Electronic Supplementary Information
Enhanced Colorimetric Immunoassay Accompanying with Enzyme
Cascade Amplification Strategy for Ultrasensitive Detection of
Low-Abundance Protein
Zhuangqiang Gao, Li Hou, Mingdi Xu & Dianping Tang*
Ministry of Education Key Laboratory of Analysis and Detection for Food Safety, Fujian Provincial Key
Laboratory of Analysis and Detection for Food Safety, Department of Chemistry, Fuzhou University, Fuzhou
350108, P.R. China.
Corresponding author information
Phone: +86-591-2286 6125; fax: +86-591-2286 6135; e-mail: [email protected] (D. Tang)
S1
■ Experimental section
ALP measurement by using conventional method. Initially, 2 μL of ALP standards with various
concentrations was added into 100-μL mixture containing 5.0 mM pNPP and 1.0 mM MgCl2 in
10% diethanolamine (DEA) buffer (pH 9.8), and then the resulting solution was incubated at 37 °C
for 30 min. After that, the absorbance at λ = 405 nm was measured by a plate reader.
ALP measurement by using enzyme cascade amplification strategy (ECAS). 2 μL of ALP with
various concentrations was initially added into 20-μL mixture containing 5.0 mM AA-P and 1.0
mM MgCl2 in carbonate buffer (pH 9.8). Then the resulting solution was incubated at 37 °C for 20
min. Afterward, 3-μL AuNP (C[AuNP] ≈ 1.92 nM), 1.5-μL HCl solution (1.0 M) and 2-μL K2PdCl6
solution (1.5 mM) were simultaneously added to the resultant solution and incubated at 37 °C for 5
min. Following that, 100 μL of substrate solution containing 7.2 M H2O2 and 0.5 mM TMB in pH
4.0 sodium citrate-phosphate buffer was added and incubated at 40 °C for 5 min for color
development. Finally, the absorbance was read at 650 nm with a plate reader.
Control testing methods for ECAS. 2 μL of ALP (1.0 U mL-1) was initially added into 20-μL
mixture containing 5.0 mM AA-P and 1.0 mM MgCl2 in carbonate buffer (pH 9.8). Then the
resulting solution was incubated at 37 °C for 20 min. Afterward, 3-μL AuNP (C[AuNP] ≈ 1.92 nM),
1.5-μL HCl solution (1.0 M) and 2-μL K2PdCl6 solution (1.5 mM) were simultaneously added to
the resultant solution and incubated at 37 °C for 5 min. Following that, 100 μL of substrate solution
containing 7.2 M H2O2 and 0.5 mM TMB in pH 4.0 sodium citrate-phosphate buffer was added and
incubated at 40 °C for 5 min for color development. Finally, the absorbance was read at 650 nm
with a plate reader.
Preparation of palladium nanostructures with nanogold cores. Palladium nanostructures with
gold nanocores were synthesized according to the literature [1] with a little modification. All
glassware used in the following procedure was cleaned in a bath of freshly prepared solution (1 : 3,
HNO3-HCl), thoroughly rinsed with double-distilled water, and dried prior to use. Initially, 8 mL of
H2O, 200 μL of 10 mM K2PdCl6 and 200 μL of 10 mM ascorbic acid were added into a 25-mL
round-bottom flask. Following that, 8 mL of gold colloids (C[AuNP] ≈ 1.92 nM, 16 nm in diameter)
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was introduced quickly into the mixture, and continuously stirred for 30 min at 750 rpm at room
temperature until the color of the mixture turned from red to dark gray. The produced palladium
nanostructures with gold nanocores (designated as palladium nanocatalysts, ~0.96 nM) were stored
at 4 °C in a dark-colored glass bottle when not in use.
Kinetic study and mechanism of palladium nanostructures relative to TMB-H2O2 system. The
steady-state kinetic studies of the as-prepared palladium nanostructures (40 μL, ~0.96 nM) were
carried out at room temperature in a cuvette cell containing 400 μL of sodium citrate-phosphate
buffer solution (pH 4.0) in the presence of TMB and H2O2. The kinetic analysis of palladium
nanocatalysts with H2O2 as the substrate were performed by using 0.5 mM TMB and
different-concentration H2O2 (0, 50, 75, 100, 250, 500, 750, 1000, 2000, 3000 mM). The kinetic
analysis of palladium nanocatalysts with TMB as the substrate were performed by using 500 mM
H2O2 and different-concentration TMB (0, 0.02, 0.04, 0.06, 0.08, 0.1, 0.2, 0.3, 0.4, 0.5 mM). All the
reactions were monitored in time scanning mode at 650 nm using a UV-vis spectrophotometer
(1102 UV-vis spectrophotometer, Techcomp, China) during a period of 5 min. The initial rate v was
calculated by calculating the slope of the tangent at t = 0 min. The apparent kinetic parameters were
calculated based on Michaelis-Menten equation, v = Vmax[S]/(Km + [S]), where v is the initial rate,
Km is the Michaelis-Menten constant, Vmax is the maximum reaction rate, and [S] is the substrate
concentration. Km and Vmax were obtained by Lineweaver-Burk plot method.
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cps/eV
3.5
3.0
2.5
N
Au
Pd
C O
2.0
Au
Pd
Au
1.5
1.0
0.5
0.0
0
2
4
keV
6
8
10
Figure S1. Energy-dispersive X-ray spectroscope (EDX) of the grown gold nanoparticles with palladium
nanostructures.
S4
Figure S2. The catalytic activity of AuNP toward TMB/H2O2 system. Error bars indicate standard deviations (n =
3). Experiments were carried out using AuNP (C[AuNP] ≈ 1.92 nM) with various volumes in 100 L of sodium
citrate-phosphate buffer solution containing 7.2 M H2O2 and 0.5 mM TMB at 40 °C for 5 min, and the absorbance
was read at 650 nm with a plate reader.
S5
Figure S3. Chemical reaction mechanism of palladium nanostructures relative to TMB-H2O2 system.
S6
Figure S4. The effects of (A) pH of sodium citrate-phosphate buffer, (B) incubation temperature, (C) H2O2
concentration and (D) incubation time on the peroxidase-like activity of PdNS. Error bars indicate standard
deviations (n = 3). Experiments (A-C) were carried out by using 5-L palladium nanostructures (~0.96 nM) in
100-L sodium citrate-phosphate buffer solution containing 0.5 mM TMB for 5 min under different pHs,
temperatures, and H2O2 concentrations, respectively. The absorbance was read at 650 nm with a plate reader. The
detail conditions for experimental A-C were as follows: (A) pH value: from 2 to 12, H2O2 concentration: 1.0 M,
Temperature: 25°C; (B) Temperature: from 10 to 60 °C, H2O2 concentration: 7.2 M, pH = 4.0; (C) H2O2
concentration: from 0 to 9 M, pH = 4.0, Temperature: 25°C. The maximum point in each curve was set as 100%.
Experiment (D) was carried out at room temperature in a cuvette cell containing 5-L palladium nanocatalyst
(~0.96 nM), 400-L sodium citrate-phosphate buffer solution, 7.2-M H2O2 and 0.5 mM TMB, and the reaction
was monitored in time scanning mode at 650 nm by using a UV-vis spectrophotometer (1102 UV-vis
spectrophotometer, Techcomp, China) during a period of 6 min.
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Figure S5. The effects of various pH values on the formation of peroxidase-like PdNS-mimics: (A) photographs
of carbonate buffer solution with different pH values + ascorbic acid + K2PdCl6 + AuNP, (B) photographs of
10-μL corresponding solution 'a' in the presence of TMB and H2O2, and (C) the corresponding absorbance
intensity of Figure S5(B). The pH optimization for the PdNS was estimated by the peroxidase-like activity of the
generated PdNS. Error bars indicate standard deviations (n = 3). Experiments were carried out at different pH
values in 100-μL carbonate buffer solution containing ascorbic acid (5 μL, 20 mM), K2PdCl6 (5 μL, 3 mM) and
10-μL AuNP [Note: The low-pH incubation solutions, pH < 9.0, were prepared by directly adding HCl into
carbonate buffer]. The reaction conditions were performed at 37 °C for 5 min. After finished, 10 μL of the
above-prepared mixture was quickly added into 100 μL of the substrate solution containing 7.2 M H2O2 and 0.5
mM TMB in pH 4.0 sodium citrate-phosphate buffer and incubated at 40 °C for 5 min for color development.
Finally, the absorbance was read at 650 nm with a plate reader.
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Figure S6. Precision and reproducibility of ECAS-CIA toward PSA standards with various concentrations.
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Table S1 Comparison of the kinetic parameters of palladium nanocatalyst, HRP and other peroxidase mimics [a]
catalyst
substance
[E]0 / M
Km / mM
Vmax / 10-8 M s-1
Kcat / s-1
TMB
2.5 × 10-11
0.434
10
4.00 × 103
H2O2
2.5 × 10-11
3.7
8.71
3.48 × 103
TMB
1.14 × 10-12
0.098
3.44
8.58 × 104
H2O2
1.14 × 10-12
154
9.78
3.02 × 104
TMB
3.43 × 10-10
0.037
6.27
1.83 × 102
H2O2
3.43 × 10-10
140.07
12.1
3.53 × 102
Prussian
TMB
3.09 × 10-10
0.307
106
3.43 × 103
blue-Fe2O3
H2O2
3.09 × 10-10
323.6
117
3.79 × 103
ZnFe2O4
TMB
3.05 × 10-18
0.85
13.31
4.36 × 1010
H2O2
3.05 × 10-18
1.66
7.74
2.54 × 1010
Platinum
TMB
8.12 × 10-11
0.120
126
1.55 × 104
nanostructures
H2O2
8.12 × 10-11
769
185
2.27 × 104
palladium
TMB
9.6 × 10-11
0.165
201
2.09 × 104
nanostructures
H2O2
9.6 × 10-11
1064
443
4.61 × 104
HRP
Fe3O4
Co3O4
[a]
refs
[2]
[2]
[3]
[4]
[5]
[6]
This work
[E]0 is the enzyme (or peroxidase mimics) concentration; Km is the Michaelis-Menten constant; Vmax is the maximal reaction velocity; and Kcat is the
catalytic constant, where Kcat=Vmax/[E]0.
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Table S2 Intra-assay precision of the ECAS-CIA
PSA concentration (ng mL-1)
mean absorbance at 650 nm
SD (n = 8)
CV (%)
0.00
0.180
0.017
9.5
0.01
0.181
0.010
5.5
0.05
0.276
0.017
6.1
0.10
0.323
0.024
7.4
0.50
0.707
0.050
7.1
1.00
0.943
0.054
5.8
5.00
1.437
0.067
4.7
10.0
1.608
0.067
4.2
20.0
1.680
0.071
4.2
50.0
1.720
0.059
3.5
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Table S3. Comparison of the Assay Results for Human Serum Specimens by Using the Developed ECAS-CIA and the Commercialized
Human PSA ELISA Kit
Method;a Concentration [mean  SD, n = 3, ng mL-1]
Sample no.b
a The
Found by the ECAS-CIA
Found by the ELISA kit
texp
1
0.737  0.06
0.841  0.13
1.31
2
1.47  0.27
1.69  0.07
1.38
3
2.10  0.29
1.97  0.12
0.72
4
1.63  0.20
1.52  0.11
0.89
5
3.40  0.33
3.65  0.18
1.13
6
2.20  0.22
2.37  0.13
1.14
7
1.30  0.21
1.18  0.10
0.94
8
1.93  0.48
2.09  0.06
0.60
9
1.84  0.23
2.06  0.11
1.46
10
2.17  0.22
2.44  0.10
1.86
11
3.53  0.63
3.07  0.20
1.21
12
2.08  0.26
2.41  0.09
2.04
13
2.14  0.26
2.30  0.08
1.00
14
2.91  0.40
2.68  0.13
0.94
15
2.24  0.27
2.57  0.16
1.80
16
1.02  0.14
1.19  0.07
1.79
17
4.60  0.76
5.13  0.21
1.17
18
3.55  0.71
4.22  0.18
1.59
19
6.44  0.94
6.79  0.31
0.62
20
5.12  0.74
5.84  0.27
1.60
21
7.00  0.90
7.42  0.45
0.74
22
10.3  1.2
9.91  0.21
0.62
23
9.57  0.86
10.8  0.3
2.29
24
8.39  0.81
7.75  0.26
1.30
25
11.6  0.7
12.3  0.5
1.59
26
14.1  1.0
13.4  0.1
1.07
27
15.6  0.9
16.4  0.3
1.55
28
18.2  1.0
18.7  0.5
0.79
29
19.3  2.0
21.3  0.8
1.57
30
4.76  0.57
4.55  0.13
0.62
31
7.06  0.80
7.88  0.31
1.66
32
10.1  0.9
8.77  0.26
2.35
33
14.4  1.0
13.2  0.52
1.74
34
17.2  1.7
17.7  0.8
0.47
35
0.167  0.022
No application
No application
36
0.462  0.044
No application
No application
regression equation (linear) for these data is as follows: y = 1.020 x + 0.052 (R2 = 0.994, n = 102) (x-axis: by the ECAS-CIA; y-axis: by the
ELISA kit).
b Samples
1−21 were clinical serum specimens, while samples 22−36 were the diluted samples by using newborn cattle serum.
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Gao, L., Zhuang, J., Nie, L., Zhang, J., Zhang, Y., Gu, N., Wang, H., Feng, J., Yang, D., Perrett, S. & Yan,
X.. Intrinsic peroxidase-like activity of ferromagnetic nanoparticles. Nat. Nanotechnol. 2, 577-583 (2007).
[3]
Mu, J., Wang, Y., Zhao, M. & Zhang, L. Intrinsic peroxidase-like activity and catalase-like activity of
Co3O4 nanoparticles. Chem. Commun. 48, 2540-2542 (2012).
[4]
Zhang, X., Gong, S., Zhang, Y., Yang, T., Wang, C. & Gu, N. Prussian blue modified iron oxide magnetic
nanoparticles and their high peroxidase-like activity. J. Mater. Chem. 20, 5110-5116 (2010).
[5]
Su, L., Feng, J., Zhou, X., Ren, C., Li, H. & Chen, X. Colorimetric detection of urine glucose based
ZnFe2O4 magnetic nanoparticles. Anal. Chem. 84, 5753–5758 (2012).
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Gao, Z., Xu, M., Hou, L., Chen, G. & Tang, D. Irregular-shaped platinum nanoparticles as peroxidase
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