Sensors and Actuators B 265 (2018) 174–181

Contents lists available at ScienceDirect

Sensors and Actuators B: Chemical

journal homepage: www.elsevier.com/locate/snb

Glucose-loaded liposomes for amplified colorimetric immunoassay of

streptomycin based on enzyme-induced iron(II) chelation reaction
with phenanthroline
Rongrong Ren, Guoneng Cai, Zhenzhong Yu, Dianping Tang ∗
Key Laboratory for Analytical Science of Food Safety and Biology (MOE & Fujian Province), State Key Laboratory of Photocatalysis on Energy and
Environment, Department of Chemistry, Fuzhou University, Fuzhou 350116, PR China

a r t i c l e i n f o a b s t r a c t

Article history: This work designed a signal-on competitive-type colorimetric immunoassay of streptomycin (STR) using
Received 25 January 2018 glucose-loaded liposome (GLL) and unique iron(II)-phenanthroline [Fe(II)-Phen] system. The assay was
Received in revised form 10 March 2018 executed based on the change in the color and absorbance of Fe(II)-Phen system, followed by enzyme-
Accepted 12 March 2018
triggered Fe(II) oxidization into Fe(III) in the presence of glucose oxidase (GOx). To construct such an
Available online 13 March 2018
assay, STR-bovine serum albumin (STR-BSA) conjugates were covalently linked with glucose-loaded lipo-
some, whereas anti-STR antibodies were coated onto a microplate. In the presence of STR, a competitive
Keywords:
immunoreaction was executed between the analyte and STR-BSA-GLL for the immobilized anti-STR.
Glucose-loaded liposome
Colorimetric immunoassay
Upon addition of Triton X-100, the carried liposome was dissociated to release the loaded glucose, which
Streptomycin residues was oxidized by GOx into gluconic acid and hydrogen peroxide (H2 O2 ). The as-generated H2 O2 oxidized
Enzymatic catalysis iron(II) into iron(III), and decreased the complexation of iron(II) with Phen, thereby resulting in the color
Iron(II)-phenanthroline colored system change and the decreasing absorbance. Under optimal conditions, the colorimetric immunoassay with
the Fe(II)-Phen system exhibited a low detection limit (LOD) of 0.4 pg mL−1 with a wide dynamic working
range from 1.0 pg mL−1 to 20 ng mL−1 STR, accompanying good reproducibility and high specificity.
© 2018 Elsevier B.V. All rights reserved.

1. Introduction candidate for qualitative or semi-quantitative detection of antibi-

The overused antibiotics in the animal husbandry and modern
agriculture bring about the existence of the residues in massive
foodstuffs, and trigger potential health hazard for people around
the world [1–3]. The important role of food safety monitoring for
antibiotic residues has heavily driven the ever-increasing demand
for exploiting highly facile, sensitive and cost-effective bioassays.
Immunoassay, based on the specific antigen-antibody reaction,
has been one of the most widely used methods [4]. Nowadays,
various immunosensing systems and strategies based on differ-
ent signal-generation principles have been intensively reported,
e.g., electrochemistry [5], electrochemiluminescence [6], chemi-
luminescence [7], fluorescence [8], and colorimetric assay [9].
Especially, the colorimetric immunoassay with simple, quick, sen-
sitive and direct readout with the naked eye has been a promising
∗ Corresponding author at: Key Laboratory of Analysis and Detection for Food
Safety (Ministry of Education), Department of Chemistry, Fuzhou University, Fuzhou
350116, PR China.
E-mail addresses: dianping.tang@fzu.edu.cn, 471598823@qq.com (D. Tang).
otic residues [10,11]. Despite some advances in this field (e.g., by
using the specific enzymatic reaction or the nanocatalysts), it is still
the requirement to exploit some new signal-transduction systems
for the development of colorimetric immunoassays.
Phenanthroline (Phen) and its derivatives with the bidentate
heterocyclic ligands have been introduced excellently in construc-
tion of the inorganic, organic and supramolecular chemistry [12].
Also, it is rigid planar and electron-conjugated heterocyclic aro-
matic, thus predestining for intense coordination ability toward
various transition metals [13]. In recent years, Phen and its
derivatives, as well as metal complexes, have been deeply-going
studied in the molecular biology, such as DNA/RNA binding [14,15],
organic-metal frameworks [16] and molecular recognition sens-
ing [17]. Taking advantages of low-lying ␲ antibonding orbitals
features, Phen displays a remarkable coordination capacity for sta-
ble transition metal ions in lower oxidation states, e.g., Ru(II) [18],
Cu(I) [19] and Co(II) [20], to form metal complexes (M-Phen) in
low energy metal-to-ligand charge transfer (MLCT) state. Inspir-
ingly, the charge transfer spectrum appears in the visible spectral
region to form the distinguishable colors. Hu et al. employed Fe(II)-
Phen reporter for the detection of alkaline phosphatase activity

https://doi.org/10.1016/j.snb.2018.03.049
0925-4005/© 2018 Elsevier B.V. All rights reserved.
 R. Ren et al. / Sensors and Actuators B 265 (2018) 174–181 175

Scheme 1. Schematic illustration of signal-on competitive-type colorimetric immunoassay for the detection of streptomycin (STR) on monoclonal anti-STR antibody-coated
microplate using glucose-loaded liposome (GLL) as the signal tracer labeled with STR-bovine serum albumin (BSA) conjugate: (A) competitive-type immunoreaction between
STR-BSA-conjugated liposome and target STR for the immobilized anti-STR antibody on the microplate followed by the glucose release from the liposome with the aid of
Triton X-100, and (B) glucose oxidase (GOx)-triggered the change of the Fe(II)-Phen system in the absorbance and visual color by the reaction of the produced H2 O2 with
iron(II).

[21]. MLCT-based chromogenic assays exhibited more portable
operation and higher stability in comparison with noble metal
nanomaterials- based colorimetric methods.
Another concern for the development of colorimetric
immunoassays is to obtain low limits of detection and quan-
tification by novel-innovative and vigorous nanostructure-based
labeling strategies. Liposomes are microscopic spherical vesicles
with a lipid bilayer phospholipid and an aqueous core. Abun-
dant biomolecules or signal tags (e.g., drugs, genes, enzymes,
quantum dot, dyes and nutrients) can be encapsulated into the
aqueous core or anchored on the surface [22]. Mei’s group uti-
lized enediol-ligands-encapsulated liposomes to design a highly
sensitive photoelectrochemical immunoassay [23]. Recently, our
group employed dopamine-loaded liposomes and Mn2+ -doped
Zn3 (OH)2 V2 O7 ·2H2 O nanobelts for in-situ amplified photoelectro-
chemical immunoassay to detect target aflatoxin B1 [24]. Inspired
by these advantages of liposomes, our motivation in this work is to
combine high-loaded liposome with MLCT-based Fe(II)-Phen chro-
mogenic system for development of high-efficient colorimetric
immunoassay.
Streptomycin (STR), a broad-spectrum antibiotic, exhibits excel-
lent antibiotic activity against a wide range of Gram-negative and
Gram-positive microorganisms by inhibiting bacterial protein syn-
thesis [25,26]. However, there have been risks of kidney damage
and permanent deafness to take the foodstuffs contained strepto-
mycin [27]. Herein, we report the proof-of-concept of high-efficient
colorimetric immunoassay for sensitive detection of STR (used as
a model analyte) by coupling with the Fe(II)-Phen chromogenic
system and liposome-based amplification strategy (Scheme 1).
The assay is carried out in anti-STR antibody-coated microplate
with a competitive-type reaction mode. Glucose-loaded liposome
(GLL) labeled with STR-bovine serum albumin (STR-BSA) conju-
gates is used as the signal-generation tag (STR-GLL). The detection
mainly consists of immunoreaction and enzyme-controlled visual
color rendering process. Upon addition of target STR, the ana-
lyte competes with STR-GLL for the coated anti-STR antibody on
the microplate. Accompanying the immunocomplex formation, the
carried STR-GLL conjugates are dissociated by Triton X-100, thus
resulting in the release of the encapsulated glucose from the lipo-
somes. The as-released glucose molecules can be oxidized into
gluconic acid and H2 O2 in the presence of glucose oxidase (GOx).
The latter (H2 O2 ) exhibits high catalytic ability to oxidize Fe(II) to
Fe(III), thereby causing the change of the complex ion [Fe(phen)3 ]2+
from orange-red to light-yellow in pH 7.4 solution. Meanwhile, the
absorbance at 510 nm decreases with the increasing H2 O2 con-
centration. By monitoring the change in the visual color on the
smartphone with the naked-eyes or the absorbance on a UV–vis
spectrometry, we can evaluate the level of target STR in the sample
qualitatively/quantitatively. The aim of this study is to explore a
signal-on competitive colorimetric immunoassay for detection of
small-molecular antibiotic residues.
2. Experimental section
2.1. Reagents and chemicals
Streptomycin-bovine serum albumin (STR-BSA) conjugates and
rabbit anti-streptomycin monoclonal antibody (mAb; 1.0 mg mL−1 )
176 R. Ren et al. / Sensors and Actuators B 265 (2018) 174–181
were purchased from Beijing Biosynth. Biotech. Co., Ltd. (Beijing,
China). Streptomycin (STR) standards with different concentrations
were achieved from Dingguo Biotech. Co., Ltd. (Beijing, China). Glu-
cose oxidase (GOx), glucose, cholesterol, hydrogenated soybean
phospholipids, phosphoethanolamine (PEA) were acquired from
Sinopharm Chem. Re. Inc. (Shanghai, China). Bovine serum albu-
min (BSA, 96–99%), Triton X-100 and Tween-20 were obtained
from Sigma-Aldrich (St. Louis, U.S.A.). 1,10-Phenanthroline mono-
hydrate (Phen), FeCl2 ·4H2 O, FeCl3 ·6H2 O, and glutaraldehyde (GA,
25 wt%) were available by Fuchen Chemicals (Tianjin, China). All
other chemicals were of analytical grade and used as received
without further purification. Deionized water generated from a
Millipore water purification system (18.25 M cm−1 , Milli-Q, Mil-
lipore) was used in all runs. A pH 7.4 phosphate-buffered saline
(PBS, 0.01 M) solution was prepared by addition 21.221 g K2 HPO4 ,
0.1362 g KH2 PO4 and 0.7455 g KCl into 1000 mL deionized water.
The washing and blocking buffers were obtained in pH 7.4 PBS
containing 0.05% Tween 20 (v/v) and 1.0 wt% BSA, respectively.
2.2. Preparation of glucose-loaded liposome (GLL)
Glucose-loaded liposome (GLL) was prepared by the reversed-
phase evaporation strategy according to the literatures [28,29].
Initially, the hydrogenated soybean phospholipids, cholesterol and
PEA (molar ratio: 50: 10: 1) were dissolved into a mixture con-
taining chloroform and methanol (5.0 mL, 3: 1, v/v). After being
sonicated for 5 min at room temperature, the mixture was evap-
orated at 45 ◦ C under the protection of nitrogen until a gel-like
suspension was obtained. Following that, the excess glucose aque-
ous solution (10 mL) was injected into the suspension using a
syringe. Afterwards, glucose-loaded liposome was formed after
sonication for 5 min at 45 ◦ C. To produce the homogeneous sus-
pension with the uniform size, the mixture was filtered several
times by using 0.4-␮m polycarbonate film. The obtained suspen-
sion was purified by a dialysis cassette (MWCO, 12–14 kDa) with
deionized water for 2 h to remove free glucose molecules. Finally,
glucose-loaded liposome was resuspension in PBS (1.0 mL, pH 7.4)
and stored at 4 ◦ C before use
2.3. Conjugation of STR-BSA with glucose-loaded liposome
(STR-GLL)
STR-BSA conjugates were linked to glucose-loaded liposome by
the cross-linkage reagent (glutaraldehyde) similar to our previous
work [24]. Initially, the above-prepared GLL suspension (1.0 mL)
was slowly dropped in the excess glutaraldehyde solution (5.0 mL,
25 wt%) under slight stirring, and then incubated for 2 h at room
temperature. Following that, the resulting suspension was dia-
lyzed in PBS (10 mM, pH 7.4) at room temperature for 24 h to
remove the unreacted glutaraldehyde. Afterwards, STR-BSA conju-
gates (300 ␮L, 1.0 mg mL−1 ) were added into the resultant mixture,
and gently shaken overnight at 4 ◦ C. During the process, STR-BSA
was conjugated to GLL via the reaction of amino group with alde-
hyde group. After ultrafiltration, STR-BSA-conjugated GLL (denoted
as STR-GLL) was dispersed into PBS (1.0 mL, 10 mM, pH 7.4) con-
taining 1.0 wt% BSA, and stored at 4 ◦ C when not in use.
2.4. Smartphone-based colorimetric immunoassay measurement
Prior to measurement, monoclonal rabbit anti-STR antibod-
ies (mAb) were coated onto a high-binding polystyrene 96-well
microtiter plates (Ref. 655061, Greiner, Frickenhausen, Germany)
by incubation them overnight at 4 ◦ C with 50 ␮L per well of mAb at
a level of 10 ␮g mL−1 in 0.05 M sodium carbonate buffer (pH 9.6).
The microplate was covered with the adhesive plastics plate seal-
ing film to prevent evaporation. On the following day, the plate was
washed three times with the washing buffer, and then incubated
with 300 ␮L per well of blocking buffer for 60 min at 37 ◦ C with
shaking. The microplate was then washed as before. Following that,
mAb-functionalized microplate was used for the detection of tar-
get STR with a competitive-type immunoassay mode as follow: (i)
50-␮L STR standard/sample and 50-␮L of STR-GLL (constant con-
centration prepared above) were simultaneously added to the well
and incubated for 40 min at 37 ◦ C with gentle shaking; (ii) after
the microplate was washed with the washing buffer, Triton X-100
(100 ␮L, 10 mg mL−1 ) solution was injected into the well to break
the liposome and release the encapsulated glucose molecules; (iii)
20 ␮L of 1.0 mg mL−1 GOx and 100 ␮L of 5.0 mM FeCl2 were trans-
ferred into each well, and reacted for 30 min at 37 ◦ C; and (iv)
100 ␮L of 15 mM phenanthroline monohydrate was introduced
and shaken (50 s) for development of visual color. Meanwhile, the
visual color and absorbance of the resulting solution was quali-
tatively/quantitatively determined on a portable smartphone and
a Tecan Infinite 200 PRO (TECAN, Switzerland), respectively. All
determinations were made at least in duplicate. The sigmoidal
curves were calculated by mathematically fitting experimental
points using the Rodbard’s four parameter function with Origin 6.0
software. Graphs were plotted in the form of absorbance against
the logarithm of STR concentration.
2.5. Food samples and extraction procedure
Food samples including honey, milk, muscle and kidney were
purchased from the local Carrefour supermarket (Fuzhou, China)
and extracted referred to our previous report [30]. Fresh milk
matrix for further addition calibration was prepared as follows:
Fresh milk sample (1.0 mL, fat percentage: 3.25%) was initially
diluted into 9.0-mL ultrapure water, and then STR standards with
different concentrations were spiked into the milk matrix. These
milk samples were assayed by using the colorimetric immunoassay
after incubation with matrix-STR for 18 min.
To spike sample into natural honey and milk, a total of 20-mL
honey or milk samples was initially placed in a polypropylene cen-
trifuge tube, and then the mixture was centrifuged for 25 min at 12
000g. The resulting supernatant was transferred to a clean tube at
a final volume of 20-mL distilled water. For the porcine muscle and
kidney, 5.0 g of the chopped sample was initially mixed with 20 mL
of methanol/water (80:20, v/v), and then sonicated for 60 min at
room temperature to assist extraction. Following that, these sam-
ples were centrifuged for 20 min at 12 000g. Finally, 3.0 mL of the
supernatant was collected and transferred into another centrifuge
tube and diluted to 15 mL of distilled water. These real samples
were prepared by spiking aliquots of STR standards into different
volumes of extraction. These spiking food samples were monitored
by using the colorimetric immunoassay.
3. Results and discussion
3.1. Characterization of glucose-loaded liposome
As described above, the glucose-loaded liposome was used as
the enhancers to increase the signal of the colorimetric assay.
To investigate the successful preparation of glucose-loaded lipo-
some, Negative-stain transmission electron microscopy (NS-TEM;
H-7650, Hitachi Instruments, Tokyo, Japan) was utilized to char-
acterize its morphology. As seen from Fig. 1A, the glucose-loaded
liposome clearly revealed a perfect spherical vesicle structure
(Fig. 1A, bottom inset) with the well-dispersed and well-defined
edges. Also, the as-prepared liposomes exhibited a pale blue opales-
cence (Fig. 1A, right inset), which was in accordance with previous
reports [31,32]. Moreover, the diameter (d) and surface charge
 R. Ren et al. / Sensors and Actuators B 265 (2018) 174–181
Fig. 1. (A) Negatively-stained TEM image of 2 wt% dispersions of the phosphotungstic/PBS acid system, high-resolution negatively-stained TEM image (bottom inset),
photograph (right inset) and DLS data (left inset) of the glucose-loaded liposome; and (B) PGM responses of (a) PBS solution, (b) Triton X–100 + PBS, (c) STR-GLL + PBS, and
(d) Triton X–100 + STR-GLL + PBS (PBS: pH 7.4, 10 mM).
zeta-potential () of glucose-loaded liposome were investigated
using dynamic light scattering (DLS; Zetasizer Nano S90, Malvern,
London, U.K.). An average diameter of approximately 199 ± 18 nm
was obtained, indicating that the GLL with a favorable size could
provide a vast space for glucose loading (Fig. 1A, left inset).
Meanwhile, a negative zeta-potential () of −19.0 ± 0.8 mV was
given, which mainly derived from the negatively charged sugar-
phosphate backbone. Logically, two puzzling questions arise to (i)
whether glucose-loaded liposome could be stable in the detection
solution before addition of Triton X-100, and (ii) Triton X-100 could
cause the release of glucose from the liposome. To verify the con-
cerns, glucose-loaded liposome was monitored by using personal
glucometer (PGM; AccuChem Active, Malaysia) in the absence and
presence of Triton X-100 (Fig. 1B). As control test, we investigated
the detection solution (pH 7.4 PBS, 10 mM), and almost no PGM
signal was acquired, indicating that the blank PBS solution could
not cause the change in the PGM signal (column ‘a’). Moreover,
addition of Triton X-100 (column ‘b’) and STR-GLL (column ‘c’)
into pH 7.4 PBS did not also trigger the increasing of PGM sig-
nal. Significantly, the signal heavily increased when Triton X-100
and STR-GLL were simultaneously present in the detection solution
(column ‘d’). These results revealed that the encapsulated glucose
molecules could be released out from the liposome through the
interaction between Triton X-100 and liposome. Therefore, Triton
X-100-triggered controllable release system from the liposome was
feasible.
3.2. Control tests and feasible analysis
Scheme 1B illustrates the assay principle based on the Triton X-
100-liposome controllable release system by oxidizing Fe(II) into
Fe(III) to inhibit the chelation of Fe(II) and Phen. First, we inves-
tigated the reaction characteristics of Phen with Fe(II) and Fe(III),
respectively, by using UV-absorption spectroscopy and the naked-
eyes (Fig. 2A) (note: 100 ␮L of 5 mM Fe2+ , 100 ␮L of 5.0 mM Fe3+ ,
100 ␮L of 15 mM Phen were used in this case). No characteristic
adsorption peak was observed from 800 nm to 300 nm at 1,10-
phenanthroline alone (curve ‘a’). Favorably, a strong characteristic
peak at 510 nm was achieved after the interaction of Phen with
Fe(II) (curve ‘b’). However, this peak disappeared upon addition of
Fe(III) into the Phen (curve ‘c’). Moreover, the visual colors changed
from orange-red to light-yellow (Fig. 2A, photographs). Further-
more, we studied the specificity of Fe(II)-Phen system against other
177
metal ions (e.g., Fe3+ , Cu2+ , Ni2+ , Mn2+ , Zn2+ , Ca2+ , Co2+ , Mg2+ ,
Al3+ and Fe2+ ) (Fig. 2B). Only Fe(II) ion could cause the strong
absorbance at 510 nm, whereas other metal ions exhibited a very
weak absorbance. As shown from the inset in Fig. 2B, other inter-
fering ions in the Phen solution displayed the close colorless. The
results indicated that the Fe(II)-Phen system could be specifically
utilized for the quantitative screening of Fe(II) ion. Thanks to these
properties, Fe(II) standards with different concentrations was mon-
itored by using the Fe(II)-Phen system under the same conditions
(100 ␮L of 15 mM Phen used in this case). As seen from Fig. 2C, the
absorbance increased with the increasing Fe(II) concentration. The
detection limit of using the Fe(II)-Phen system was 6.0 ␮M. Such an
obvious change in the absorbance and color could provide a prereq-
uisite for the development of the Fe(II)-Phen system by controlling
the concentration of Fe(II) ion.
As is well-known, Fe(II) ion can be consumed by the classical
redox reaction (H2 O2 + Fe2+ + H+ → Fe3+ + H2 O). The oxidizer (H2 O2 )
can originate from glucose oxidase toward the catalytic oxidiza-
tion of glucose molecules. In this regard, Triton X-100-responsive
glucose release from the liposome can be readily integrated with
the Fe(II)-Phen system by using glucose oxidase (GOx). To demon-
strate this issue, several control tests were investigated under
different conditions. As seen from curve ‘a’ in Fig. 2D, the absence
of GOx between two systems could not decrease the absorbance
of the Fe(II)-Phen system relative to (Fe2+ + Phen) alone (Fig. 2A,
curve ‘b’), even if the GLL-Triton system and the Fe(II)-Phen sys-
tem simultaneously existed in the detection solution. When GOx
was added to these two systems, however, the absorbance obvi-
ously decreased (Fig. 2D, curve ‘c’ vs. curve ‘a’). Moreover, the color
changed from orange-red to light-red (Fig. 2D, photograph ‘c’ vs.
photograph ‘a’). These results indicated that GOx could be used
as the bridge-linker between two systems. For comparison, we
also monitored the effects of other three chemicals (e.g., STR-GLL,
Phen and Fe2+ ) on the rounded system [Fig. 2D, curve ‘b’; (STR-
GLL + Triton X-100) + GOx + (Fe(II) + Phen)]. Curve ‘b’ in Fig. 2D gives
UV–vis absorption spectroscopy of this system in the absence of
STR-GLL, indicating that Triton X-100 and GOx could not stimulate
the change of the Fe(II)-Phen system. Moreover, the simultaneous
existence of Fe(II) and Phen could induce the appearance of char-
acteristic peak at a 510 nm regardless of the presence of STR-GLL,
Triton X-100 and GOx (Fig. 2D, curves ‘d–e’). These results suggested
the feasibility of our designed visual system.
178 R. Ren et al. / Sensors and Actuators B 265 (2018) 174–181

Fig. 2. (A) UV–vis absorption spectra of (a) Phen, (b) Fe2+ + Phen and (c) Fe3+ + Phen; (B) the specificity of the Fe(II)-Phen system toward different metal interfering ions (insets:
the corresponding photographs); (C) the Fe(II)-Phen system toward Fe(II) response in the absorbance (inset: the corresponding linear plots); and (D) UV–vis absorption spectra
of (a) STR-GLL + Triton X–100 + Fe(II) + Phen, (b) Triton X–100 + GOx + Fe(II) + Phen, (c) STR-GLL + Triton X–100 + GOx + Fe(II) + Phen, (d) STR-GLL + Triton X–100 + GOx + Fe(II),
and (e) STR-GLL + Triton X–100 + GOx + Phen (note: These chemicals were dispersed in pH 7.4 PBS, 10 mM).

3.3. Evaluation of experimental conditions
In this work, the assay mainly consists of three steps: the
competitive-type immunoreaction, the catalytic reaction of GOx
between the GLL-Triton system and Fe(II)-H2 O2 system, and the
colored reaction in the Fe(II)-Phen system. To achieve an optimum
analytical performance, the experimental conditions should be
optimized during the whole measurement. In this case, 5.0 ng mL−1
STR was used as an example. Typically, it takes some times for
the antigen-antibody reaction. A short incubation time did not
facilitate the detection of low-concentration analyte due to the
formation of the small-amount immunocomplexes. As shown in
Fig. 3A, the absorbance of the colorimetric immunoassay increased
with the increasing reaction time, and tended to level off after
40 min. A long incubation time did not cause the significant increase
in the absorbance. To save the assay time, 40 min was used for the
antigen-antibody reaction.
At this condition, the formed immunocomplexes with glucose-
loaded liposomes were used for the glucose release with the
assistance of Triton X-100 and the H2 O2 generation by the added
GOx. the as-released glucose from the liposome by Triton X-
100 and the as-produced H2 O2 by the added GOx would directly
affect the progression of Fe(II)-Phen system. In this study, the
glucose release and the H2 O2 generation were simultaneously
implemented. Fig. 3B represents the effects of different reaction
times on the absorbance of the colorimetric assay. An optimal
absorbance in this system was obtained after 30 min, indicating
that the reaction reached a dynamic equilibrium. So, 30 min could
be utilized for the catalytic reaction of GOx, which adequately
ensured the formation of H2 O2 for the following redox reaction.
Certainly, an important issue on the development of the colori-
metric immunoassay lies in the colored time. As indicated from
Fig. 3C, the absorbance increased the increment of the chelation
time, and reached a plateau after 50 s with a rapid colored reaction.
To reduce the whole detection time, 30 min and 50 s were employed
for the enzymatic catalytic reaction and the colored progression,
respectively.
3.4. Analytical performance of Fe(II)-Phen-based colorimetric
immunoassay
To evaluate the sensitivity and dynamic measurement range
of the developed colorimetric immunoassay, STR standards with
different levels were tested in the 96-well microtiter plates by
using Fe(II)-Phen-based chromogenic system accompanying the
signal-amplified strategy of glucose-loaded liposome under the
optimum conditions. Fig. 4A gives UV–vis absorption spectra of
the colorimetric immunoassay toward STR standards with differ-
ent concentrations. Owing to the high-loading capacity of liposome
toward glucose molecules, the absorbance at 510 nm increased
with the increasing of STR amount in the sample. Moreover,
the color of the detection solution changed from light-yellow to
 R. Ren et al. / Sensors and Actuators B 265 (2018) 174–181 179

Fig. 3. Effects of (A) immunoreaction time, (B) the catalytic time of GOx between the GLL-Triton system and Fe(II)-H2 O2 system, and (C) the color-developed time in the
Fe(II)-Phen system on the absorbance of the colorimetric immunoassay (5.0 ng mL−1 STR used in all cases).

Fig. 4. (A) Absorbance intensity and (B) calibration plots of Fe(II)-Phen-based colorimetric immunoassay toward different-concentration STR standards (inset: the corre-
sponding linear plots and the corresponding photograph); (C) the specificity of Fe(II)-Phen-based colorimetric immunoassay against 5.0 ng mL−1 STR, 5.0 ng mL−1 kanamycin
(Kana), 5.0 ng mL−1 oxytetracycline (OTC), 5.0 ng mL−1 chloram phenicol (CAP) and 5.0 ng mL−1 gentamicin sulphate (GS).

orange-red (Fig. 4B, inset). A good linear correlation between the
absorbance at 510 nm and the logarithm of STR concentration was
achieved in the dynamic range from 1.0 pg mL−1 to 20 ng mL−1 with
a limit of detection (LOD) of 0.4 pg mL−1 on the basis of 3␴/slope
(where ␴ stands for the standard deviation for 10 determinations
in the blank sample) (Fig. 4B). Obviously, the LOD of our strategy
was comparable with other detection methods and schemes, e.g.,
surface plasmon resonance-based immunosensor (0.37 ng mL−1 )
[33], optical immunobiosensor (4.1 ng mL −1 ) [34] and fluorescence
immunoassay (2.01 ng mL−1 ) [35]. Moreover, the developed colori-
metric immunoassay could accomplish all the steps within 1.5 h per
sample, and the cost was 0.67 RMB for each sample. Then, Fe(II)-
Phen-based colorimetric immunoassay was simple, rapid and low
cost for the detection of STR residues.
3.5. Precision, reproducibility and selectivity
The reproducibility and precision of the Fe(II)-Phen-based
colorimetric immunoassay were investigated by repeatedly assay-
ing three high-middle-low STR standards including 10 pg mL−1 ,
15 ng mL−1 and 50 ng mL−1 , using identical batches of STR-GLL,
respectively. Results revealed that the coefficients of variation (CVs)
of the intra-assay with this method were 7.3, 4.1, and 6.7%, whereas
the CVs of the inter-assay with various batches were 8.9, 10.3, and
7.6% toward the aforementioned concentrations. The results indi-
cated that the reproducibility of the colorimetric immunoassay was
acceptable.
Next, the specificity of Fe(II)-Phen-based colorimetric
immunoassay was also monitored by challenging this system
against other antibiotics including kanamycin (Kana), oxytetra-
cycline (OTC), chloram phenicol (CAP) and gentamicin sulphate
(GS), respectively. The results are recorded in Fig. 4C. As seen from
Fig. 4C, almost no obvious response was observed toward other
antibiotics, while the significant response was obtained toward
target STR. Moreover, the coexistence of the interfering antibiotics
caused no obvious change in the absorbance relative to target STR
alone. So, the selectivity of the colorimetric immunoassay was
satisfactory.
3.6. Analysis of real samples and method validation
To further monitor the possibility of the newly developed
colorimetric immunoassay for testing of real samples, spiked
samples of STR including milk, honey, muscle and kidney have
been prepared referring to our previous report [36]. Follow-
ing that, these as-prepared samples were determined by using
the colorimetric immunoassay and the results were compared
the high-performance liquid chromatograph (HPLC) method.
Experimental results are listed in Table 1. The recoveries were
85.6–119.4% for colorimetric immunoassay. The regression equa-
tion on the basis of the average values for each sample between two
methods could be fitted to y = 1.0036 x − 0.1664 (R2 = 0.9908, n = 14;
where x and y stand for the results obtained by the colorimetric
immunoassay and HPLC, respectively). No significant differences
between two methods, thereby indicating that the colorimetric
immunoassay was capable of detecting STR in real sample.
4. Conclusions
In summary, this work constructed a new signal-on colori-
metric immunoassay protocol for the qualitative or quantitative
detection of small-molecular antibiotic residues (streptomycin
used as a model analyte) with a competitive-type assay format.
The signal was amplified by glucose-loaded liposome with high
carried capacity and high-efficient GOx catalytic property. The
Fe(II)-Phen system was used as the visually colored chromogen
180 R. Ren et al. / Sensors and Actuators B 265 (2018) 174–181

Table 1
Comparison of the results obtained by the colorimetric immunoassay and HPLC for STR samples spiked in foodstuffs.

Substrate Sample no. Spiked STR (ng mL−1 ) Methods; recovery (mean ± SD, ng mL−1 , n = 3)

Colorimetric immunoassay HPLC

Milk 1a 0 0.04 ± 0.02 (−) 0.02 ± 0.01 (−)

2 2 1.91 ± 0.13 (95.5%) 1.94 ± 0.08 (97%)
3 5.0 4.28 ± 0.14 (85.6%) 5.09 ± 0.23 (101.8%)
4 15 17.37 ± 0.17 (115.6%) 16.91 ± 0.41 (112.7%)

Honey 5a 0 0.03 ± 0.02 (−) 0.04 ± 0.01 (−)

6 2 2.26 ± 0.14 (113%) 1.97 ± 0.09 (98.5%)
7 5.0 6.32 ± 0.14 (126.4%) 4.9 ± 0.36 (98%)
8 15 14.90 ± 0.22 (99.3%) 14.65 ± 0.23 (97.7%)
9a 0 –b –

Muscle 10 2 2.31 ± 0.14 (115.5%) 1.96 ± 0.10 (98%)

11 5.0 5.97 ± 0.16 (119.4%) 4.98 ± 0.28 (99.6%)
12 15 14.98 ± 0.25 (99.9%) 14.75 ± 0.29 (98.3%)
13a 0 – –
Kidney 14 2 1.93 ± 0.12 (95.5%) 1.84 ± 0.13 (92%)
15 5.0 4.78 ± 0.11 (95.6%) 5.11 ± 0.23 (102.2%)
16 15 16.37 ± 0.13 (109.1%) 17.31 ± 0.31 (115.4%)
a
Without spiking STR samples.
b
Not detected.

for the development of the colorimetric immunoassay. Compared
with conventional competitive-type colorimetric immunoassays,
highlights of this work can be simply summarized as follows:
(i) introduction of the Fe(II)-Phen system can induce the genera-
tion of the signal-on assay mode relative to traditional signal-off
competitive immunoassays; (ii) this method can efficiently com-
bine with nanoliposome label and natural enzyme for the signal
amplification; and (iii) the colored procedure is relatively simple
and rapid (1.5 h per sample on the whole assay). Additionally, the
visible bright-coloured Fe(II)-Phen chelation system was readily
triggered by the catalytic product of GOx toward the abundant
glucose released from liposomes. Inspiringly, the work provides a
simple, sensitive, low-cost and visual readout with the naked-eyes
for the detection of the streptomycin. Future work should focus
on the practical application of the colorimetric immunoassay in
foodstuff.
Acknowledgements
This work was financially supported by the National Natural
Science Foundation of China (Grant nos. 21675029 & 21475025),
the National Science Foundation of Fujian Province (Grant no.
2014J07001), and the Program for Changjiang Scholars and Innova-
tive Research Team in University (Grant no. IRT15R11). Also thanks
Dr. Jiayao Liao for HPLC measurement for real sample (College of
Chemistry, Southwest University, Chongqing, China)
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Biographies
Rongrong Ren received her B.S. degree from Fuzhou University in 2016. She is per-
suing her M.S. degree at Fuzhou University in the group of Prof. Dianping Tang.
She is majoring in nanomaterials and biosensing in Institute of Nanomedicine and
Nanobiosensing, Department of Chemistry, Fuzhou University, Fuzhou, PR China.
Guoneng Cai received he BS degree from Fuzhou University in 2016. He is per-
suing her M.S. degree at Fuzhou University in the group of Prof. Dianping Tang.
He is majoring in nanomaterials and biosensing in Institute of Nanomedicine and
Nanobiosensing, Department of Chemistry, Fuzhou University, Fuzhou, PR China.
Zhenzhong Yu received he BS degree from Fuzhou University in 2016. He is per-
suing her M.S. degree at Fuzhou University in the group of Prof. Dianping Tang.
He is majoring in nanomaterials and biosensing in Institute of Nanomedicine and
Nanobiosensing, Department of Chemistry, Fuzhou University, Fuzhou, PR China.
Dianping Tang is a Full Professor for Analytical Chemistry at the Fuzhou University,
received his PhD from the Southwest University of China in 2008. His research inter-
ests mainly include Clinical/Environmental immunoassays, Biological and Chemical
microsensors. Importantly, he was selected amongst the 100 most influential Peo-
ple in the Analytical Scientist in 2013. He is currently a member within the editorial
board of “Analytical Chemistry”, “Scientific Reports” and “Microchimica Acta”. Up
to now, more than 100 publications under his authorship appeared in reviewed
international scientific journals.