were negative showed in Figure S5, 6B with two red bands appeared at test zone and control zone. However, surimi products were positively detected by the ...
Analytical and Bioanalytical Chemistry
Electronic Supplementary Material
Nano-gold capillary immunochromatographic assay for parvalbumin
Shuyuan Du, Hong Lin, Jianxin Sui, Xiudan Wang, Limin Cao
1
Fabrication of CICA The stepwise procedures for fabrication of the CICA is illustrated in Fig. S1. The epoxy group was firmly fixed on the wall of the glass capillary by pretreatment (Fig. S1a, b). PV and goat anti-rabbit IgG were successfully immobilized on the inner wall of the capillary by their interaction with epoxy groups, which were introduced through the modification of the capillary, therefore the test zone and control zone were constructed at different ends, respectively (Fig. S1c). Subsequently, BSA was used to block the non-specific sites (Fig. S1d).Optimization of analytical conditions To increase the sensitivity and stability of CICA, the GPTMS modification time, the color development time, the working concentrations of coating PV (test zone) and the coating goat anti-rabbit IgG (control zone) were optimized in detail (Fig. S3. Table S1). Calibration curve for PV detection The relative brightness of the test zone positively correlates with the parvalbumin concentration ranging from 50 ng/mL to 1000 ng/mL that prepared in phosphate buffered saline (PBS) (Fig. S4). Specificity of CICA In order to evaluate the specificity of the CICA, human serum albumin, egg albumin, extracts from several aquatic foods and other foods including beef, pork, and chicken were tested using the CICA. The CICA results for detecting negative control (human serum albumin and egg albumin ranging from 1 to 1000 μg/mL) and livestock meat were negative showed in Figure S5, 6B with two red bands appeared at test zone and control zone. However, surimi products were positively detected by the CICA (Fig.S6 A) with one red band appeared at control zone.
2
SDS- PAGE analysis of extracts The SDS-PAGE pattern showed that several proteins existed in the crude extracts of Scophthalmus maximus (Linnaeus) (Fig. S7A), however, only protein bands with molecular masses of about 9-14 kDa were recognized as PV. The high purity of the PV from Scophthalmus maximus (Linnaeus) samples was obtained after heating at 98℃ over 5 min. The presence of PV was also detected in 4 fish products (Fig. S7B), but for pork, beef and chicken there was no PV band in SDS-PAGE pattern (Fig. S7C).
Fabrication cost estimation The fabrication cost showed in Table S2 was estimated to be ~$0.53 with the main material of CICA.
3
Si Si Si
Si O
OH
Si
O
H O /H SO 2
2
2
4
Si
OH KOH/HCl
Si
O
OH
Si
O Si
Si
O Si
Si
c
O Si
O Si
Si
O Si
Si
O Si
O O
O
GPTMS
OH
a
Ag
Si
O
O O
b
O
OH
NH
O
OH
Si BSA
O
O
Ag
NH
Si Si
d
O Si O Si O Si
O O O
OH OH OH
NH
Ag
NH NH
Fig. S1 Stepwise procedures for the fabrication of CICA: (a) pretreatment to activate hydroxyl groups, (b) introduction of epoxy groups by modification with GPTMS, (c) the immobilization of PV and anti-PV antibody, and (d) BSA blocking
4
Au
Au
Au Ab
BSA
Fig. S2 The scheme of immobilizing the antibody to the gold nanoparticle
5
a -5
Relative brightness
-10 -15 -20 -25 -30 -35 0
b
5
10
15
20
25 30 Time (h)
35
3
5
40
45
50
-8
Relative brightness
-12 -16 -20 -24 -28 -32 0
1
2
4
6
7
Time (min) Fig. S3 The influence of GPTMS modification time (A) and color development time (B) on CICA results
6
-5
Relative brightness
-10
-15 y = a + b*x Instrumental
Equation Weight
-20
-25
Residual Sum of Squares
18.2464
Pearson's r
0.98981
Adj. R-Square
0.97683 Value
B
-30 1.50
B
1.75
2.00
2.25
Intercept Slope
2.50
Standard Error
-60.9291 19.3739
2.75
2.31863 1.05341
3.00
3.25
LogC(ng/mL)
Fig. S4 The response curve of the relative brightness vs. the concentrations of PV under optimal conditions
7
a
b
Control zone
Test zone Control
1
10 100 1000 μg/mL Control
1 10 100 1000 μg/mL
Fig. S5 Assay results for negative control detection: scanned image of the CICA showing the color intensity for PBS spiked with human serum albumin (A) and egg albumin (B) with concentration ranging from 1 to 1000 μg/mL
8
a
fish ballⅠ
cuttlefish ball
fish ballⅡ
fish tofu
Control zone
Test zone Control
0
5 15
0
5
15
0
5
15 0
5
15
control
15 min
b pork
beef
chicken
Control zone
Test zone min
0
5 15
0
5
15
0
5
Fig. S6 CICA results of extracts from (a) fish products and (b) livestock meat, crude extracts and extracts after heating at 98°C for 5 and 15 min, from left to right
9
a
Mark a b
c d
b
e
116.0
116.0
66.2
66.2
45.0
45.0
35.0
35.0
25.0
25.0
fish Mark ballⅠ
fish ballⅡ
cuttlefish ball
fish tofu
18.4
18.4
14.4 PV
14.4 PV kDa
kDa 0 5 15 0 5 15 0 5 15 0
0 5 10 15 25 min c
Mark
beef
pork
5 15
chicken
116.0 66.2 45.0 35.0 25.0 18.4 14.4 kDa 0 5
15
0
5
15 0
5
15 min
Fig. S7 SDS-PAGE analysis of extracts from (A ) Scophthalmus maximus (Linnaeus), (B ) fish products, and (C ) livestock meat. (A) Lane a: crude extracts, Lane b: extracts after heating at 98℃ for 5 min, Lane c: extracts after heating at 98℃ for 10 min, Lane d: extracts after heating at 98℃ for 15 min, Lane e: extracts after heating at 98℃ for 25 min, (B) (C) crude extracts and extracts after heating at 98℃ for 5 min 10 and 15 min, from left to right
min
Coated PV and goat antiRelative brightness rabbit IgG concentration (mg/mL) Test zone CV(%) Control zone CV(%) 0.1 -20.29 3.03 -13.41 4.87 0.2
-30.65
7.61
-21.91
1.39
0.3
-35.68
7.08
-31.69
2.34
0.4
-43.82
5.30
-32.72
4.34
0.5
-45.33
3.63
-33.91
6.47
Table S1 Optimization of PV and goat anti-rabbit IgG coating concentration on test zone and control zone was selected by detecting bank
11
Material
Cost/Volume
Cost per test
$ 50/100 mL
Volume/ Test 25 μL
Colloidal gold Parvalbumin (0. 2mg/mL)
$ 180/100 mL
3 μL
$ 0.054
anti-parvalbumin antibody (10 mg/mL)
$ 600/mL
0.5 μL
$ 0.30
Goat anti-rabbit IgG (0.3 mg/mL)
$ 100/2mL
3 μL
$ 0.056
$ 0.0125
glass capillary ($ 3/500 tubes)
$ 0.006
chemicals
$ 0.10
Total
~$ 0.53
Table S2 The fabrication cost estimation of CICA per test
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