Electronic Supplementary Material
Zinc oxide nanoparticle-enhanced ultrasensitive chemiluminescence immunoassay for
CEA the carcinoma embryonic antigen
Souvik Pala and Sunil Bhanda,*
a
Biosensor Lab. Department of Chemistry, BITS, Pilani –KK Birla Goa Campus, Goa
403726, India
*
Corresponding author, Email: sunil17_bhand@yahoo.com, sunilbhand@goa.bits-pilani.ac.in,
Ph: +91-832-2580332, Fax: + 91-832-2557030/33
Materials and instrumentation
Mouse monoclonal anti-carcino embryonic antigen CEA antibody ( 1° Ab), rabbit polyclonal
anti-carcino embryonic antigen CEA antibody (2° Ab), goat polyclonal secondary antibody to
rabbit IgG – H&L (HRP) (3° Ab – HRP) , carcino embryonic antigen CEA protein (CEA) and
goat F(ab) polyclonal secondary antibody to rabbit IgG – H&L (FITC) (3° Ab – FITC) were
procured from Abcam (UK) (http://www.abcam.com/). Human Serum, 30 % aqueous solution
was purchased from Calbiochem (USA) (http://www.calbiochem.com). Zinc oxide
nanopowder (<100 nm particle size), bovine serum albumin (BSA), tween 20, luminol were
purchased from Sigma-Aldrich (USA) (http://www.sigmaaldrich.com/india.html). 384 well
polystyrene
microtiter
plates
were
purchased
from
Nunc
(Denmark)
(http://www.sigmaaldrich.com/labware/labware-products.html?TablePage=103658794).
For
CL measurements, Victor™ X4 2030 multiplate reader from Perkin Elmer (USA) was used
(http://www.perkinelmer.com/catalog/product/id/2030-0040). For the handling of CEA
standard solution, biological safety cabinet (Labgard ES, Class II, Type B2) from Nuaire
(USA) was used (http://www.nuaire.com/products/biological-safety-cabinets.html). Quartz
crystal microbalance (QCM) study was performed using EQCN-700 system integrated with
flow cell model FC-6 (M/s Elchema, Potsdam, New York, USA) (http://www.elchema.com/).
Fluorescence images were acquired on inverted microscope (IX-71 Olympus, Japan)
(http://www.olympusamerica.com/seg_section/product.asp?product=1023)
charged
coupled
device
(CCD),
Hamamatsu
coupled
with
(Japan)
(http://www.hamamatsu.com/us/en/index.html). Water produced in a Milli-Q system
(Millipore, Bedford, MA, USA) (www.emdmillipore.com) was used for preparing all the
1
solutions. Automated eppendorf Xplorer plus (8-channel) (Eppendrof AG, Germany) was
used for liquid and sample pipetting (http://www.eppendorf.com/IN-en/).
Optimization of experimental conditions
Optimization of ionic strength and pH of buffer
Several physicochemical factors that influenced the CL- ELISA performance were studied.
To evaluate the influence of buffer ionic strength and buffer pH, standard curves were
prepared. A constant concentration of 3° Ab-HRP was added to serial dilutions (from 100 to 1
mM PBS) of a concentrated buffer. Increment in signal intensity was found up to 10 mM. A
slight decrease in signal intensity was observed for 50 mM and 100 mM. Influence of pH on
the 1° Ab and 2° Ab was studied in the range 5.0 to 9.0. The optimum results for the ionic
strength and pH of PBS were found at 10 mM, pH 7.4 (Figure S1 and S2).
Signal intensity (ADU)
1600
1200
800
400
5
6
7
8
9
pH
Figure S1: Optimization of pH of buffer (5.0 – 9.0) for ZnONPs based CEA immunoassay; in
triplicate.
Signal intensity (ADU)
2000
1600
1200
800
400
0
20
40
60
80
100
Ionic strength (mM)
Supplementary Figure S2: Optimization of ionic strength of buffer (1 – 100 mM) for ZnONPs
based CEA immunoassay; in triplicate.
2
Optimization of 3° Ab-HRP and ZnONPs
The principle of the ZnO-NPs based immunoassay was based on a sandwich immunoassay as
described in earlier section. Effective 3° Ab dilution is very important to produce reproducible
results and reduce non-specific bindings, which might produce false signals. To examine
appropriate amount of 3° Ab-HRP, various 3° Ab-HRP dilutions were tested. It was found
that a 3° Ab-HRP dilution of 1:2000 produced better signal intensity against 1° Ab (0.2
µg·mL-1) (Figure S3). Further, the amount of ZnO-NPs – CEA probe particles was also
optimized as 2 mg·mL-1, having maximum signal intensity (Figure S4).
Signal intensity (ADU)
21000
1:100
1:200
1:500
1:1000
1:2000
1:4000
18000
15000
12000
9000
6000
3000
0
10
20
30
40
Time (min)
Figure S3: Optimization of pAb-HRP (1:100 – 1:4000 dilutions) for ZnONPs based CEA
immunoassay.
Signal intensity (ADU)
24000
20000
16000
12000
8000
4000
0
1
2
3
4
-1
5
Amount of ZnO-NPs (mgmL )
Figure S4: Optimization of amount of ZnONPs (1 – 5 mg mL-1) for ZnONPs based CEA
immunoassay.
3
Selectivity studies
In the presence of possible interferences, such as glucose (Glu, 5 mM), ascorbic acid (AA, 10
µM), glycine (Gly), bovine serum albumin (BSA, 380 µg mL-1) and mixture, the developed
immunosensor was studied for its selectivity in the incubation solution with 20 ng mL-1 CEA.
The % I was calculated from the signal intensity values in the presence of the interference and
without interference (Figure S5).
60
-1
Selectivity study against 20 ng.mL of CEA
% Inhibition
50
40
30
20
10
ur
e
BS
A
M
ixt
Gl
y
AA
Gl
u
CE
A
AA
Gl
u
+C
EA
+C
EA
Gl
y+
CE
BS
A
A
+C
EA
0
Figure S5: Selectivity study using glucose (Glu, 5 mM), ascorbic acid (AA, 10 µM), glycine
(Gly), bovine serum albumin (BSA, 380 µg mL-1) and mixture against CEA (20 ng·mL-1).
50
-1
Selectivity study against 2 ng.mL of CEA
% Inhibition
40
30
20
10
Gl
y
AA
Gl
u
CE
A
BS
A
M
ixt
ur
e
Gl
u
+C
EA
AA
+C
EA
Gl
y+
CE
BS
A
A
+C
EA
0
Figure S6: Selectivity study using glucose (Glu, 0.5 mM), ascorbic acid (AA, 1 µM), glycine
(Gly), bovine serum albumin (BSA, 38 µg mL-1) and mixture against CEA (2 ng·mL-1).
In the presence of possible interferences, such as glucose (Glu, 0.5 mM), ascorbic acid (AA, 1
µM), glycine (Gly), bovine serum albumin (BSA, 38 µg mL-1) and mixture, the developed
immunosensor was studied for its selectivity in the incubation solution with 2 ng mL-1 CEA.
4
The % I was calculated from the signal intensity values in the presence of the interference and
without interference (Figure S6).
In the presence of possible interferences, such as glucose (Glu, 0.05 mM), ascorbic acid (AA,
0.1 µM), glycine (Gly), bovine serum albumin (BSA, 3.8 µg mL-1) and mixture, the
developed immunosensor was studied for its selectivity in the incubation solution with 0.2 ng
mL-1 CEA. The % I was calculated from the signal intensity values in the presence of the
interference and without interference (Figure S7).
40
-1
Selectivity study against 0.2 ng.mL of CEA
% Inhibition
30
20
10
ur
e
BS
A
M
ixt
Gl
y
AA
Gl
u
CE
A
AA
Gl
u
+C
EA
+C
EA
Gl
y+
CE
BS
A
A
+C
EA
0
Figure S7: Selectivity study using glucose (Glu, 0.05 mM), ascorbic acid (AA, 0.1 µM), glycine
(Gly), bovine serum albumin (BSA, 3.8 µg mL-1) and mixture against CEA (2 ng·mL-1).
Signal intensity (ADU)
mAb-16PHA-ZnO-NPs;
mAb coated microwell plate
20000
15000
10000
5000
0
1
2
3
4
Time (Weeks)
Figure S8: Comparison of stability of 1° Ab coated ZnONPs and microwell plate at storage
condition 4 °C, 10 mM pH 7.4 PBS.
5
Table S1: Recovery of [CEA] from different serum samples as determined by CEA – ZnONPs nano-immunosensor to assess the recovery efficiency.
ZnO-NPs – CEA immunoassay
[CEA] added
(ng·mL-1 )
[CEA] Found
(ng·mL-1 )
Mean S.D.
Microplate ELISA
R.E.
(%)
Recovery
(%)
[CEA] Found
(ng·mL-1 )
Mean S.D.
R.E.
(%)
Recovery
(%)
0.01
0.00988
0.00019
1.2
98.8
0.0104
0.0002
- 4.0
104
0.05
0.0487
0.00252
2.6
97.4
0.0453
0.00252
9.4
90.6
0.0283
- 10.14
110.14
1
0.986
0.007
1.4
98.6
1.1014
2.5
2.49
0.0264
1.0
99.6
2.50
0.0152
0.0
100.0
10
10.05
0.02
- 0.5
100.5
10.47
0.379
- 4.7
104.7
15
14.993
0.041
0.047
99.95
N.D.#
-
-
20
19.81
0.02
0.95
99.05
N. D.
-
-
# Not determined
Found [CEA] ng.mL
-1
20
ZnO-NPs - CEA ELISA
Conventional ELISA
16
12
8
4
Correlation coefficient = 0.99998
0
0
4
8
12
16
-1
Added [CEA] ng.mL
20
Figure S9: Correlation curve between two methods (Conventional ELISA and ZnO-NPs –
CEA ELISA) with Pearson’s correlation coefficient (r) = 0.99998.
Figure 10: Theoretically calculated correlation coefficient (r) between two methods
(Conventional ELISA and ZnO-NPs – CEA ELISA).
6