Magnetic bead-based mimic enzyme-chromogenic substrate and silica nanoparticles signal amplification system for avian influenza A (H7N9) optical immunoassay

Article abstract of DOI:10.1039/c7ra06273g

This work reports on a simple, feasible and optical color-enhanced colorimetric immunoassay with hemin (a horseradish peroxidase mimic enzyme) catalyzed color reaction and multifunctional colored silica nanoparticles for qualitative and quantitative deter

Full text of DOI:10.1039/c7ra06273g

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PAPER
Magnetic bead-based mimic enzyme-chromogenic
substrate and silica nanoparticles signal
amplification system for avian influenza A (H7N9)
optical immunoassay†
Cite this: RSC Adv., 2017, 7, 41989
a
a
b
a
a
a
a
Dan Su, Hanyun Li, Jinlin Li, Yali Liu, Mi Peng, Bingwei Feng, Pengfei Xu
a
and Yonggui Song
*
This work reports on a simple, feasible and optical color-enhanced colorimetric immunoassay with hemin
a horseradish peroxidase mimic enzyme) catalyzed color reaction and multifunctional colored silica
(
nanoparticles for qualitative and quantitative determination of avian influenza A (H7N9) virus (H7N9 AIV)
at an ultralow concentration by using the magnetic nanobeads (MBs)-mimic enzyme-multifunctional
silica nanoparticles color-enhanced chromogenic substrate system. In this work, MBs were fabricated by
co-immobilizing target recognition molecules (mAb–MB), which were used as both separation and
enrichment carriers. The multifunctional colored silica nanoparticles were synthesized by doping Cy2
(
red organic dye) into silica nanoparticles using an inverse microemulsion method, and then made
a covalent modification of pAb and glucose oxidase (GOx). In the presence of H7N9 AIV, GOx catalyzed
glucose to gluconic acid and hydrogen peroxide (H ). The latter can oxidize 4-aminoantipyrine
4-AAP) to red products and the reaction is catalyzed by hemin. The chromogenic compound and the
2 2
O
(
Received 5th June 2017
Accepted 8th August 2017
red-silica nanoparticles (Red-SiNPs) together cause the color of the solution to change from colorless to
red. Through this color-enhanced immunoassay, we can detect H7N9 AIV with the naked eye and UV
detector. And the immunosensor provides a simple and reliable platform with high sensitivity and
selectivity which shows great potential in early diagnosis of diseases and in public health security.
DOI: 10.1039/c7ra06273g
rsc.li/rsc-advances
6,7
matrixes. Usually, high sensitivity for ELISA is achieved by
1
. Introduction
using an indicator system that results in the amplication of the
8,9
Since human infections with novel H7N9 AIV were rst docu- product to be measured, e.g., enzyme labels or nanolabels.
1
mented in Shanghai City and Jiangsu Province in 2013, human Undoubtedly, enzyme labels are utilized more widely than other
2,3
cases continued to be reported in China in 2014 and 2016,
which raise signicant concerns that H7N9 AIV is a likely horseradish peroxidase (HRP), can cause the conversion of 10
labeling strategies, since a single enzymatic molecule, e.g.,
7
10
candidate for the next pandemic strains. Meanwhile, fast and substrate molecules per minute. Meanwhile using H
2 2
O as
sensitive virus detection techniques are urgently needed to substrate, HRP can catalyze the oxidation of numerous
11
provide immediate and appropriate clinical strategies to control hydrogen donors (DH ). Despite some advances in this eld,
2
the spread of the virus. Up to now, traditional virus detection there are some limitations, such as use of an expensive protease
methods have been employed for inuenza virus detection, like HRP, harsh reaction conditions, short period of stability
4
including virus isolation, polymerase chain reaction (PCR) and and lack of easy operation methods to combine visual qualita-
5
enzyme-linked immunosorbent assay (ELISA). Among these tive identication and quantitative analysis methods. Moreover,
methods, ELISA based on a specic antigen–antibody interac- for optical detection, too light a color is the important reason
tion is a very powerful technique for the determination of for the unsatisfactory detection limit.
clinically important analytes in
a
variety of biological
To solve the above problems, a magnetic nanobeads (MBs)-
mimic enzyme chromogenic substrate-multifunctional silica
nanoparticles color-enhanced system-based colorimetric
immunoassay (MB–MEMSCI) has been designed. In this work,
MBs were used as separation and enrichment carriers, and
hemin was used in place of HRP to catalyze hydrogen peroxide
and simultaneously to produce a color reaction. Moreover,
a multifunctional bright red silica nanoparticle modied with
a
National Pharmaceutical Engineering Center for Solid Preparation in Chinese Herbal
Medicine, Jiangxi University of Traditional Chinese Medicine, 56 Yangming Road,
Nanchang 330006, China. E-mail: songyonggui1999@163.com; Fax: +86 791
8
7119632; Tel: +86 791 87119632
b
Nanchang Institute for Food and Drug Control, Nanchang 330038, China
†
Electronic supplementary information (ESI) available. See DOI:
0.1039/c7ra06273g
1
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antibody and GOx was prepared. The experimental results show (0.2 g) were dissolved in 1000 mL of double-distilled water.
that the catalytic activity of hemin can match that of HRP, and it Phosphate-buffered saline (PBS) solutions with various pH
also has the advantages of low cost, good stability of physical values were prepared by using Na HPO and KH PO , and 0.1 M
2
4
2
4
and chemical properties, easy preservation and mild reaction NaCl was used as the supporting electrolyte. Clinical serum
conditions. To model the MB–MEMSCI in terms of immuno- samples were made available by Jiangxi Provincial Hospital of
reactions, we employed H7N9 AIV as our target analyte, with Traditional Chinese Medicine, China. And all experiments were
specic monoclonal antibody (mAb) and rabbit-derived poly- in accordance with the guidelines of the National Institute of
clonal antibody (pAb) as the capture and detection antibodies, Food and Drug, Nanchang, China, and approved by the insti-
respectively. Herein, the assay is carried out on a magnetic tutional ethical committee (IEC) of Jiangxi University of Tradi-
immunosensing probe by using polyclonal anti-H7N9 AIV/GOx- tional Chinese Medicine.
labeled Red-SiNPs as the signal-transduction tag with a sand-
wich-type immunoassay format. In the presence of target
H7N9 AIV, the carried GOx accompanying the sandwiched
immunocomplex initially catalyzes glucose to gluconic acid and
2.2 Preparation of mAb–MB conjugates
hydrogen peroxide, and then the regenerated hydrogen Before conjugation with mAb, MBs were initially separated
ꢂ
peroxide is catalyzed by hemin to produce the colored product. using an external magnet and then dried in a vacuum at 80 C
By monitoring the obvious color change together caused by the for 1 h. Following that, 50 mg of MB was added into 1 mL of
red product and the multifunctional Red-SiNPs with the naked anhydrous ethanol and the resulting mixture was sonicated
eye and UV absorption, we can not only qualitatively but also for 10 min at room temperature (RT) to obtain a homoge-
quantitatively determine ultralow concentrations of target neous suspension. Aerward, 30 mL of APTES (98 wt%) was
H7N9 AIV in samples with wide linear range, low detection injected in the mixture and continuously stirred for 6 h at RT.
limit, high selectivity and long-term stability. Meanwhile, the During this process, aminated MB was formed based on the
3
handy operational MB–MEMSCI has good accuracy to detect reaction between –OCH and –OH on the MB. The functional
H7N9 AIV in real samples.
MB was separated and redispersed into 1 mL of PBS (pH 7.4)
containing 300 mL of glutaraldehyde (25 wt%). The suspen-
sion was stirred vigorously for 6 h at RT. Aer magnetic
separation, the precipitate was dissolved into 1 mL of
carbonate buffer (pH 9.6) solution containing 100 mg of mAb
2. Materials and methods
2.1 Materials and reagents
Inactivated H7N9 AIV, H5N1 AIV, pseudo rabies virus (PRV), antibody and shaken on a shaker (MS, IKA GmbH, Staufen,
ꢂ
Newcastle disease virus (NDV) and murine origin H7N9 Germany) overnight at 4 C. The excess mAb antibody was
hemagglutinin (HA) specic mAb and rabbit-derived pAb were removed by magnetic separation. Subsequently, the mAb–MB
obtained from Wuhan Institute of Virology, Chinese Academy of conjugates were treated with 10 wt% BSA–PBS (1.0 mL, pH 7.4) at
ꢂ
Sciences. The inuenza A H9N2 virus and enterovirus 71 viruses 4 C for 2 h to block the unreacted and nonspecic sites. Finally,
ꢁ
1
(
EV71) were obtained from Academy of Military Medical 100 mL of sodium cyanoborohydride (25 mg mL ) was injected
Sciences, Beijing. 3-Aminopropyltriethoxysilane (APTES, into the suspension in order to reduce the resultant Schiff bases.
8 wt%), hemin and glucose oxidase (GOx) were obtained from The as-prepared mAb–MB conjugates were collected by using an
9
Sigma-Aldrich (Shanghai, China). Glucose was purchased from external magnet and dispersed into 1 mL of PBS (pH 7.4) con-
Alfa Aesar (Shanghai, China). 3-[2-(2-Aminoethylamino)ethyl- taining 1.0 wt% BSA and 0.1 wt% sodium azide.
amino]propyltrimethoxysilane (APTMS), tetraethyl orthosilicate
(
TEOS),
dimethylaminopropyl)carbodiimide hydrochloride (EDC) and
-(N-morpholino)ethanesulfonic acid (MES) were obtained from
N-hydroxysuccinimide
(NHS),
1-ethyl-3,3-(3-
2.3 Synthesis of Red-SiNPs
2
Aladdin Industrial Inc. (Shanghai, China). Bovine serum Red-SiNPs were synthesized according to an inverse micro-
albumin (BSA) was obtained from Sinopharm Chemical emulsion method described in a previous paper with slight
1
3,14
Reagent Co. Ltd (Shanghai, China). 4-Aminoantipyrine (4-AAP), modication.
The details of the procedure are described in
phenol, glutaraldehyde (25 wt%), Triton X-100, cyclohexane, the following: 8 mL cyclohexane, 2 g Triton X-100, 2 mL 1-
ꢁ1
hexanol, and ammonia (25–28 wt%) were obtained from Fuchen hexanol, 150 mL Cy2 (100 mg mL ) and 400 mL of water were
Chemicals (Tianjin, China). Colloidal gold (AuNP) of 15 nm in added into a 25 mL Erlenmeyer ask and stirred for 15 min to
12
diameter was synthesized according to our previous report.
ensure the water was completely dispersed into cyclohexane.
MB (particle size ꢀ100 nm) in an aqueous suspension with Aerwards, 100 mL TEOS and 20 mL APTMS were added to this
ꢁ1
a concentration of 25 mg mL were obtained from Chemical inverse microemulsion system followed by 100 mL ammonia
GmbH (Berlin, Germany). Cy2 was supplied by Zhejiang Shun- (25–28 wt%) to catalyze the hydrolysis of TEOS. Aer stirring
long Chemical Co. Ltd (Zhejiang, China). All reagents used for for 48 h, the microemulsion was broken by adding 10 mL
all experiments were of analytical grade and were used without acetone. The Red-SiNPs were separated from the supernatant
further purication. Ultrapure water (18.2 MU cm) was puried by centrifugation at 8000 rpm for 10 min and washed with
by a Millipore-Q system. In the preparation of a carbonate buffer ethanol three times followed by washing twice with ultrapure
solution of pH 9.6, Na
2
CO
3
(1.59 g), NaHCO
3
(2.93 g), and NaN
3
water.
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2.4 Covalent immobilization of antibody and GOx onto Red-
antibody complex; (ii) 50 mL of GOx–Red-SiNPs-pAb as prepared
SiNPs surface
above was injected into the centrifuge tube and incubated for
another 30 min at 4 C to form the sandwiched immunocom-
ꢂ
3
1
0 mg Red-SiNPs were suspended in 20 mL ultrapure water, and
.4 mL acetic acid and 200 mL APTMS were added into the Red-
plex; (iii) 50 mL of glucose (4 mM) in pH 7.0 PBS was added into
ꢂ
the centrifuge tube and incubated for 30 min at 37 C on the
SiNPs solution for the post-coating treatment. The APTMS was
allowed to hydrolyze under stirring for 1 h at room temperature.
Aer the hydrolysis reaction of APTMS, amino groups were
introduced onto the surface of Red-SiNPs. The amino-modied
ꢁ
1
shaker for enzymatic reaction; (iv) 70 mL of 1.0 mg mL 4-AAP,
ꢁ
1
7
0 mL of 0.12 M phenol, 35 mL of 0.05 mg mL heme chloride
solution and 350 mL buffer solution (K HPO : NaOH,
2
4
pH ¼ 10.6) were added to the centrifuge tube in turn; and (v) the
color of the solution was observed and at the same time the
absorbance was recorded at l ¼ 505 nm with a Lambda 35
UV-visible spectrophotometer at room temperature. Note that
the resulting mixture was separated with an external magnet
and washed with pH 7.4 PBS aer steps (i) and (ii). Control tests
with normal (negative) samples and the evaluations for clinical
specimens were performed accordingly. All measurements were
Red-SiNPs (Red-SiNPs-NH ) were isolated by centrifuging at
2
8
000 rpm for 5 min and washing three times with DMF.
Carboxyl-modied Red-SiNPs were synthesized by reacting Red-
SiNPs-NH with glutaric anhydride. 20 mg Red-SiNPs-NH were
dispersed in 5 mL DMF containing 200 mg glutaric anhydride,
and then the solution was stirred under N gas for 6 h. These
carboxyl-modied Red-SiNPs were centrifuged at 8000 rpm for
0 minutes and washed with PBS (10 mM, pH 7.3). The pAb and
2
2
2
1
ꢂ
done at room temperature (25 ꢃ 1.0 C).
GOx were directly immobilized onto the functionalized Red-
SiNPs with well-established EDC/NHS coupling chemistry. The
immobilization protocol was as follows: 20 mg Red-SiNPs- 2.7 The procedure of H7N9 AIV optical detection by MB–
COOH were resuspended in 5 mL MES (0.1 M, pH 6.7). The MEMSCI
Red-SiNPs-COOH suspension was mixed with 5.0 mL of pAb
In order to get a more clear visual effect and a low detection
limit, optical detection and UV detection of H7N9 AIV are
ꢁ
1
ꢁ1
(1.0 mg mL ) and 50 mL of GOx (1.0 mg mL ) solution, fol-
lowed by the addition of 1 mL of 10 mM EDC and 10 mM NHS
carried out separately. The optical detection scheme is given in
solution. Aer 2 h incubation at room temperature, the free pAb
ꢁ1
Fig. 2. For a typical sandwich assay, 20 mL MB–mAb (1 mg mL
)
ꢂ
and GOx were removed by centrifugation at 8000 rpm at 4 C for
was mixed with 1 mL H7N9 AIV solution in a centrifuge tube.
5
minutes and washed with PBS. The modied Red-SiNPs were
ꢂ
Aer incubation for 30 min at 37 C with gentle shaking, the
resuspended in 3 mL of PBS containing 1% BSA to block non-
specic adsorption sites on the nanoparticles. The prepared
resultant GOx- and antibody-conjugated Red-SiNPs (GOx–Red-
mAb–MB–H7N9 AIV complex was separated magnetically and
the clear supernatant was discarded. The immune complex was
then washed with PBS three times to remove any unbound
species. The mAb–MB–H7N9 AIV complex was then dispersed
in 20 mL PBS (pH 7.3), transferred to a well of a 96-well micro-
ꢂ
SiNPs-pAb) were dispersed in 1 mL of PBS and stored at 4 C
for subsequent use.
ꢁ
1
titer plate, and 20 mL GOx–Red-SiNPs-pAb (10 mg mL ) was
added to the well and the mixture was subjected to react for
2.5 Monitoring of GOx activity using the hemin/4-AAP/
15 min at room temperature. GOx–Red-SiNPs-pAb formed
phenol system
a sandwich structure with H7N9 AIV and MB–mAb via immune
reaction. The immune sandwich complex was separated
magnetically and the supernatant was discarded. The immune
complex was then washed with PBS to effectively remove
unbound GOx–Red-SiNPs-pAb. Then 20 mL of glucose (4 mM) in
pH 7.0 PBS was added into the centrifuge tube and incubated
Fig. 1a displays the assay mechanism of the hemin/4-AAP/
phenol system. Initially, 10 mL of GOx with specied concen-
ꢁ
1
tration (from 0 to 1000 mg mL ) was added in 50 mL of PBS
(
0.5 mM, pH 7.0) containing 4 mM glucose. The resulting
ꢂ
solution was incubated for 30 min at 37 C. Then 70 mL of 0.1%
4
ꢁ
1
-AAP, 70 mL of 0.123 M phenol, 35 mL of 0.05 mg mL heme
ꢂ
for 30 min at 37 C on the shaker for enzymatic reaction.
chloride solution and 350 mL buffer solution (K HPO : NaOH,
2
4
ꢁ
1
Simultaneously, 28 mL of 1.0 mg mL 4-AAP, 70 mL of 0.12 M
pH ¼ 10.6) was added into the above-prepared mixture and
reacted for 10 min at room temperature. Aer interaction, the
color of the solution was observed and at the same time the
absorbance was recorded at l ¼ 505 nm with a Lambda 35 UV-
visible spectrophotometer.
ꢁ
1
phenol, 35 mL of 0.05 mg mL heme chloride solution and
40 mL buffer solution (K HPO
1
2
4
: NaOH, pH ¼ 10.6) were added
to the centrifuge tube in turn. The nal color of the suspension
solution in the well was recorded with a digital camera.
3
. Results and discussion
2.6 Immunoassay for target H7N9 AIV using MB–MEMSCI
3.1 Characterizations of the multifunctional Red-SiNPs
Fig. 1b presents the immunoassay process toward target H7N9
AIV by coupling with the hemin/4-AAP/phenol system. The The inverse microemulsion method was chosen to prepare Red-
detailed procedure is summarized as follows: (i) 50 mL of H7N9 SiNPs in this study. This method is a simple and diverse prep-
AIV standards or samples with different concentrations and aration method for synthesis of silica nanoparticles in the
ꢁ1
2
5 mL of mAb–MB suspension (6 mg mL ) were initially added laboratory, and its use makes it easy to control the morphology
into a 0.5 mL centrifuge tube, and the mixture was then incu- of silica nanoparticles. The physical images of Red-SiNPs
ꢂ
bated for 30 min at 37 C on a shaker to form the antigen– showed that they were of bright color and formed a good
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Fig. 1 Schematic illustration of (a) H
2
O
2
-stimulated colorimetric assay for monitoring of GOx activity and (b) principle of the colorimetric MB–
MEMSCI.
dispersion in aqueous solution (Fig. 3A and B). The size and measurements. Fig. 3D displays the zeta potential of Red-SiNPs,
morphology of Red-SiNPs were characterized by SEM (Fig. 3C). Red-SiNPs-NH and Red-SiNPs-COOH as a function of pH. The
2
The nanoparticles had uniform particle size and all Red-SiNPs isoelectric point (IEP) of Red-SiNPs was at pH 5.0 (curve a).
showed a spherical shape and smooth surface. The average When Red-SiNPs were in the environment of a neutral solution,
diameter of the nanoparticles determined by SEM was approx- the surface potential of Red-SiNPs was about ꢁ8 mV (curve a).
imately 40 ꢃ 5 nm and the size distribution was also quite The particle surface had a negative charge, because of the
2
uniform. The presence of chemical group on the outermost presence of hydroxyl groups; the IEP of Red-SiNPs-NH was
layer of Red-SiNPs was conrmed by zeta potential determina- shied to pH 8.2 (curve b). The increase of zeta potential was
tion. Zeta potential measurement was carried out using a Zeta- attributed to the increasing number of protonated amine group
sizer. For the determination of zeta potential, the pH of the on the surface of Red-SiNPs-NH . Compared to Red-SiNPs-NH ,
2
2
sample was adjusted by the addition of 0.01 M HCl or 0.01 M the IEP of Red-SiNPs-COOH was shied to pH 3.4 (curve c),
NaOH. All values shown in this work were the average of three which was ascribed to negative charge of the carboxyl group,
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Fig. 2 Schematic view of optical MB–MEMSCI for rapid detection of H7N9.
Fig. 3 Digital photos of Red-SiNPs (A) and their aqueous solution (B). SEM image of Red-SiNPs (C). Zeta potential (D) of Red-SiNPs (a), Red-
SiNPs-NH (b), Red-SiNPs-COOH (c). FTIR spectra (E) of Red-SiNPs (c), Red-SiNPs-NH (b), and Red-SiNPs-COOH (a).
3
3
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which made the modication and the bioconjugation of the 3.3 Control tests for the MB–MEMSCI
nanoparticles easier. Chemical composition on the coating
For the successful development of MB–MEMSCI, one of the very
important preconditions was whether the hemin/4-AAP/phenol
system could be smoothly progressed in the presence of GOx, as
depicted in Fig. 1a. To demonstrate this concern, several relative
control tests were put into effect under the different conditions by
using UV-visible absorption spectroscopy (Fig. S1 (ESI†)) and
colorimetric measurement (see Fig. S1 (ESI†)) (note: 10 mL of
layer of Red-SiNPs was also examined by FTIR (as shown in
Fig. 3E). Dried sample was measured using the KBr pellet
ꢁ
1
method in the range of 400–4000 cm . Strong IR absorption
ꢁ
1
bands in the region 800–1200 cm , corresponding to the Si–
O–Si of the silica core, were found in spectra of both Red-SiNPs
(
curve a) and Red-SiNPs-NH
3090 cm in the Red-SiNPs-NH spectrum was assigned to
2
(curve b). A new band at
ꢁ1
ꢀ
2
ꢁ1
ꢁ1
1
4
mg mL GOx, 50 mL of 1 mM glucose, 70 mL of 1.0 mg mL
-AAP, 70 mL of 0.12 M phenol, 35 mL of 0.05 mg mL heme
the N–H of the silica surface. Compared to Red-SiNPs, the
ꢁ1
FTIR spectrum of Red-SiNPs-NH had a signicant difference
2
2 4
chloride solution and 350 mL buffer solution (K HPO : NaOH,
ꢁ1
in the region 3000–3500 cm . These results were consistent
with the results of zeta potential. APTMS was thus believed to
be successfully introduced onto the surface of the Red-SiNPs.
The chemical composition of Red-SiNPs-COOH was also
examined using its FTIR spectrum. The stretching band of
C]O (which is the characteristic band of carboxyl group)
pH ¼ 10.6) were used in this case). As shown from curve a in Fig
S1,† a strong absorption peak at 505 nm was observed aer the
added GOx reacted with the mixture containing glucose, hemin,
4-AAP and phenol. The reason might be most likely as a conse-
quence of the fact that the H O generated through the catalytic
2
2
reaction of enzymatic product oxidized 4-AAP to form a dye
compound. Moreover, the newly formed dye compound could
cause the change in the color of the mixed solution from colorless
to pink (photograph a in Fig. S1 (ESI†)). Further, we also found
that the absorption increased with increasing GOx concentration
under the same conditions (see Fig. 4D). To further investigate
the feasibility of the immunoassay system, we also used
UV-visible absorption spectroscopy and the visible color of
different components in the absence of GOx, glucose, hemin, or
ꢁ1
appears at 1705 cm . Both the symmetric and asymmetric
ꢁ
1
bands of COO arise in this spectrum at 1616 cm
1
and
ꢁ1
510 cm , respectively. The spectra indicate the presence of
C]O (the characteristic band of COOH) which arises from the
modication of COOH by glutaric anhydride.
3.2 Principle of the MB–MEMSCI
4-AAP. For comparison, we initially monitored the mixture con-
In this work, mAb is immobilized on the MB by using glutar-
aldehyde as cross-linkage reagent (mAb–MB), which is used as
the immunosensing probe for the capture of target H7N9 AIV.
MB is not only used as a substrate for the conjugation of mAb
antibody but also enables the rapid separation and purica-
tion of bionanocomposites aer synthesis. Red-SiNPs-COOH
heavily functionalized with GOx and pAb antibody (GOx–
Red-SiNPs-pAb) is formed possibly owing to the covalent
taining GOx, glucose, and hemin (i.e., in the absence of 4-AAP). As
seen from curve c in Fig. S1 (ESI†), no absorption peak
appeared at 505 nm. Meanwhile, the mixture was colorless
(
photograph c in the inset of Fig. S1 (ESI†)). Favorably, when
glucose (curve b in Fig. S1 (ESI†) and photograph b in the inset
of Fig. S1 (ESI†)), hemin (curve d in Fig. S1 (ESI†) and photo-
graph d in the inset of Fig. S1 (ESI†)), or GOx (curve e in Fig. S1
(
ESI†) and photograph e in the inset of Fig. S1 (ESI†)) was
2
binding between –COOH and free –NH of the proteins to form
absent in the detection system, the absorption peak and the
color were almost the same as that of curve c and photograph
c, respectively. The results revealed that (i) hemin could not
cause the appearance of the absorption peak at 505 nm aer
C]N. The as-prepared GOx–Red-SiNPs-pAb is employed as the
signal-transduction tag (detection antibody) for the construc-
tion of the MB–MEMSCI. In the presence of target H7N9 AIV,
the sandwiched immune-conjugates can be formed between
MB–mAb and GOx–Red-SiNPs-pAb. Accompanying the GOx–
Red-SiNPs-pAb, the carried GOx can trigger the enzymatic
catalytic reaction to produce the colored product. Initially, the
GOx-biocatalyzed oxidization toward the added glucose leads
to the formation of gluconic acid and hydrogen peroxide
incubation with the 4-AAP/phenol system without H
2 2
O , (ii)
H O could be provided by GOx toward the catalytic reaction of
2
2
glucose, and (iii) the cascade reaction could be successfully
carried out only in the simultaneous presence of glucose/GOx/
hemin/4-AAP/phenol system. Hence, we might suspect that
when GOx was conjugated onto the detection antibody, the
mimic enzyme-chromogenic strategy could be employed for
the development of the sandwiched immunoassay by moni-
toring the change in the absorbance or color.
2 2
(H O ) with the participation of oxygen. The generated
hydrogen peroxide is catalyzed by hemin in the 4-AAP and
phenol solution to produce the colored product which makes
the solution change from colorless to red. The change in the
color/absorbance indirectly depends on the concentration of
target H7N9 AIV in the sample. By monitoring the shi in the
absorbance, we can quantitatively determine the concentra-
3.4 Evaluation and characteristics of the MB–MEMSCI
system
tion of target H7N9 AIV in the sample. We can also qualita- In the MB–MEMSCI system, the cascade reaction mainly con-
tively judge the H7N9 AIV level by evaluating the change in the sisted of two steps: (i) the catalytic reaction of GOx toward
visible color. In contrast, GOx–Red-SiNPs-pAb cannot be glucose and (ii) the redox reaction between hydrogen peroxide
conjugated onto the functionalized mAb–MB in the absence of and hemin in the 4-AAP/phenol system. Signicantly, the H O2
2
target H7N9 AIV; therefore, it cannot trigger the progression of in the 4-AAP/phenol system toward hemin was specic and
MB–MEMSCI.
selectable. As shown from Fig. 4A, a signicant change in the
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2
+
Fig. 4 Comparison of the absorbance intensity of the hemin/4-AAP/phenol system after interaction with different interfering components (Zn
,
2ꢁ
2+
2+
2+
ꢁ
3+
ꢁ
3ꢁ
+
2ꢁ
+
CO
3
, Mg , Cu , Ca , NO
3
, Fe , Cl , PO
4
, K , SO
4
, Na , cysteine and lysine) (A). Calibration plots of the hemin/4-AAP/phenol system
toward H O
2 2
standards with various final concentrations (inset: corresponding linear plots) (B). Effect of different catalytic times between GOx
and glucose on the absorbance of the hemin/4-AAP/phenol system (C), and catalytic reactivity of GOx with different concentrations in the
hemin/4-AAP/phenol system (D).
absorbance was observed at 505 nm with H O against 20-fold monitored the effect of different incubation times between GOx
2
2
2
+
2ꢁ
2+
2+
ꢁ1
higher interfering components (e.g., Zn , CO , Mg , Cu , and glucose on the absorbance (note: 10 mL of 1 mg mL GOx,
, Na , cysteine and 50 mL of 1 mM glucose, 70 mL of 1.0 mg mL 4-AAP, 70 mL of
lysine). Although Cu ion could cause an increase in the 0.12 M phenol, 35 mL of 0.05 mg mL heme chloride solution
3
+
2
+
ꢁ
3+
ꢁ
3ꢁ
+
2ꢁ
ꢁ1
Ca , NO
3
, Fe , Cl , PO
4
, K , SO
4
2
+
ꢁ1
absorbance, the density was less. Hence, the hemin/4-AAP/ and 350 mL buffer solution (K
2
HPO
4
: NaOH, pH ¼ 10.6) were
phenol system could be used for the detection of H . To used in this case). As indicated in Fig. 4C, the absorbance
2 2
O
realize the subsequent MB–MEMSCI, the system must have the increased within the initial 30 min and then tended to slightly
ability to exactly differentiate H O with various concentrations. decrease. The reason might be attributed to the fact that H O2
2
2
2
Next, we investigated the analytical performance of the hemin/ can be decomposed by light irradiation. Therefore, 30 min was
-AAP/phenol system toward H O with various concentrations selected for the enzymatic reaction in this work. The enzymatic
4
2
2
under the same conditions. As shown in Fig. 4B, the absorbance reactivity of GOx in the cascade reaction system was also
increased with increasing H concentration. Moreover, studied. Various amounts of GOx were added to the system, and
a linear tendency could be tted in the working range of 7 mM to the absorption spectra were recorded. In this case, 50 mL of
2 2
O
ꢁ
1
5
00 mM, as shown from the inset in Fig. 4B, and the detection 1 mM glucose, 70 mL of 1.0 mg mL 4-AAP, 70 mL of 0.12 M
ꢁ
1
limit could be lowered to 2.5 mM. The results revealed that the phenol, 35 mL of 0.05 mg mL heme chloride solution and
system was feasible for quantitative monitoring of H O in the 350 mL buffer solution (K HPO : NaOH, pH ¼ 10.6) were used
2
2
2
4
sample. For bioactive GOx enzyme, however, the catalytic effi- for detection of 10 mL of GOx with various concentrations. As
ciency toward glucose relied on the catalytic time and temper- seen from Fig. 4D, the absorbance increased with an increase of
ꢂ
ature to some extent. Usually, normal body temperature (37 C) GOx concentration in the sample. At a low concentration of
is suitable for enzymatic reaction. At this condition, we GOx, the absorbance exhibited a strong shi, and the detection
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ꢁ1
limit was ꢀ1 mg mL GOx. On the basis of these results, we 3.6 Reproducibility, selectivity and stability of colorimetric
might draw a conclusion that the designed cascade reaction immunoassay using MB–MEMSCI
strategy based on the hemin/4-AAP/phenol system could be
The reproducibility and precision of the colorimetric immu-
utilized for the determination of GOx by coupling with the
change in the color or absorbance, and simultaneously, the
results revealed that the system was feasible for quantitative
monitoring of H7N9 AIV in the sample, because its concentra-
tion changes can indirectly lead to concentration changes of
GOx.
noassay using MB–MEMSCI were evaluated by calculating the
intra- and inter-batch variation coefficients (CVs). Results
revealed that the CVs of the intra-assay with this method were
between 4.0% and 7.5% (n ¼ 5) in all cases. The batch-to-batch
reproducibility of MB–MEMSCI was also monitored by assaying
ꢁ1
3
5 ng mL H7N9 AIV (as an example) for six times on different
days, and the obtained CV was about 7.8%. The low CVs might
be attributed to the specic antigen–antibody reaction and the
strong interaction in the hemin/4-AAP/phenol system. Thus, the
precision and reproducibility of the colorimetric immunoassay
were acceptable.
Further, the specicity of the colorimetric immunoassay was
also investigated for other viruses, such as H5N1 AIV, PRV, NDV,
H2N9 AIV and EV71. The selectivity of the GOD/NECF biosensor
3
.5 Analytical performance of colorimetric immunoassay
using MB–MEMSCI
To further investigate the capability of the developed MB–
MEMSCI for colorimetric immunoassay, the as-prepared GOx–
Red-SiNPs-pAb was employed as secondary antibody for the
detection of target H7N9 AIV with a sandwich-type immuno-
assay format on MB–mAb by using the hemin/4-AAP/phenol
system. Under the optimal conditions, H7N9 AIV standards
with different concentrations were monitored based on the
designed immunoassay protocol. As shown in Fig. 5A and B, the
increase of absorbance is proportional to the H7N9 AIV antigen
was also studied. Fig. 5C(a) presents the absorbance of the
ꢁ1
biosensor with 40 ng mL
H7N9 AIV, including a 5-fold
concentration of interfering substances, such as H5N1 AIV,
PRV, NDV, H2N9 AIV and EV71. No obvious interference was
observed, and the results clearly indicated the high specicity of
the colorimetric immunoassay using the MB–MEMSCI. The
stability of the colorimetric immunosensor was also tested, as
shown in Fig. S2 (ESI†). When the immunosensor was not in
ꢁ
1
concentration in the range of 0.01–50.0 ng mL , and the
ꢁ
1
detection limit was 3.5 pg mL (S/N ¼ 3) which is obviously
superior to that of the colorimetric immunoassay without color
enhanced multifunctional silica nanoparticles (Fig. S5A and B)
ꢂ
use, it was stored in PBS (pH 7.4) at 4 C. The absorbance
(ESI†). Moreover, Table S1 (ESI†) shows the linear range and
response of the MB–MEMSCI only had a change of 5.0% aer 30
days. This indicates the effective retention of the activity of the
immobilized H7N9 AIV antibody, and further conrmed the
reaction conditions of mimic enzyme are not harsh.
15–22
detection limit of previously reported immunosensors
and
Table S2 (ESI†) shows the colorimetric immunoassay without
color enhanced multifunctional silica nanoparticles. Compared
to other methods, the presented immunosensor has a relatively
large linear range and low detection limit. We suspect that
improvement of the sensitivity might be attributed to the highly
efficient mimic enzyme-chromogenic substrate system as
3.7 The development of H7N9 AIV optical detection by MB–
MEMSCI
a result of the strong reaction in the hemin/4-AAP/phenol In order to get a more clear visual effect and low detection limit,
system and the color enhancement of the multifunctional optical detection and UV detection of H7N9 AIV are carried out
SiNPs.
separately. In this method, we used the naked eye to detect
Fig. 5 (A) Absorbance of the colorimetric MB–MEMSCI by coupling with the 4-AAP/phenol strategy toward different concentration H7N9 AIV
standards. (B) Calibration plots of the colorimetric MB–MEMSCI by coupling with the 4-AAP/phenol strategy toward different concentration
ꢁ
1
ꢁ1
H7N9 AIV standards. (C) Specificity of the colorimetric MB–MEMSCI and against H7N9 AIV (40 ng mL ), H5N1 AIV (200 ng mL ), PRV
ꢁ ꢁ1 ꢁ1 ꢁ1
1
(
200 ng mL ), NDV (200 ng mL ), H9N2 AIV (200 ng mL ) and EV71 (200 ng mL ) (a); and specificity of the optical MB–MEMSCI, from left to
ꢁ1
ꢁ1
ꢁ1
ꢁ1
ꢁ1
ꢁ1
right: PBS (0.01 mol mL ), H7N9 (5 ng mL ), H5N1 AIV (25 ng mL ), PRV (25 ng mL ), NDV (25 ng mL ), H9N2 AIV (25 ng mL ) and EV71
ꢁ
1
(
25 ng mL ) (b).
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H7N9 AIV by MB–MEMSCI. mAb–MBs were added to H7N9 AIV made the optical sandwich assay more sensitive. Taking into
solution; the H7N9 AIV was enriched and isolated in a magnetic account the impact of blood collection on patients in clinical

eld and transferred to wells of a 96-well microtiter plate. GOx– applications and cost, we chose 1 mL as the sample volume for
Red-SiNPs-pAb, acting as an indicator of the presence of H7N9 this test.
AIV, was added and reacted for 15 min. When the sample
solution contained enough H7N9 AIV, mAb–MBs and GOx–Red- 3.8 Specicity of H7N9 AIV optical detection by MB–
SiNPs-pAb were effectively combined on the surface of H7N9 MEMSCI
AIV, forming red sandwich complexes through double antibody
sandwich binding reaction, and producing red compounds in
the glucose/hemin/4-AAP/phenol system to amplify the detec-
tion signal. The red suspension was clearly observed in the well
with the naked eye and GOx–Red-SiNPs-pAb played a role of
color indicator.
To demonstrate the potential of our optical assay in biomarker
discrimination, we examined the specicity of this optical MB–
MEMSCI. The procedure for the specicity test was the same as
mentioned in Section 2.7. H7N9 AIV, H5N1 AIV, PRV, NDV,
H9N2 AIV and EV71 were all detected by this optical sandwich
assay method. The concentration ratio of H7N9 AIV and inter-
Aiming to prove the superiority of Red-SiNPs in visual
ferents was 1 : 5, and PBS was used as negative control. As
detection, pAb-AuNPs and pAb-Red-SiNPs were both used to
shown in Fig. 5C(b), a red signal was only observed for H7N9
detect H7N9 AIV, and the preparation of pAb-AuNPs is shown in
AIV but not for H5N1 AIV, PRV, and NDV, conrming this
optical assay could distinguish H7N9 AIV from other
biomarkers. This result showed that this method had good
specicity for H7N9 AIV.
ESI (Fig. S3 (ESI†)).
The procedure was the same as mentioned in Section 2.7.
H7N9 AIV was detected by our sandwich assay method using
pAb-AuNPs and pAb-Red-SiNPs separately, and PBS was used as
negative control. As shown in Fig. S4 (ESI†), when pAb-AuNPs
3.9 Stability of H7N9 AIV optical detection by MB–MEMSCI
was used as signal label, the positive and negative results
were difficult to distinguish with the naked eye. Red plaque was
observed when pAb-Red-SiNPs were used as signal label to
detect H7N9 AIV. This result showed that Red-SiNPs were more
suitable to be used as signal label in optical immunoassay.
We examined the sensitivity of this optical sandwich assay
method and the detection limit for H7N9 AIV. The positive and
negative results were distinguished by observing the bottom of
the well with the naked eye. Red suspension in the well repre-
The stabilities of mAb–MBs and GOx–Red-SiNPs-pAb were
ꢁ
1
studied by the detection of H7N9 AIV solution (8 ng mL ) with
ꢂ
the Red-SiNPs based optical MB–MEMSCI aer storing at 4 C
for 1, 10, 20 and 30 days. These two kinds of immune nano-
particles were found to retain similar reaction activity aer
ꢂ
storage at 4 C for at least 30 days (as shown in the inset of
Fig. S2 (ESI†)).
sents positive result, and brown plaque in the bottom of the well 4. Detection of H7N9 AIV in real
with colorless transparent liquid represents negative result. The
samples
original H7N9 AIV solution was diluted to ten concentrations
ꢁ1
ꢁ1
along a gradient from 10 ng mL to 0.1 ng mL using 0.85% It is reported that avian inuenza virus (AIV) could replicate in
23,24
stroke-physiological saline solutions. In the optical sandwich the organs of infected humans or animals,
so detection
assay, pAb–MBs and pAb-Red-SiNPs were sequentially mixed with methods with good anti-interference ability and high specicity
different concentrations of H7N9 AIV solution. The lowest are needed to detect AIV in complex samples such as tissues and
concentration of H7N9 AIV which can produce a positive result is feces. Taking advantages of the very good separation and
dened as the detection limit of this method. Fig. S6A (ESI†) enrichment ability of MBs, we investigated the capability of this
shows that a red turbid solution was observed when the concen- method for complex samples. An amount of H7N9 AIV was
ꢁ1
tration of H7N9 AIV was as low as 0.5 ng mL . Brown color of added to chicken serum and ground chicken liver to prepare
ꢁ
1
pAb–MBs was observed in the well containing 0.4 ng mL H7N9 H7N9 AIV positive samples. The insoluble solids were removed
ꢁ1
AIV solution. The low detection limit (0.5 ng mL ) is due to the by centrifuging. And the negative samples were prepared in the
enrichment effect of pAb–MBs and the function of vision signal same way without adding H7N9 AIV. As shown in Fig. 6, the
enhancement from the Red-SiNPs in the red reaction product H7N9 AIV ultraviolet absorption (Fig. 6A) and optical detection
solution. The sensitivity of the optical MB–MEMSCI is related to (Fig. 6B) also had high signal-to-noise ratio (p < 0.05) and
the volume of the sample and we can improve the detection recognition even in these media, which suggested the feasibility
sensitivity by increasing the sample volume. In order to prove this, of the immunosensor in complex matrices of real samples
we conducted a control experiment, while the other conditions directly with strong anti-interference ability. And the detection
were unchanged; 400 mL H7N9 AIV solutions of different ability in real samples has been veried by the avian inuenza
concentrations were detected with this sandwich assay. As virus (H7N9) Real Time RT-PCR Kit (Table S3 (ESI†)). By
shown in Fig. S6B (ESI†), when the concentration of H7N9 AIV comparing with the current standard method, we conrmed the
ꢁ1
was equal to or greater than 2 ng mL , the red color of Red- accuracy (Table S3 (ESI†)) and practicability (Table S4 (ESI†)) of
SiNPs and the red enzyme-catalyzed reaction solution started this MB–MEMSCI.
to be observed. In this experiment, the detection limit for
H7N9 AIV is 2 ng mL . By increasing the volume of H7N9 AIV robes in sandwich assay method, Red-SiNPs are cheaper and
solution, more H7N9 AIV was enriched by IgG-MBs, and this more stable than gold nanoparticles. Moreover, for bright color
Compared with gold nanoparticles, the most used nanop-
ꢁ1
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Fig. 6 Comparison of the absorbance of the colorimetric MB–MEMSCI in the presence (positive) and absence (negative) of H7N9 AIV in different
media (A), and comparison of the color of the optical MB–MEMSCI in the presence (positive) and absence (negative) of H7N9 AIV in different
media (B). Note: significant differences were considered significant when test P values were less than 0.05 (P < 10.05).
of Red-SiNPs, our sandwich assay method can be read out easily
by the naked eye.
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