Highly Uniform Gold Nanobipyramids for Ultrasensitive Colorimetric Detection of Influenza Virus

Article abstract of DOI:10.1021/acs.analchem.6b03711

Gold nanoparticles (AuNPs) have been frequently utilized for the construction of diverse colorimetric biosensors. Normally, AuNPs with sharp edges could have better sensitivity. However, the poor monodipersity of AuNPs with sharp edges seriously confines their utility for colorimetric biosensing. Herein, we demonstrate the utility of highly uniform gold nanobipyramids (Au NBPs) for ultrasensitive colorimetric detection of H₅N₁ virus. The proposed method is based on the fact that alkaline phosphatase (ALP) could catalyze the decomposition of 4-aminophenyl phosphate (4-APP) to generate 4-aminophenol (4-AP), which would then reduce silver nitrate to metal silver and then deposited on Au NBPs. The metal silver shell coated on the Au NBPs changed the refractive index of gold and thus resulted in a blue shift of longitudinal localized surface plasmon resonance (LSPR) and accompanied a vivid color change. This method exhibited a higher sensitivity than that of other Au NPs such as gold nanorods due to the high-index-faceted on the tips of the Au NBPs. This method was used to detect the activity of ALP. It exhibited a linear range of 0.1-5 mU/mL with a limit of detection (LOD) of 0.086 mU/mL. Finally, the proposed method was used in immunoassay to detect H₅N₁ virus. The results showed that the corresponding linear range for the detection of H₅N₁ virus antigen was 0.001-2.5 ng/mL, and the LOD was determined to be 1 pg/mL, which is more sensitive than those in most of the colorimetric biosensors reported previously.

Full text of DOI:10.1021/acs.analchem.6b03711

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Article
Highly uniform gold nanobipyramids for
ultrasensitive colorimetric detection of influenza virus
Shaohua Xu, Wenjun Ouyang, Peisi Xie, Yi Lin, Bin Qiu, Zhenyu Lin, Guonan Chen, and Longhua Guo
Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b03711 • Publication Date (Web): 26 Dec 2016
Downloaded from http://pubs.acs.org on December 26, 2016
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Highly uniform gold nanobipyramids for ultrasensitive colorimetric
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Shaohua Xu, Wenjun Ouyang, Peisi Xie, Yi Lin, Bin Qiu, Zhenyu Lin*, Guonan Chen and Longhua
Guo*
Institute of Nanomedicine and Nanobiosensing, Ministry of Education Key Laboratory of Analysis and Detection for Food
Safety, Fujian Provincial Key Laboratory of Analysis and Detection Technology for Food Safety, College of Chemistry, Fuꢀ
zhou University, Fuzhou, 350116, China.
*Eꢀmail: guolh@fzu.edu.cn; zylin@fzu.edu.cn
ABSTRACT: Gold nanoparticles (AuNPs) have been frequently utilized for the construction of diverse colorimetric biosensors.
Normally, AuNPs with sharp edges could have better sensitivity. However, the poor monodipersity of AuNPs with sharp edges
seriously confines their utility for colorimetric biosensing. Herein, we demonstrate the utility of highly uniform gold nanobipyraꢀ
mids (Au NBPs) for ultraꢀsensitive colorimetric detection of H N virus. The proposed method is based on the fact that alkaline
5
1
phosphatase (ALP) could catalyze the decomposition of 4ꢀAminophenyl phosphate (4ꢀAPP) to generate 4ꢀaminophenol (4ꢀAP),
which would then reduce silver nitrate to metal silver, and then deposited on Au NBPs. The metal silver shell coated on the Au
NBPs changed the refractive index of gold and thus resulted in a blue shift of longitudinal localized surface plasmon resonance
(LSPR) and accompanied a vivid color change. This method exhibited a higher sensitivity than that of other Au NPs such as gold
nanorods due to the highꢀindexꢀfaceted on the tips of the Au NBPs. This method was used to detect the activity of ALP. It exhibited
a linear range of 0.1 ~ 5 mU/mL with a limit of detection (LOD) of 0.086 mU/mL. Finally, the proposed method was used in imꢀ
munoassay to detect H N virus. The results showed that the corresponding linear range for the detection of H N virus antigen was
5
1
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1
0
.001 ~ 2.5 ng/mL, and the LOD was determined to be 1 pg/mL, which is more sensitive than those in most of the colorimetric
biosensors reported previously.
Keywords: colorimetric biosensor; gold nanobipyromid; localized surface plasmon resonance; influenza virus.
1
. Introduction
Of all kinds of noble metal nanomaterials, gold nanorods (Au
NRs) show great potential for the fabrication of different kinds
of biosensors and chemical sensors due to their highly controlꢀ
Rapid identification and quantification of contagious virus
is great significant in prompt treatment and effective manageꢀ
ment for illness. Detection of pathogen with high sensitivity
and selectivity was regarded as the key for preventing probꢀ
lems from further spreading . However, current approaches
for ultrasensitive detection of biomarkers often need sophistiꢀ
cated instruments and professional operating, such as those
approaches based on fluorescence , electrochemistry , elecꢀ
trochemiluminescence , and surface enhanced raman scatterꢀ
ing (SERS) . In contrast, colorimetric assay shows obvious
superiority for its simplicity, convenience, low cost and the
lable shape and tunable localized surface plasmon resonance
14,15,16,17
(
LSPR) peak from visible to nearꢀinfrared range
. Howꢀ
1
,2
ever, disadvantages still exist. Firstly, the relatively wide size
and shape distributions of Au NRs may make the longitudinal
18,19,20
plasmon peaks inhomogeneously broadened
; secondly,
3
4,5
the round end shape of Au NRs offers a relatively small local
electric field enhancement which limits the application of
6
20
7
plasmon enhanced spectroscopy . As an alternative, Au nanoꢀ
bipyramids (Au NBPs) have received much attention because
of their intriguing optical properties. For example, both the
extinction crossꢀsections and local electricꢀfield enhancements
8,9,10
possibility to be readout by naked eyes
. Nevertheless, the
relatively low sensitivity of conventional colorimetric biosenꢀ
sors limits the widespread application. Hence, it is significant
to exploit a sensitive and selective colorimetric approach.
21,22
of Au NBPs are much larger than those of Au NRs . As a
result, Au NBPs exhibit a higher sensitivity to refractive index
23,24
and a larger figure of merit . Moreover, the LSPR properꢀ
ties of Au NBPs can also be turned from visible to nearꢀ
Noble metal nanomaterials have attracted great interest owꢀ
11,12,13
ing to their excellent physical and chemical properties
.
25
infrared region , enabling them a promising nanomaterial for
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different applications. For example, Zhang et al. demonstratꢀ
ed Au NBPs can be used as saturable absorbers for ultrafast
Aldrich (USA). Cetanecyltrimethyl ammonium chloride
(CTAC) was obtained from BBI Life Sciences (Shanghai,
China). Sodium orthovanadate (Na VO ) was obtained from
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pulsedꢀlaser application; Liu et al. used Au NBPs to enhance
the circular dichroism (CD) signal for DNA detection. The
results showed that the plasmonic CD exhibits good sensitivity
and reproducibility. Although these methods have shown adꢀ
vanced applications, the shortcomings are also obvious, such
as a broaden LSPR peak or insensitivity to the targets. The
main reason is that the Au NBPs used for those applications
are not uniform. It is only until recently that the synthesis of
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Macklin (Shanghai, China). ALP (25.8 U/mL) was obtained
from Worthington (USA). 4ꢀAPP (Santa Cruz Biotechnology,
Inc. USA) was used throughout the work. Mouse antiꢀH N
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hemagglutinin monoclonal antibody (Ab ), biotinylated rabbit
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antiꢀH N hemagglutinin polyclonal antibody (Ab ) and H N
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hemagglutinin standard were purchased from Sino Biological
Inc. (Beijing, China). Streptavidin alkaline phosphatase was
purchased from Promega (USA). All highꢀbinding polystyrene
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highly homogenous Au NBPs have been reported .
9
6ꢀwell singleꢀbreak strip plates were purchased from Thermo
Enzymeꢀlinked immunosorbent assay (ELISA) is known for
its high selectivity due to the specific recognition between
antibody and antibody and has been widely employed in cliniꢀ
environmental monitoring
and so on. Most of the commercial ELISAs currently
use horseradish peroxidase (HRP) as the enzyme label and
3,3',5,5'ꢀtetramethylbenzidine (TMB) as the chromogenic subꢀ
strate. The targets are usually quantified by the optical density
Fisher Scientific (USA). All other reagents were of analytical
grade without further purification, and millipore purification
systemꢀbased ultrapure water was used throughout this study
(18.2 Mꢁ·cm, MilliꢀQ, Millipore). All photographs were recꢀ
orded by a Canon EOS 600D digital camera (Japan). The huꢀ
man serum specimens were obtained from the Hospital of
Fuzhou University (Fujian, China).
29,30
31,32
cal diagnosis
safety
,
,
food
3
3,34
of a yellow compound (TMB ). Human eyes are insensitive
2.2 Synthesis and purification of Au NBPs.
oxi
to optical density changes of the same color, so it is not suitaꢀ
ble for sensitive detection of targets by the naked eyes. To
improve the visual detection sensitivity, it is key to develop an
immunoassay approach that shows multicolor changes. To
meet this challenge, many investigations have been done. For
example, Roberto de la Rica et al. employed hydrogen perꢀ
oxide as the reductant for the growth of gold nanoparticles,
and an ultraꢀlow detection limit of 10 g/mL was achieved for
the detection of a target protein; Zhang et al designed a gold
nanoparticleꢀaggregation based sensor for the detection of
H N virus. The detection limit was as low as 25 pg/mL. All
the aboveꢀmentioned approaches have exhibited good sensitivꢀ
ity. Regretfully, the accuracy for the determination of targets
with the naked eyes could still be a big challenge.
Au NBPs were synthesized according to the typical seedꢀ
25
mediated method . The freshly prepared iceꢀcold NaBH
4
(0.01 M, 0.6 mL) was added to the mixed solution containing
HAuCl (0.01 M, 0.5 mL), trisodium citrate (0.01 M, 1 mL),
4
and water (38.5 mL) under vigorous stirring. Then the orange
red seed was aged at least 5 h in dark before use. A volume of
gold seed solution (5.0 mL) was injected to the growth soluꢀ
tion, which was prepared in advance by mixing CTAB (0.1 M,
3
5
ꢀ18
36
2
00 mL), HAuCl (0.01 M, 10 mL), AgNO (0.01 M, 2 mL),
4
3
hydrochloric acid (HCl, 1 M, 4 mL), and Vc (0.1 M, 1.6 mL),
followed by gentle shaking for about 2 min. The color of the
mixed solution turned to purple red after the addition of seed.
Then the mixture was left for undisturbed at water bath overꢀ
night (30 ℃). The longitudinal plasmon wavelength of the asꢀ
prepared Au NBPs was around 750 nm.
7
9
In this work, we developed a colorimetric immunoassay
method for the ultrasensitive detection of H N virus. This
method was based on the growth of silver shell on the Au
NBPs in the presence of silver ion and 4ꢀaminophenol (4ꢀAP)；
ꢀAP was produced via the catalysis of 4ꢀaminophenyl phosꢀ
phate monosodium salts (4ꢀAPP) by alkaline phosphatase
(ALP). Owning to the sharp tips of the Au NBPs, significant
response was observed in a target concentration as low as 1.0
pg/mL. More interestingly, a multiple of nakedꢀeye distinꢀ
guishable colors were observed in response with different conꢀ
centrations of H N virus antigen. As a consequence, our proꢀ
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The purification of Au NBPs was followed by a threeꢀstep
20
method . Firstly, the resulting Au NBPs sample (200 mL) was
centrifuged at 10000 rpm for 15 min. The precipitate was reꢀ
dispersed in CTAC (0.08 M, 150 mL), followed by the addiꢀ
4
tion of AgNO (0.01M, 40 mL) and Vc (0.1 M, 20 mL); then
3
the resultant solution was kept in an oil bath (65 ℃, 4 h) to
produce Au@Ag coreꢀshell nanorods. The asꢀprepared
Au@Ag coreꢀshell nanorods was centrifuged at 6000 rpm for
1
1
5 min, and the precipitate was redispersed by CTAB (0.05 M,
50 mL). The solution was kept undisturbing for at least 4 h.
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posed approach may be used for the semiꢀquantitative deterꢀ
mination of the H N virus in biological samples with the naꢀ
The rodꢀlike Au@Ag nanomaterial precipitated, while other
shaped nanoparticles stayed in the supernatant. Then the suꢀ
pernatant was discarded, and the precipitate was redispersed in
water (100 mL). The redispersed Au@Ag coreꢀshell nanorods
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ked eye.
2
. Experimental section
solution was gently mixed with NH •H O (25%, 10 mL) and
3
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2
.1 Materials and regents
Chloroauric acid tetrahydrate (HAuCl •4H O), cetyltrimeꢀ
thylammonium bromide (CTAB), ammonium hydroxide
NH •H O, 25%)，sodium citrate, silver nitrate (AgNO ), and
hydrogen peroxide (H O , 30%) were purchased from Siꢀ
nopharm Chemical Reagent Co. Ltd. (Shanghai, China).
Ascorbic acid (Vc) and diethanolamine (DEA) were obtained
from Aladdin (Shanghai, China). Bovine serum albumin
BSA) and Tween 20 were purchased from Glenview (USA).
Sodium borohydride (NaBH ) was purchased from Sigmaꢀ
H O (5%, 1 mL) and kept undisturbing for 4 h. The Ag segꢀ
2 2
ments wrapped on the Au NBPs were etched and AgCl precipꢀ
itate was formed during the process. Finally, the supernatant
was taken out carefully and centrifuged at 8000 rpm for 15
min. After that, the product was redispersed with a CTAB
solution (0.05 M, 100 mL) for further use.
4
2
(
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2.3 Colorimetric determination of ALP
ALP was carefully diluted by DEA buffer (pH 7.6) to difꢀ
ferent concentration (0.1 ~ 50 mU/mL). Au NBPs (50 ꢂL, 1.0
a.u. at 750 nm), 4ꢀAPP (50 ꢂL, 2.0 mM in 0.4 M pH 9.8 DEA
(
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buffer), and AgNO (50 ꢂL, 10 mM) were added to a 96ꢀwell
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ence of ALP, and the produced 4ꢀAP could reduce silver niꢀ
trate to silver monomer. The silver monomer was then deposꢀ
ited on the surface of Au NBPs, which results in a LSPR peak
blueꢀshifted and meanwhile the color of the solution changed
from brownish red to green and even red in response with
different thickness of silver deposition. No immunocomplex
formed without target antigen. Thus, both the LSPR and soluꢀ
tion color were unaltered. Owing to the vivid color response,
the target concentration could be easily distinguished with the
naked eyes or detected with a spectrometer by the LSPR peak
shifts.
plate (as the substrate solution). ALP standards with various
concentrations in DEA buffer (10 ꢂL) were added simultaneꢀ
ously to the substrate solution and incubated for 35 min at 37
℃
. Na VO (50 ꢂL, 10 mM) was added to the resulting soluꢀ
3 4
tion to inhibit the activity of ALP. Such solution was then
monitored by a Microplate Spectrophotometer (Multiskan GO,
Thermo Scientific, USA) or distinguished with the naked eyes.
2
.4 Detection of H N virus
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Before detection, a functionalized procedure was performed
to immobilize the capture antibody on a 96ꢀwell plate. Briefly,
capture antibody (100 ꢂL, 0.5 ꢂg/mL) in carbonate buffer (pH
9
.6) was added to the 96ꢀwell plate and incubated over night at
4
℃. Then the plate was washed by 0.05 % tween 20ꢀTrisꢀHCl
buffer (TBST buffer) three times. After that, bovine serum
albumin (300 ꢂL, 3%, BSA) was injected into each well and
incubated 1 h at 37 ℃ to block the plate. During these proꢀ
cesses, the capture antibody was immobilized on the plate.
Different concentrations of H N virus standards or serum
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samples (100 ꢂL) were added to each plate and incubated at
least 60 min at 37 ℃. After washing the plate, biotinylated
detection antibody (100 ꢂL, 0.5 ꢂg/mL) was added to each
well and incubated for another 60 min at 37 ℃, followed by
the addition of avidinꢀALP (100 ꢂL) and incubation for 30
min at 37 ℃. Au NBPs (50 ꢂL, 1.0 a.u. at 750 nm), 4ꢀAPP (50
ꢂL, 2.0 mM in 0.4 M pH 9.8 DEA buffer), and AgNO (50 ꢂL,
3
Scheme 1 Schematic illustration of the principle of the
proposed immunosensor
1
0 mM) were added to the wells and incubated for 35 min at
3
7 ℃. Finally, Na VO (50 ꢂL, 10 mM) was added to each
3
4
well to stop the reaction and then the UVꢀvis spectra were
measured by a microplate spectrophotometer in a range of 300
To verify the reduction of silver nitrate by 4ꢀAP, a transmisꢀ
sion electron microscope (TEM) study was carried out. As
shown in Figure 1A, B, and C, the original Au NBPs were
standard bipyramid shape with a brownish red color (Figure
1A). After the deposition of Ag, the bipyramid nanostructures
were cylinder like shape and the solution color was green
−
1000 nm.
3
. Results and Discussion
3
.1 Characterization of Au NBPs and principle of the pro-
posed biosensor
In this work, the colorimetric signals mainly result from enꢀ
zymeꢀinduced silver deposition on the surface of the Au
NBPs. Therefore, one key factor of this assay is the synthesis
of Au NBPs with high monodispersity. However, the purity of
asꢀgrown Au NBPs usually is no more than 60% by the seedꢀ
(
Figure 1B). When almost all silver precursors were reduced,
the nanostructures were “walnutꢀlike”. The solution color was
red (Figure 1C). Simultaneously, the LSPR peak blueꢀshifted
from 750 nm to 580 nm (Figure 1D). It is worth to note that
this silver deposition process is significantly different from
that of Au NRs, which is one side deposition on the
2
8
mediated growth method , which limits the application of this
promising nanomaterial for biosensing. After threeꢀstep purifiꢀ
cation, Au NBPs with high monodispersity were obtained in
this study. As shown in Figure S1A, the asꢀgrown Au NBPs
exhibited two approximate intensity LSPR peaks at ~550 nm
and ~750 nm, respectively, indicating that many spherical
particles and impurities coexist. After the threeꢀstep purificaꢀ
tion, the intensity at ~550 nm significantly decreased and the
LSPR peak slightly blue shifted, demonstrating that the sphere
nanoparticles and impurities are removed. The transmission
electron microscope (TEM) image shown in Figure S1B furꢀ
ther verifies the high monodispersity of Au NBPs after purifiꢀ
cation.
37,38
surface . In this work, Ag is deposited uniformly around the
Au NBPs side and formed a “walnutꢀlike” nanostructure. This
is because the side surface of Au NBPs is highꢀindexꢀfaceted,
and the deposition of Ag on the side facet may lower the surꢀ
39
face energy . The surfactant used in this work is Tween 20
rather than CTAC or CTAB, which may be another reason for
the formation of “walnutꢀlike” Au NBPs@Ag. The CTAB
molecule could selectively bind to [110] facets strongly and
40
thus minimizes the overall surface energy . As a result, a difꢀ
ferent growth rate is presented between [110] and [100] facets
of gold nanorods, and then an anisotropic coating is formed.
While in this case, none CTAB was existed around the Au
NBPs, thus the silver coating process was homogeneous.
As shown in Scheme 1, when target antigen (e.g. H N ) exꢀ
5
1
isted, the capture antibody (Ab ), antigen, and detection antiꢀ
1
To further verify the production of 4ꢀAP by ALP hydrolysꢀ
ing 4ꢀAPP, a control study was performed. As shown in Figure
1E, when only 4ꢀAPP was mixed with silver nitrate and Au
NBPs, the color remained unchanged, and no LPSR peak shift
was observed. After the addition of ALP, the color turned to
body (Ab ) formed a sandwiched immunocomplex (Ab ꢀ
2
1
antigenꢀAb ) via antibodyꢀantigen interaction. Then the sandꢀ
2
wiched immunocomplex was conjugated with ALP through
biotinꢀavidin interaction. 4ꢀAPP was hydrolyzed in the presꢀ
3
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green immediately, and the LSPR peak blueꢀshifted signifiꢀ
cantly. In addition, both the solution color and the LSPR peak
were similarly when ALP and 4ꢀAPP were replaced by 4ꢀAP.
These results demonstrate that only 4ꢀAP can lead to the silver
deposition, while 4ꢀAPP cannot reduce silver nitrate to silver
element.
working temperature of 37 ℃ . Under this condition, some
experimental parameters were optimized, including DEA conꢀ
centration, 4ꢀAPP concentration, Ag precursor concentration,
and the reaction time. The amount of LSPR peak shifts (ꢃλmax
)
were employed as the criterion to evaluate the performance of
these conditions. As shown in Figure 2A, the value of ꢃλmax
increased gradually in case that the concentrations of DEA
were below 0.35 M, and reached a platform when the concenꢀ
trations of DEA were over 0.35 M. Therefore, 0.35 M DEA
was chosen as the optimum condition. Similarly, the effect of
ꢀAPP and Ag concentrations were also studied. As shown in
Figure 2B and C, ꢃλmax increased gradually when the concenꢀ
tration of 4ꢀAPP and Ag were below 2 mM and 10 mM, reꢀ
spectively. However, when the 4ꢀAPP concentration was largꢀ
er than 2 mM, ꢃλmax would not increase any more. On the
contrary, ꢃλmax decreased with the further increase of 4ꢀAPP
concentration. We deduced that the heterogeneous nucleation
in case of a relatively high concentration of 4ꢀAPP could be
the main reason to cause the decrease of ꢃλmax. It can be seen
from Figure 1C that heterogeneous nucleation was obvious
when a high concentration of substrate was presented. It is
easy to understand that when a high concentration of 4ꢀAPP
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4
+
Figure 1 (A) UVꢀvis absorption spectra of (1) 50 ꢂL 1 mM 4ꢀAP
+
was presented, more Ag would be reduced to Ag simultaneꢀ
ously, while a high concentration of Ag tended to induce hetꢀ
erogeneous nucleation.
+
+
50 ꢂL 10 mM Ag + 50 ꢂL Au NBPs, (2) 10 ꢂL10 mU/mL ALP
+
+ 50 ꢂL 2 mM 4ꢀAPP + 50 ꢂL 10 mM Ag + 50 ꢂL Au NBPs, (3)
50 ꢂL 10 mM 4ꢀAPP + 50 ꢂL 10 mM Ag + 50 ꢂL Au NBPs, (4)
+
5
0 ꢂL Au NBPs. (B) TEM and photographs of the Au NBPs in
the absence (B) and in the presence of different thickness of silver
deposition (C, D) and their corresponding UVꢀvis absorption
spectra (E). (F) UVꢀvis absorption spectra of silver nitrate reduced
by different concentration of 4ꢀAP and deposited on Au NBPs.
The UVꢀvis extinction spectra of the nanostructures deposꢀ
ited with different thickness silver are shown in Figure 1 F.
The longitudinal and transverse LSPR peaks of the starting Au
NBPs were located at ~750 nm and ~520 nm, respectively.
After the deposition of Ag, both the longitudinal and the
transverse LSPR peaks blue shifted, owing to the surrounding
refractive index change of Au NBPs. And two new peaks at
around 350 nm and 400 nm emerged; this may be attributed to
the transverse multipolar plasmon mode of the Au NBP@Ag
nanostructures and the plasmon resonance of bulk Ag, respecꢀ
3
9
tively.
Figure 2 Effect of (A) DEA concentration, (B) 4ꢀAPP concentraꢀ
tion, (C) Ag concentration and (D) the reaction time on the LSPR
peak blueꢀshift of Au NBPs (All the concentration of ALP used in
this was 5.0 mU/mL, n=3).
+
In a word, the principle of this work could be divided into
two parts: firstly, the formation of sandwiched immunocomꢀ
plex by antibodyꢀantigen interaction; secondly, 4ꢀAPP was
hydrolyzed by ALP and 4ꢀAP subsequently generate, then
silver was produced and deposited on Au NBPs surface with
the help of 4ꢀAP and accompanied with LSPR peak blueꢀshifts
and color changes.
Finally, the reaction time was investigated. The value of
ꢃλmax gradually increased when the reaction time was less than
5 min, and reached a platform when the reaction time was
3
between 35 – 45 min. Small increase was observed when the
reaction time was over 45 min (Figure 2 D). We inferred that a
long reaction time would also induce the formation of heteroꢀ
geneous nucleation. In this case, most of the silver was coated
onto the newly formed silver nanoparticle seeds, while few of
them were deposited on Au NBPs@Ag. As shown in Figure
S2, the longitudinal LSPR peak almost no shifted and the abꢀ
sorption around 490 nm increasing when the reaction time was
3
.2 Optimization of experimental conditions
In this work, ALP played two important roles, on one hand,
it acted as a “bridge”, which connected the immune response
system with the signal indication unit; on the other hand, as an
enzyme hydrolyzed 4ꢀAPP to produce 4ꢀAP to participate in
the chromogenic reaction. Thus, ALP plays a very important
role in this system. According to Gao et al’s work, ALP
should be better carried out at an effective pH of 9.8 and a
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over 45 min. Therefore, 35 min was chosen as the optimal
reaction time.
concentration of ALP, and that means the color change more
obviously and more easily detected by naked eye for the same
concentration of ALP.
3
.3 Detection of ALP
3
.4 Detection of H N virus
5 1
Under the optimum conditions, different concentrations of
+
ALP were incubated with 4ꢀAPP, Ag and Au NBPs. The soꢀ
lution exhibits a significant color changes from brownish red
to green to dark blue with the increase concentration of ALP
It is well known that ALP has been widely used as an enꢀ
zyme label in conventional ELISA. Thus, next we explored
the application of the proposed detection strategy for immunoꢀ
assays. The detection of H N virus was selected as a demonꢀ
stration. As shown in Scheme 1, in our detection scheme, ALP
was labelled on the detection antibody by the avidinꢀbiotin
system. Thus, the more H N viruses existed in the sample
solution, the more Ab ꢀALP would adsorb on the ELISA miꢀ
croplate. As a result, the amount of 4ꢀAPP hydrolyzed by ALP
was in proportional with the concentration of H N viruses
existed in the sample solution. As shown in Figure 4A and B,
with the increase of H N virus concentration, the solution
color changed from brownishꢀred to green, dark blue and dark
red. Meanwhile, the LSPR peak of the solution blue shifted
gradually from 755 nm to 550 nm. When the concentration of
the virus protein exceeded 5 ng/mL, no more LSPR peak blueꢀ
shift was observed, instead, a little LSPR peak redꢀshift was
appeared. This should mainly come from the evolution of the
shape of the nanostructures instead of the optical properties of
Ag . Nevertheless, the LSPR blue shifted value ꢃλ has a
liner range with virus concentration at the concentration rangꢀ
ing from 1pg/mL to 2.5 ng/mL. The LOD was determined to
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(
Figure 3A). Continuous blue shift of the extinction peaks
5
1
were observed when the concentration of ALP was increased
from 0.1 to 10 mU/mL. The vivid color changes could be easiꢀ
ly distinguished with the naked eye. Thus, semiꢀquantitative
detection of ALP with the naked eye was accomplished. In
addition, the UVꢀvis spectra of the solution containing differꢀ
ent concentration of ALP were recorded (Figure 3B). The lonꢀ
gitudinal LSPR blue shifted from 755 nm to 595 nm with the
ALP concentration increase from 0.1 to 10 mU/mL. The caliꢀ
bration curve exhibited a liner range from 0.1 to 5 mU/mL
5
1
2
5
1
5
1
(
Figure 3C), and the detection limit (LOD) for ALP was 0.086
mU/mL (3σ /slope, σ represents the standard deviation of
blank samples).
b
b
39
max
be 1pg/mL (3σ /slope). Compared with other methods for the
b
detection of influenza virus, the proposed method not only
displayed an ultralow LOD but also possessed an additional
advantage of semiꢀquantitative detection of the target with the
naked eye (Table S1). Additionally, compared with other noꢀ
ble metal nanomaterials used for colorimetric analysis, the
proposed method has a significantly lower LOD and more
colorful displays for visual detection (Table S2).
Figure 3 Detection of ALP activity using the proposed colorimetꢀ
ric biosensor. (A) and (B) are photographs and UVꢀvis spectra in
response to different concentrations of ALP; the concentration of
ALP in (A) is 0, 0.1, 0.5, 1.0, 2.5, 5.0, 8.0, 10.0 mU/mL (from left
to right). (C) is the relationship between longitudinal LSPR blueꢀ
shift value (ꢃλmax) and ALP activity, and the inset in (C) was the
calibration curve between ꢃλmax and ALP activity. (D) The typical
UVꢀvis spectra of Au NBPs and Au NRs. (E) The comparison of
analytical performance of Au NBPs and Au NRs for the detection
of ALP activity.
Figure 4 Detection of H N virus using the proposed colorimetric
5
1
immunosensor. Photographs (A) and corresponding UVꢀvis specꢀ
tra (B) of the sensor in the presence of different concentrations of
H N virus antigens. (C) is the relationship between ꢃλ and the
5
1
max
antigen concentration.
For comparison, we also investigated the different analytical
performance between Au NBPs and Au NRs (the UVꢀvis
spectra were shown in Figure 3D). As shown in Figure S3A in
the Supporting Information, the color changes between these
two nanomaterials were similar. But, it is worth to note that
Au NBPs was much more sensitive than Au NRs when detectꢀ
ing low concentration of ALP. As shown in Figure 3E, the Au
NBPs LSPR peak blue shifted more than Au NRs at the same
We further investigated the selectivity of the proposed bioꢀ
sensor. Alphaꢀfetoprotein (AFP), carcinoꢀembryonic antigen
(CEA), human serum albumin (HSA) and H N virus were
selected as the competitors. As shown in Figure 5, other proꢀ
teins caused no obvious peak shift and only H N induced a
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significant LSPR peak shift and corresponding vivid color
change. These results indicated that the proposed assay had a
good selectivity.
In conclusion, we developed an ultrasensitive colorimetric
immunoassay method based on targetꢀguided deposition of
silver on highly monodispersed Au NBPs. The deposition of
silver on Au NBPs generated significant blue shift of the
LSPR band. As a result, vivid colors were observed correꢀ
sponding to different thickness of silver coating. This pheꢀ
nomenon was utilized for the detection of ALP and H N viꢀ
In order to evaluate the potential applicability of the proꢀ
posed method for clinical diagnostics, mimetic samples conꢀ
5
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taining the normal serum and different concentration of H N1
5
rus. The corresponding LODs were 0.086 mU/mL for ALP
and 1.0 pg/mL for H N virus antigen. Compared with other
antigen were tested. As shown in table 1, four mimetic samꢀ
ples were investigated; the results revealed that the proposed
assay has a good accuracy and precision with the RSD range
of 2.07 ~ 9.41%; and with good recoveries range of 99.00% ~
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noble metal nanostructures, colorimetric sensors based on
highly monodispersed Au NBPs showed much better sensitiviꢀ
ty. In addition, the vivid colors of the proposed sensing strateꢀ
gy enabled us to semiꢀquantitatively detect the analytes with
the naked eye.
1
02.0%. The corresponding UVꢀvis spectra of the mimetic
samples were shown in Figure S4. Moreover, in order to
demonstrate the universality of the proposed sensor, the proꢀ
posed approach was also demonstrated to detect AFP (Figure
S5). Both the solution color and the LSPR spectra are changed
significantly with the variations of AFP concentration. These
results strongly indicated that our method could be employed
for the detection of other biomarkers based on ALPꢀbased
ELISA strategy.
ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website.
AUTHOR INFORMATION
Corresponding Author
Institute of Nanomedicine and Nanobiosensing; Ministry of Eduꢀ
cation Key Laboratory of Analysis and Detection Technology for
Food Safety; College of Chemistry, Fuzhou University, Fuzhou,
3
50116, China. Tel: 086ꢀ591ꢀ22866164. Fax: 086ꢀ591ꢀ22866135.
Eꢀmail: guolh@fzu.edu.cn; zylin@fzu.edu.cn.
Notes
The authors declare no competing financial interest
ACKNOWLEDGMENT
This project was financially supported by the Natural Science
Foundation of China (21575027, 21675028, 21375021, and
2
1575025) and the Program for Changjiang Scholars and Innovaꢀ
tive Research Team in University (No. IRT1116).
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