Microchimica Acta Electronic Supplementary Material
A triple immunochromatographic test for simultaneous determination of cardiac troponin I, fatty acid binding protein, and C-reactive protein biomarkers Nadezhda A. Byzova1, Anatoly V. Zherdev1, Yury Yu. Vengerov1, Тatyana A. Starovoitova2, Boris B. Dzantiev1,* 1
A.N. Bach Institute of Biochemistry, Federal Research Center «Fundamentals of
Biotechnology», 119071 Moscow, Russia 2
N. I. Pirogov City Clinical Hospital No. 1, 117049 Moscow, Russia
*
Corresponding author: Boris B. Dzantiev;
Tel. +7 495 954 31 42; Fax +7 495 954 28 04; e-mail:
[email protected].
Table S1. Analytical parameters of the ELISA for TnI, FABP and CRP Analyte
Immobilized
Biotinylated
Detection limit,
Operational
antibodies
antibodies
ng·mL–1
range, ng·mL–1
TnI
TnI-C4
TnI-C19
5.0
7.2–100
TnI
TnI-C19
TnI-C4
3.1
5.4–33
FABP
FABP-F5
FABP-F10
100
150–1,000
FABP
FABP-F10
FABP-F5
6.3
9.8–45
CRP
CRP-11
CRP-12
3400
5,000–50,000
CRP
CRP-12
CRP-11
600
1,000–15,000
1
Fig. S1. Electronic microphotography of the gold nanoparticles. Images were obtained using a CX-100 electron microscope under accelerating voltage 80,000 V and enlargement 3,300,000.
2
2
50
Line intensity, arb. unirts
40
30
20
1 10
0 0,0 0
0,5 0.5
1,0 1.0
1,5 1.5
2,0 2.0
C (Tween 20), %
Fig. S2. The effect of Tween 20 concentration in the buffer used when applying ТnI-С4–AuNP conjugate to the membrane. The concentrations of TnI in the serum were 1.0 (1) and 50 (2) ng·mL–1. TnI-C19 antibodies were immobilized in the test zone from the solution with a concentration of 1.0 mg·mL–1. The D520 of the ТnI-С4–AuNP conjugate was 4.0. The measurements were made in triplicate.
3
Table S2. The detection limits and colouration intensities of the binding zones for FABP and CRP with various concentrations of Tween 20 detergent in the buffer used for antibody–AuNP conjugates applying to the PT-R7 membrane Analyte
Assay parameters
Concentration of Tween 20, %
FABP
0.05
1.0
3.9
3.8
at serum FABP concentration of 30 ng·mL–1
33.6
36.1
at serum FABP concentration of 120 ng·mL
80.5
83.2
Detection limit, ng·mL–1
600
600
at serum CRP concentration of 4 μg·mL–1
79.3
77.9
at serum CRP concentration of 32 μg·mL–1
103.7
108.2
Detection limit, ng·mL–1 Colouration intensities of the binding zone for FABP, arb. units:
CRP
Colouration intensity of the binding zone for CRP, arb. units:
4
A
B 4
4 3
80
3
60
Line intensity, arb. unirts
Line intensity, arb. unirts
2 1 40
20
60
40
20
0
0 0,01 0.01
2 1
0,1 0.1
1
10
1
100
10
100 -1
-1
C (FABP), ng mL
C (cTnI), ng mL
C
100
2 3 4
Line intensity, arb. unirts
80
1 60
40
20
0 0,01 0.01
0,1 0.1
1
10 -1
C (CRP), g mL
Fig. S3. Dependence of test zone colouration intensity on the concentration of (a)ТnI, (b) FABP and (c) CRP in the serum for conjugates (a)ТnI-С4–AuNP, (b) FABP-F5–AuNP and (c) CRP11–AuNP with individual D520 of (1) 2.0, (2) 3.0, (3) 4.0 and (4) 5.0 mixed in equal volumes. Mixture consumption equalled 16.0 μL·cm–1
5
A
B 20
Line intensity, arb. unirts
Line intensity, arb. unirts
80
60
40
20
0
15
10
5
0 0,1 0.1
1
10
100 100
0
1
2
3 -1
-1
C (TnI), ng mL
C (TnI), ng mL
C
D 15
Line intensity, arb. unirts
Line intensity, arb. unirts
80
60
40
10
5
20
0
0 1
10
100
0
5
-1
E
10
15 -1
C (FABP), ng mL
C (FABP), ng mL
F 100
50
Line intensity, arb. unirts
Line intensity, arb. unirts
80
60
40
20
40
30
20
10
0
0 0,1 0.1
1
0,0 0
10 -1
C (CRP), g mL
0,2 0.2
0,4 0.4
0,6 0.6
0,8 0.8
1,0 1.0
-1
C (CRP), g mL
Fig. S4. Calibration curves for the detection of (A, B) TnI, (C, D) FABP and (E, F) CRP in serum using the triple immunochromatographic test system. A, C, E = approximations in semilogarithmic axes, B, D, F = linear approximation for initial parts of the curves 6
