
PBS, pH 7.4 4685 46,319
94,567 0.5 mg/mL
STI−DBPMedia for STI − 1.0
2.0 mg/mL
mg/mL 46,319
94,567 Deionized water
Media forDBP immobilization
STI−DBP im- 10 mM 2.0 PBS,
mg/mL
Deionized pH 7.4
water 25,16394,567
4685 Deionized
Media for STI−DBP im- 10 mM 2.0 PBS,
mg/mL pH 7.4 94,567
4685 Deionized
Mediamobilization
for STI−DBP im- 10Deionized
mMCNPC PBS, 15 pH
water 7.4 8630 4685
25,163 Deionized
water
Mediamobilization
forWorking
STI−DBP im-
membrane 10Deionized
mM PBS, water pH 7.4 4685
25,163 Deionized
Milliporewater
180
mobilization Deionized
CNPC
Milliporewaterwater
15
180 25,163
8630
23,64525,163 water
mobilization
Working membrane Deionized
CNPC 15 8630 water
Millipore 180
Working membrane CNPC
Millipore
Glass-fiber 15
180 42,618 8630
23,645 Millipore 180
Working membrane Sample pad CNPC
Millipore 180 15 8630
23,645 Millipore 180
Glass-fiber
Working membrane Millipore
PTR7 180
Glass-fiber 17,25523,645
42,618 Millipore 180
Sample pad Millipore
Glass-fiber 180 23,645
42,618 Glass-fiber
Sample pad Glass-fiber
10 mM PTR7
borate buffer, 42,618
17,255 Glass-fiber
Sample pad Glass-fiber
PTR7 41,84542,618
17,255 Glass-fiber
Sample pad 10 mM borate PTR7
pH 9.0 17,255 Glass-fiber
10 mM borate PTR7buffer,
buffer, pH
pH 17,255
41,845
10 mM10boratemM9.0 buffer, pH
carbonate 41,845
10 mM borate 9.0 buffer, pH 17,02041,845 10 mM borate buffer,
Diluting medium 10 mMbuffer, 9.0
carbonate
pH 10.0
buffer, 41,845
9.0 109.0
pH mM bo-
10 mM carbonate buffer, 17,020 10 mM bo-
Diluting medium 10 mM10carbonate
mM Tris-HCl
pH 10.0 buffer, 20,78417,020 10 mM
rate bo-
buffer,
Diluting medium 10 mM buffer,
carbonate
pH 10.0 pH 8.8buffer, 17,020 10 mM
rate bo-
buffer,
Diluting medium 10 mM pH 10.0
Tris-HCl buffer, 17,020 rate
pH buffer,
9.0
Diluting medium 10 mM 10 mMpHPBS, 10.0pHbuffer,
Tris-HCl 7.4 10,94020,784 rate
pHbuffer,
9.0
10 mM Tris-HCl
pH 8.8 buffer, 20,784 pH 9.0
Conditions for 10 mM 0.25 Tris-HCl
pHmg/mL 8.8 buffer, 20,784 AccordspHto9.0
pH 8.8pH 20,784
25,53710,940
GAMI immobilization 10 1010
mM
mMmM pHPBS, 8.8pH
PBS,
PBS, pH7.4
7.4
7.4 10,940 earlier data
Conditions for GAMI 10 mM0.25 PBS,
mg/mL pH 7.4 10,940 Accords to
Conditions for GAMI 10 mM0.25PBS,mg/mL pH 7.4 10,940
25,537 Accords to
Conditions
immobilizationfor GAMI
3.7. Analysis of Natural10 0.25
mM
Water mg/mL
PBS,
Samples pH 7.4 25,537 Accords
earlier to
data
Conditions
immobilizationfor GAMI 0.25 mg/mL
10 mM PBS, pH 7.4 25,537 Accords
earlier data to
immobilization
The concentration 10
of mM
dibutylPBS, pH
phthalate7.4 as well as 25,537
other phthalic acid earlier
esters in data
natural
immobilization 10 mM PBS, pH 7.4 earlier data
waters Charaсteristics
3.6. Analytical varies depending on the geographical
in Optimized Conditions location in a different concentration range–
3.6. Analytical Charaсteristics
from several ng/mL [57]
in Optimized
to several µg/mL
Conditions
[58]. Thus, to determine low concentrations
3.6. Analytical
Under optimizedCharaсteristics in Optimized
conditions (Table 3), Conditions
a calibration curve has been obtained, shown
3.6. Analytical
Under
of DBP
Charaсteristics
optimized
in natural
in
conditions Optimized
water, the (Table
use of a3),
Conditions
a calibration
calibration curvecurve
as wellhas been obtained,
as instrumental shown
processing
Under
in Figure optimized
6.the
Two detectionconditions
options (Table 3), a calibration
are possible–visual (bycurve has been obtained,
the disappearance of shown
a band in
Under
of optimized
data is conditions
required. In (Table
addition, 3),
thea calibration
analysis of curve
samples
in Figure 6. Two detection options are possible–visual (by the disappearance of a band in has
withbeen obtained,
relatively low shown
content
in
theFigure
test of 6.
zone Two detection
inis the options
presence of DBP)are possible–visual
and instrumental, (by the
tobaseddisappearance
onsensitivity
registeredandof a band
coloration in
in
theFigure
test zone6.DBP
Two preferably
detection
in the provided
options
presence of DBP) without
are and their dilution
possible–visual
instrumental, (by keep
the the
disappearance
based on registeredof a to
bandavoid
coloration in
the test false-negative
intensities.zone Thein cut-off
the presence
level
results. of
for DBP)
visual and instrumental,
detection was 1500 based
ng/mL on registered
when the coloration
band disap-
the test zone in the presence of DBP) and instrumental,
intensities. The cut-off level for visual detection was 1500 ng/mL when the band disap- based on registered coloration
intensities.
peared; the The cut-off
negative levelisfor
result whenvisualthedetection
intensity was 1500 ng/mL
of staining was lesswhenthan the band
2700 disap-
arbitrary
intensities.
peared; the The cut-off
negative levelisfor
result whenvisualthedetection
intensity was 1500 ng/mL
of staining was lesswhenthan the band
2700 disap-
arbitrary
peared;
units. thelevel
This negative
has beenresult is when the
established as aintensity
relative of staining
cut-off was less
of visual than 2700
detection when arbitrary
the test
peared; the negative result is when the intensity of staining was
units. This level has been established as a relative cut-off of visual detection when the test less than 2700 arbitrary
units.
line isThis level by
not seen hasthe been established
naked eye. Foras a relative cut-off
instrumental of visualof
registration detection
the assay when the test
results, the
units.
line isThis level by
not seen hasthe been established
naked eye. Forasinstrumental
a relative cut-off of visualof
registration detection
the assay when the test
results, the
line is not limit
calculated seen of bydetection
the naked eye. For
(LOD) was instrumental
33.4 ng/mL, registration
the limit of of the assay results,
quantification (LOQ) the
was
line is not seen by the naked eye. For instrumental registration
calculated limit of detection (LOD) was 33.4 ng/mL, the limit of quantification (LOQ) was of the assay results, the
Biosensors 2022, 12, x FOR PEER REVIEW 12 of 16

Biosensors 2022, 12, 1002 12 of 16

the coefficient of variation in experiments during the day did not exceed 11.5%, and in
day-to-day experiments it was no more than 12.8%.
(a) (b)
Figure 6. LFIA of DBP in the buffer. (a) The linear range of the calibration curve. R2= 0,98
(b) The appearance of the test strips when analyzing samples with different content of D
centrations of DBP from right to the left were 0, 47, 94, 188, 375, 750, 1500 ng/mL.

3.7. Analysis of Natural Water Samples

The concentration of dibutyl phthalate as well as other phthalic acid esters in
waters varies depending on the geographical location in a different concentration
from several ng/mL [57] to several µg/mL [58]. Thus, to determine low concentra
DBP in natural water, the use of a calibration curve as well as instrumental proce
the data is required. (a) In addition, the analysis of samples (b) with relatively low co
DBP6. is
Figure
Figure LFIA
6. preferably
LFIAof DBP
of DBPin theprovided
in buffer.
the buffer. without
(a) The
(a)linear their
range
The linear dilution
of the
range to curve.
calibration
of keepR2=
the calibration the0,984
curve. sensitivity
R2(n==0.984
3), and t
(b)(nThe appearance of the test strips when analyzing samples with different content of DBP. Con-
false-negative results.
= 3), (b) The appearance of the test strips when analyzing samples with different content of DBP.
centrations of DBP from right to the left were 0, 47, 94, 188, 375, 750, 1500 ng/mL.
Concentrations of DBP from
The test strips prepared right to thebyleft the
werechosen
0, 47, 94, 188, 375, 750,
optimal 1500 ng/mL.
protocol were applied to det
3.7.inAnalysis
spring
The test strips prepared by the chosen optimal protocol were applied tonatural
water
of Natural (Figure
Water 7).
SamplesAs mentioned above, the dilution of detect DBPwater wit
indecreases
spring coloration
The concentration
water (Figure inAs
of 7). the
dibutyl test zone
phthalate
mentioned as(Table as 1,
wellthe
above, strip
other
dilution 3). Therefore,
phthalic waterwater
acid esters
of natural in samples we
natural
with buffer
waters varies
decreases
without depending
coloration
dilution. in on the
the
Analysis test geographical
zone location
(Table 1,water
of spring strip 3). in a different
Therefore,
samples concentration
water
showed samples range–
were used
good reproducibility a
from several
without ng/mL
dilution. [57]
Analysis to several
of springµg/mL
water [58]. Thus,
samples to
showeddetermine
recovery of DBP in added-found experiments (Table 4), which demonstrated the good low concentrations
reproducibility and of
high
recovery
DBP of DBP
in natural in added-found
water, the use of a experiments (Tableas4),well
calibration curve which demonstratedprocessing
as instrumental the absence ofof
ofdata
the
the
the influence
influence of the of
is required.
the matrix.
matrix.
In addition, the analysis of samples with relatively low content of
DBP is preferably provided without their dilution to keep the sensitivity and to avoid
false-negative results.
The test strips prepared by the chosen optimal protocol were applied to detect DBP
in spring water (Figure 7). As mentioned above, the dilution of natural water with buffer
decreases coloration in the test zone (Table 1, strip 3). Therefore, water samples were used
without dilution. Analysis of spring water samples showed good reproducibility and high
recovery of DBP in added-found experiments (Table 4), which demonstrated the absence
of the influence of the matrix.

Figure The
Figure7. 7. appearance
The of the of
appearance testthe
strips when
test analyzing
strips whenspring water with
analyzing added
spring DBP.with
water Concentra-
added DBP.
tions of DBP from right to the left were 0, 16, 47, 94, 188, 375, 750, and 1500 ng/mL.
trations of DBP from right to the left were 0, 16, 47, 94, 188, 375, 750, and 1500 ng/mL.
Table 4. Determination of DBP in spring water samples by the developed lateral flow assay.

Found DBP/Recoveries
Added DBP
Water 1 Water 2 Water 3
Figureng/mL
7. The appearance
ng/mL
of the test %
strips whenng/mL
analyzing spring %
water withng/mL
added DBP. Concen-
%
trations of DBP from right to the left were 0, 16, 47, 94, 188, 375, 750, and 1500 ng/mL.
50 43.7 87.4 ± 4.9 43.35 86.7 ± 5.8 39.4 78.5 ± 7.2
100 75.0 75.0 ± 5.9 110.4 110.4 ± 7.4 80.7 80.7 ± 11.2
200 230 115.0 ± 6.5 186.4 93.2 ± 9.4 230 115.0 ± 13.5

Table S1 integrates data about the possibilities and limitations of different immunoas-
say formats that were realized for DBP detection (Supplementary Materials). As can be
seen, instrumental techniques for testing in laboratory conditions dominate among these
developments. Often, they demonstrated low detection limits, but these improvements
accord to the concentrations being much lower than MRLs for phthalates. The main trend
of the last few years has been to replace the traditionally used peroxidase as a label in
Biosensors 2022, 12, 1002 13 of 16

ELISA with new markers [57,59–62]. The proposed LFIA fills an empty niche of simple
on-site tests, meeting practical requirements for sensitivity.

4. Conclusions
A simple and rapid assay of dibutyl phthalate in natural waters has been developed.
Under chosen conditions the developed LFIA technique allowed for the LOD of 33.4 ng/mL.
The factors influencing the coloration intensity of the test zone have been established, and
the conditions for reliable determination of dibutyl phthalate in spring water samples have
been selected. The advantages of the developed test system are rapidity, simplicity, and the
possibility to visually detect the presence and evaluate instrumentally the content of dibutyl
phthalate in natural waters. The effectiveness of the development has been demonstrated
in “added-found” experiments. The obtained results are promising background for dibutyl
phthalate assays in other samples. This development solves specific problems associated
with the demand for monitoring phthalates in water samples. On the other hand, it is
not limited to the target analyte, but considers issues that arise when carefully choosing
the conditions of immunochromatographic analysis. It has been shown that varying the
environment of immobilization and the interaction medium can significantly improve
the characteristics of the system. The article, in our opinion, systematizes approaches to
overcome the “underwater rocks” under the development of immunochromatographic
test-systems.

Supplementary Materials: The following supporting information can be downloaded at: https://
www.mdpi.com/article/10.3390/bios12111002/s1, Table S1: Examples of DBP immunoassays in
different samples.
Author Contributions: Conceptualization, A.N.B., N.S.K. and K.V.S.; methodology, A.N.B., N.S.K.
and M.Y.R.; software, N.S.K. and K.V.S.; validation, A.N.B., N.S.K. and M.Y.R.; formal analysis,
K.V.S.; investigation, A.N.B., N.S.K. and M.Y.R.; resources, A.V.Z. and B.B.D.; data curation, K.V.S.;
writing—original draft preparation, A.N.B. and N.S.K.; writing—review and editing, A.N.B., N.S.K.
and A.V.Z.; visualization, A.N.B. and N.S.K.; supervision, A.V.Z. and B.B.D.; project administration,
A.N.B. and A.V.Z.; funding acquisition, K.V.S. All authors have read and agreed to the published
version of the manuscript.
Funding: This work was financially supported by the Russian Science Foundation (grant 21-14-
00306, development and characterization of immunochromatographic technique for DBP detection),
and by the Ministry of Science and Higher Education of the Russian Federation (synthesis and
characterization of the nanoparticles).
Informed Consent Statement: Not applicable.
Data Availability Statement: Data are contained within the article. Initial data of instrumental
measurements are available on request from the corresponding author.
Acknowledgments: The authors are grateful to S.M. Pridvorova from A.N. Bach Institute of Bio-
chemistry (Research Center of Biotechnology of the Russian Academy of Sciences, Moscow, Russia)
for the support with TEM images.
Conflicts of Interest: The authors declare no conflict of interest.

References
1. Berenstein, G.; Hughes, E.A.; Zalts, A.; Basack, S.; Bonesi, S.M.; Montserrat, J.M. Environmental fate of dibutylphthalate in
agricultural plastics: Photodegradation, migration and ecotoxicological impact on soil. Chemosphere 2022, 290, 133221. [CrossRef]
[PubMed]
2. Erna, M.; Harazono, A.; Miyawaki, E.; Ogawa, Y. Developmental toxicity of mono-n-benzyl phthalate, one of the major metabolites
of the plasticizer n-butyl benzyl phthalate, in rats. Toxicol. Lett. 1996, 86, 19–25. [CrossRef]
3. Martino-Andrade, A.J.; Chahoud, I. Reproductive toxicity of phthalate esters. Mol. Nutr. Food Res. 2010, 54, 148–157. [CrossRef]
[PubMed]
4. Foster, P.; Lake, B.; Thomas, L.V.; Cook, M.; Gangolli, S. Studies on the testicular effects and zinc excretion produced by various
isomers of monobutyl-o-phthalate in the rat. Chem. Biol. Interact. 1981, 34, 233–238. [CrossRef]
Biosensors 2022, 12, 1002 14 of 16

5. Sedha, S.; Lee, H.; Singh, S.; Kumar, S.; Jain, S.; Ahmad, A.; Bin Jardan, Y.A.; Sonwal, S.; Shukla, S.; Simal-Gandara, J.; et al.
Reproductive toxic potential of phthalate compounds—State of art review. Pharmacol. Res. 2021, 167, 105536. [CrossRef] [PubMed]
6. Zhang, Y.; Jiao, Y.; Li, Z.; Tao, Y.; Yang, Y. Hazards of phthalates (PAEs) exposure: A review of aquatic animal toxicology studies.
Sci. Total Environ. 2021, 771, 145418. [CrossRef] [PubMed]
7. Benson, R. Hazard to the developing male reproductive system from cumulative exposure to phthalate esters—Dibutyl phthalate,
diisobutyl phthalate, butylbenzyl phthalate, diethylhexyl phthalate, dipentyl phthalate, and diisononyl phthalate. Regul. Toxicol.
Pharm. 2009, 53, 90–101. [CrossRef]
8. Latini, G.; Del Vecchio, A.; Massaro, M.; Verrotti, A.; De Felice, C. Phthalate exposure and male infertility. Toxicology 2006, 226,
90–98. [CrossRef]
9. Giribabu, N.; Reddy, P.S. Protection of male reproductive toxicity in rats exposed to di-n-butyl phthalate during embryonic
development by testosterone. Biomed. Pharmacother. 2017, 87, 355–365. [CrossRef]
10. Wang, S.W.; Wang, S.S.W.; Wu, D.C.; Lin, Y.C.; Ku, C.C.; Wu, C.C.; Chai, C.Y.; Lee, J.N.; Tsai, E.M.; Lin, C.L.; et al. Androgen
receptor-mediated apoptosis in bovine testicular induced pluripotent stem cells in response to phthalate esters. Cell Death Dis.
2013, 4, e907. [CrossRef]
11. He, L.; Gielen, G.; Bolan, N.S.; Zhang, X.; Qin, H.; Huang, H.; Wang, H. Contamination and remediation of phthalic acid esters in
agricultural soils in China: A review. Agron. Sustain. Dev. 2015, 35, 519–534. [CrossRef]
12. Yan, Y.; Zhu, F.; Zhu, C.; Chen, Z.; Liu, S.; Wang, C.; Gu, C. Dibutyl phthalate release from polyvinyl chloride microplastics:
Influence of plastic properties and environmental factors. Water Res. 2021, 204, 117597. [CrossRef] [PubMed]
13. Ye, X.; Wang, P.; Wu, Y.; Zhou, Y.; Sheng, Y.; Lao, K. Microplastic acts as a vector for contaminants: The release behavior of dibutyl
phthalate from polyvinyl chloride pipe fragments in water phase. Environ. Sci. Pollut. Res. 2020, 27, 42082–42091. [CrossRef]
[PubMed]
14. SANPIN 1.2.3685-21; Hygienic Standards and Requirements for Ensuring the Safety and (or) Harmlessness of Environmental
Factors for Humans, Federal Service for Supervision of Consumer Rights Protection and Human Welfare. Ministry of Justice of
Russia: Moscow, Russia, 2021.
15. Dada, E.O.; Ikeh, R.K. Phthalate and Metal Concentrations in Drinking Water in Lagos, Nigeria. J. Health Pollut. 2018, 8, 180603.
[CrossRef] [PubMed]
16. US EPA United States Environmental Protection Agency (US). National Primary Drinking Water Regulations; Federal Register:
Washington, DC, USA, 1991; Volume 56, pp. 30266–30281.
17. Wang, W.; Leung, A.O.W.; Chu, L.H.; Wong, M.H. Phthalates contamination in China: Status, trends and human exposure-with
an emphasis on oral intake. Environ. Pollut. 2018, 238, 771–782. [CrossRef]
18. Song, P.; Jiang, N.; Zhang, K.; Li, X.; Li, N.; Zhang, Y.; Wang, Q.; Wang, J. Ecotoxicological evaluation of zebrafish liver (Danio
rerio) induced by dibutyl phthalate. J. Hazard. Mater. 2022, 425, 128027. [CrossRef]
19. Xu, Y.; Weng, R.; Lu, Y.; Wang, X.; Zhang, D.; Li, Y.; Qiu, J.; Qian, Y. Evaluation of Phthalic Acid Esters in Fish Samples Using Gas
Chromatography Tandem Mass Spectrometry with Simplified QuEChERS Technique. Food Anal. Methods 2018, 11, 3293–3303.
[CrossRef]
20. González-Sálamo, J.; Socas-Rodríguez, B.; Hernández-Borges, J. Analytical methods for the determination of phthalates in food.
Curr. Opin. Food Sci. 2018, 22, 122–136. [CrossRef]
21. Salazar-Beltrán, D.; Hinojosa-Reyes, L.; Ruiz-Ruiz, E.; Hernández-Ramírez, A.; Guzmán-Mar, J.L. Phthalates in Beverages and
Plastic Bottles: Sample Preparation and Determination. Food Anal. Methods 2018, 11, 48–61. [CrossRef]
22. Di Nardo, F.; Chiarello, M.; Cavalera, S.; Baggiani, C.; Anfossi, L. Ten Years of Lateral Flow Immunoassay Technique Applications:
Trends, Challenges and Future Perspectives. Sensors 2021, 21, 5185. [CrossRef]
23. Wei, C.; Ding, S.; You, H.; Zhang, Y.; Wang, Y.; Yang, X.; Yuan, J. An Immunoassay for Dibutyl Phthalate Based on Direct Hapten
Linkage to the Polystyrene Surface of Microtiter Plates. PLoS ONE 2011, 6, e29196. [CrossRef] [PubMed]
24. Tang, M.; Wei, J.; Du, H.; Zhang, J.; Yang, D.; Peng, Y. Synthesis of an artificial antigen and preparation of a polyclonal antibody
for the sensitive determination of phthalate esters by enzyme-linked immunoassay. Anal. Methods 2015, 7, 3402–3410. [CrossRef]
25. Zeng, Q.; Wei, C.; Wu, Y.; Li, K.; Ding, S.; Yuan, J.; Yang, X.; Chen, M. Approach to distribution and accumulation of dibutyl
phthalate in rats by immunoassay. Food Chem. Toxicol. 2013, 56, 18–27. [CrossRef] [PubMed]
26. Baranovskaya, V.; Berlina, A.; Eremin, S. A Fluorescence Polarization Immunoassay Procedure for Determining Dibutyl Phthalate
in Water. J. Anal. Chem. 2022, 77, 466–472. [CrossRef]
27. Li, J.; Jin, H.; Wei, M.; Ren, W.; Wang, J.; Zhang, Y.; Wu, L.; He, B. Dual mode competitive electrochemical immunoassay for
dibutyl phthalate detection based on PEI functionalized nitrogen doped graphene-CoSe2/gold nanowires and thionine-Au@Pt
core-shell. Sens. Actuators B 2021, 331, 129401. [CrossRef]
28. Zhang, M.-C.; Wang, Q.-E.; Zhuang, H.-S. A novel competitive fluorescence immunoassay for the determination of dibutyl
phthalate. Anal. Bioanal. Chem. 2006, 386, 1401–1406. [CrossRef]
29. Zhang, M.C.; Wang, Q.E.; Zhuang, H.S. Determination of dibutyl o-phthalate by antigen-coated competitive fluorescence
immunoassay. Anal. Lett. 2007, 40, 127–137. [CrossRef]
30. Tian, X.; Dong, Y.; Wang, Y.; Song, Z.; Meng, M.; Eremin, S.A.; Deng, C.; Yin, Y.; Xi, R. Quantification of Diethyl Phthalate by a
Rapid and Homogenous Fluorescence Polarization Immunoassay. Anal. Lett. 2015, 48, 2843–2855. [CrossRef]
31. Hermanson, G. Bioconjugate Techniques, 2nd ed.; Academic Press: London, UK, 2008.
Biosensors 2022, 12, 1002 15 of 16

32. Wang, Y.; He, C.-H.; Zheng, H.; Zhang, H.-B. Characterization and Comparison of Fumonisin B1-Protein Conjugates by Six
Methods. Int. J. Mol. Sci. 2012, 13, 84. [CrossRef]
33. Chalmers, J.M.; Griffiths, P.R. Handbook of Vibrational Spectroscopy; Wiley: Hoboken, NJ, USA, 2002; Volume 4.
34. Ranaldi, S.; Belle, V.; Woudstra, M.; Rodriguez, J.; Guigliarelli, B.; Sturgis, J.; Carriere, F.; Fournel, A. Lid opening and unfolding
in human pancreatic lipase at low pH revealed by site-directed spin labeling EPR and FTIR spectroscopy. Biochemistry 2009, 48,
630–638. [CrossRef]
35. Ramesh, S.; Yin, T.S.; Liew, C.-W. Effect of dibutyl phthalate as plasticizer on high-molecular weight poly(vinyl chloride)–lithium
tetraborate-based solid polymer electrolytes. Ionics 2011, 17, 705. [CrossRef]
36. Rajamanikyam, M.; Vadlapudi, V.; Parvathaneni, S.P.; Koude, D.; Sripadi, P.; Misra, S.; Amanchy, R.; Upadhyayula, S.M. Isolation
and characterization of phthalates from Brevibacterium mcbrellneri that cause cytotoxicity and cell cycle arrest. EXCLI J. 2017,
16, 375. [PubMed]
37. Bhatnagar, B.S.; Bogner, R.H.; Pikal, M.J. Protein Stability During Freezing: Separation of Stresses and Mechanisms of Protein
Stabilization. Pharm. Dev. Technol. 2007, 12, 505–523. [CrossRef]
38. Ielo, I.; Rando, G.; Giacobello, F.; Sfameni, S.; Castellano, A.; Galletta, M.; Drommi, D.; Rosace, G.; Plutino, M.R. Synthesis,
Chemical–Physical Characterization, and Biomedical Applications of Functional Gold Nanoparticles: A Review. Molecules 2021,
26, 5823. [CrossRef]
39. Tabatabaei, M.S.; Islam, R.; Ahmed, M. Applications of gold nanoparticles in ELISA, PCR, and immuno-PCR assays: A review.
Anal. Chim. Acta 2021, 1143, 250–266. [CrossRef] [PubMed]
40. Posthuma-Trumpie, G.A.; Korf, J.; van Amerongen, A. Lateral flow (immuno) assay: Its strengths, weaknesses, opportunities and
threats. A literature survey. Anal. Bioanal. Chem. 2009, 393, 569–582. [CrossRef]
41. Li, X.; Zhu, Q.; Xu, F.; Jian, M.; Yao, C.; Zhang, H.; Wang, Z. Lateral flow immunoassay with peptide-functionalized gold
nanoparticles for rapid detection of protein tyrosine phosphatase 1B. Anal. Biochem. 2022, 648, 114671. [CrossRef]
42. Wang, Z.; Yu, W.; Xie, R.; Yang, S.; Chen, A. A strip of lateral flow gene assay using gold nanoparticles for point-of-care diagnosis
of African swine fever virus in limited environment. Anal. Bioanal. Chem. 2021, 413, 4665–4672. [CrossRef]
43. Lu, X.; Mei, T.; Guo, Q.; Zhou, W.; Li, X.; Chen, J.; Zhou, X.; Sun, N.; Fang, Z. Improved performance of lateral flow immunoassays
for alpha-fetoprotein and vanillin by using silica shell-stabilized gold nanoparticles. Microchim. Acta 2018, 186, 2. [CrossRef]
44. Panferov, V.G.; Safenkova, I.V.; Zherdev, A.V.; Dzantiev, B.B. Post-assay growth of gold nanoparticles as a tool for highly sensitive
lateral flow immunoassay. Application to the detection of potato virus X. Microchim. Acta 2018, 185, 506. [CrossRef]
45. Urusov, A.E.; Petrakova, A.V.; Zherdev, A.V.; Zvereva, E.A. Indirect labeling of antibodies as a universal approach to increase
sensitivity of lateral flow tests: A case study for mycotoxins detection. Open Biotechnol. J. 2019, 13, 113–121. [CrossRef]
46. Berlina, A.N.; Zherdev, A.V.; Xu, C.; Eremin, S.A.; Dzantiev, B.B. Development of lateral flow immunoassay for rapid control and
quantification of the presence of the colorant Sudan I in spices and seafood. Food Control 2017, 73, 247–253. [CrossRef]
47. Hendrickson, O.D.; Byzova, N.A.; Zvereva, E.A.; Zherdev, A.V.; Dzantiev, B.B. Sensitive lateral flow immunoassay of an antibiotic
neomycin in foodstuffs. J. Food Sci. Technol. 2021, 58, 292–301. [CrossRef] [PubMed]
48. Zvereva, E.A.; Byzova, N.A.; Hendrickson, O.D.; Popravko, D.S.; Belichenko, K.A.; Dzantiev, B.B.; Zherdev, A.V. Immunochro-
matographic Detection of Myoglobin as a Specific Biomarker of Porcine Muscle Tissues in Meat Products. Appl. Sci. 2020, 10, 7437.
[CrossRef]
49. Dykman, L.; Khlebtsov, N. Gold nanoparticles in biomedical applications: Recent advances and perspectives. Chem. Soc. Rev.
2012, 41, 2256–2282. [CrossRef] [PubMed]
50. Hermanson, G.T. Chapter 20—Antibody Modification and Conjugation. In Bioconjugate Techniques, 3rd ed.; Hermanson, G.T., Ed.;
Academic Press: Boston, MA, USA, 2013; pp. 867–920. [CrossRef]
51. Charbaji, A.; Smith, W.; Anagnostopoulos, C.; Faghri, M. Zinculose: A new fibrous material with embedded zinc particles. Eng.
Sci. Technol. Int. J. 2021, 24, 571–578. [CrossRef]
52. Kim, J.-H.; Lee, Y.-J.; Ahn, Y.-J.; Kim, M.; Lee, G.-J. In situ detection of hydrogen sulfide in 3D-cultured, live prostate cancer cells
using a paper-integrated analytical device. Chemosensors 2022, 10, 27. [CrossRef]
53. Berlina, A.N.; Taranova, N.A.; Zherdev, A.V.; Vengerov, Y.Y.; Dzantiev, B.B. Quantum dot-based lateral flow immunoassay for
detection of chloramphenicol in milk. Anal. Bioanal. Chem. 2013, 405, 4997–5000. [CrossRef]
54. Berlina, A.N.; Bartosh, A.V.; Zherdev, A.V.; Xu, C.; Dzantiev, B.B. Development of Immunochromatographic assay for determina-
tion of tetracycline in human serum. Antibiotics 2018, 7, 99. [CrossRef]
55. Berlina, A.N.; Komova, N.S.; Zherdev, A.V.; Dzantiev, B.B. Combination of phenylboronic acid and oligocytosine for selective and
specific detection of lead (ii) by lateral flow test strip. Anal. Chim. Acta 2021, 1155, 338318. [CrossRef]
56. Urusov, A.E.; Zherdev, A.V.; Dzantiev, B.B. Influence of the compounds used in immunochromatographic test-system on its
analytical characteristics: Multi-factor optimization of the system for aflatoxin B1. Iran. (Iran.) J. Energy Environ. 2015, 6, 63–71.
[CrossRef]
57. Zhu, F.; Zhang, H.; Qiu, M.; Wu, N.; Zeng, K.; Du, D. Dual-label time-resolved fluoroimmunoassay as an advantageous approach
for investigation of diethyl phthalate & dibutyl phthalate in surface water. Sci. Total Environ. 2019, 695, 133793. [CrossRef]
[PubMed]
Biosensors 2022, 12, 1002 16 of 16

58. Adeniyi, A.A.; Okedeyi, O.O.; Yusuf, K.A. Flame ionization gas chromatographic determination of phthalate esters in water,
surface sediments and fish species in the Ogun river catchments, Ketu, Lagos, Nigeria. Environ. Monit. Assess. 2011, 172, 561–569.
[CrossRef] [PubMed]
59. Xiong, D.; Zhu, N.; Zhu, F.; Yakubu, S.; Lv, J.; Liu, J.; Zhang, Z. Investigation and risk assessment of dibutyl phthalate in a typical
region by a high-throughput dual-signal immunoassay. J. Hazard. Mater. 2022, 425, 127991. [CrossRef] [PubMed]
60. Meng, H.; Yao, N.; Zeng, K.; Zhu, N.; Wang, Y.; Zhao, B.; Zhang, Z. A Novel Enzyme-Free Ratiometric Fluorescence Immunoassay
Based on Silver Nanoparticles for the Detection of Dibutyl Phthalate from Environmental Waters. Biosensors 2022, 12, 125.
[CrossRef]
61. Kuang, H.; Liu, L.; Xu, L.; Ma, W.; Guo, L.; Wang, L.; Xu, C. Development of an Enzyme-Linked Immunosorbent Assay for
Dibutyl Phthalate in Liquor. Sensors 2013, 13, 8331–8339. [CrossRef]
62. Zhang, Z.; Zhu, N.; Zou, Y.; Wu, X.; Qu, G.; Shi, J. A novel, enzyme-linked immunosorbent assay based on the catalysis of
AuNCs@BSA-induced signal amplification for the detection of dibutyl phthalate. Talanta 2018, 179, 64–69. [CrossRef]