Electronic Supplementary Material
Electrochemical immunoassay for the prostate specific antigen using a reduced graphene
functionalized with a high molecular-weight silk peptide
Yanying Wanga,b, Ying Qua,Guishen Liuc, Xiaodong Houc, Yina Huangc,
Wangze Wuc, Kangbing Wub*, Chunya Lia*
a Key Laboratory of Analytical Chemistry of the State Ethnic Affairs Commission, College of
Chemistry and Materials Science, South-Central University for Nationalities, Wuhan 430074,
China
b Key Laboratory for Large-Format Battery Materials and System, Ministry of Education, School of
Chemistry and Chemical Engineering, Huazhong University of Science and Technology, Wuhan
430074, China
c Chaozhou Quality and Measurement Supervision and Inspection Institute, Chaozhou 521011,
China
d The central hospital of Wuhan, Wuhan 430014, China
* Corresponding Author. E-mail: lichychem@163.com.
Table S1 Comparison of the sensing performance of the developed immunosensor with previous
reports.
Electrode
SP-rGO/GCE
Electrochemical
Detection limit
Linear range
technique
(ng·mL‾1)
(ng·mL‾1)
Differential pulse
voltammetry
0.053
0.1 – 5.0
5.0 – 80.0
Ref.
This work
Potentiometry
0.04
0.1 – 50
[1]
Ag@MSNs/GCE
Amperometry
0.015
0.05 – 50.0
[2]
ILs/CNTs/GCE
Differential pulse
0.02
0.02 – 1
[3]
AuNP/PEI-PSAA/Au
electrodes
1 – 40
voltammetry
SPE-nAu
Linear sweep
0.76
1 - 35
[4]
0.25
-
[5]
voltammetry
Pt/SWCNTs
Differential pulse
voltammetry
Fig. S 1 Morphologic characterization of the SP-rGO nanocomposites (a) and the rGO nanosheets
(b) with transmission electron microscopy.
From the comparison of TEM images of SP-rGO and rGO we can obviously see that
modification has no significant on the morphology of nanosheets.
Fig. S 2 X-ray diffraction characterization of the GO (a), the rGO (b) and the SP-rGO (c).
Disappearing of the sharp peak located around 10° indicates that the reduction of the GO to the
rGO.
Fig.S3 X-ray photoelectron spectroscopy characterizations of the SP-rGO (a) and the r-GO (b).
Existence of N1s in the SP-rGO nanocomposites means the conjugation of silk peptides onto the
rGO nanosheets.
Fig. S 4 FTIR spectra of the GO (a), the SP (b) and the SP-rGO (c).
Some characteristic peaks of the SP and the GO were observed in the SP-rGO, especially vC-N at
1385.69 cm-1, indicate the conjugation of the SP and the rGO.
Fig. S 5 Roman spectra of the GO (a) and the SP-rGO (b).
The Raman spectra of graphene oxide nanosheets (a) and high molecular-weight silk
peptide/reduced graphene oxide nanosheets (b) were shown in Fig. S 5. G-band located at 1591
cm-1 is a result of in-plane vibrations of SP2 bonded carbon atoms. The peak appeared at 1351 cm-1
is known as the D-band which is due to out of plane vibrations attributed to the presence of
structural defects. Comparing the ratio of the intensitiy of D/G, it is clearly to see that the ID/IG of
the GO nanosheets is obviously higher than that obtained from SP-rGO nanosheets. The result
demonstrates that the reduction of the graphene oxide nanosheets to be the reduced graphene oxide
nanosheets have been successfully achieved.
Fig. S 6 Effects of pH values (A), incubation temperature (B) and incubation time (C) on the peak
current response of the immunosensor towards 50.0 ng·mL-1 PSA.
Based on the current response of the immunosensor towards 50.0 ng·mL-1 PSA, the influences of
pH values (A), incubation temperature (B) and incubation time (C) were investigated and shown in
Fig. S 6. The peak current difference (∆I) before and after the immunosensor being incubated into
PSA solution was used as a criterion to evaluate their effects. pH value of the buffer solution greatly
affected the immunosensor for selective recognition of PSA. From Fig. S 6 (A), it can be seen that
the current response sharply increase with the pH values varied from 4.0 to 6.0. With further
increasing of pH values, the current response decreased reversely. A maximum current response was
obtained at pH value of 6.0, therefore, it was chosen for the further experiments to obtain high
sensitivity.
Incubation temperature can have strong effects on the bioactivity of anti-PSA and may influence
the specific binding between the immunosensor and PSA, thus further affect the current response of
the immunosensor. As shown in Fig. S 6 (B), it was found that the ΔI values of 50.0 ng·mL–1 PSA
increases with the variation of incubation temperature from 20 ºC to 35 ºC. However, a slight
decrease was observed when the incubation temperature was varied from 35 ºC to 45 ºC, meaning
that a high temperature may cause the ineffective binding of PSA. Therefore, 35 ºC was selected as
the optimized incubation temperature for the selective recognition of PSA by the immunosensor.
The incubation time for the binding reaction between anti-PSA and PSA is also critically
important for the immunoassay. Fig. S 6 (C) shows the dependence of the current response on the
incubation time. The current response of the immunosensor almost reaches the maximum value
after being incubated into 50.0 ng·mL-1 of PSA for 30 min. The result indicates that the binding
reaction can be finished when the incubation time is 30 min. In order to reduce the analytical time
and obtain relatively high sensitivity, therefore, 30 min was employed as the incubation time in the
following experiments.
Fig. S 7 Current response of the immunosensor to (a) 10 ng·mL-1 PSA and (b) 10 ng·mL-1 PSA in
the presence of 100.0 ng·mL-1 AFP, 100.0 ng·mL-1 human IgG, 1.0 μg·mL-1 BSA, 1.0
μg·mL-1 Vc, 1.0 μg·mL-1 L-Cysteine, and 1.0 μg·mL-1 L-Lysine. Error bars represent standard
deviation (n =3).
References
[1] B. Zhang, B. Q. Liu, G. N.Chen, D. P.Tang, Biosens. Bioelectron. 53 (2014) 465.
[2] H. Wang, Y. Zhang, H. Q. Yu, D. Wu, H. M. Ma, H. Li, B. Du, Q. Wei, Anal. Biochem. 434
(2013) 123.
[3] A. Salimi, B.Kavosi, F. Fathi, R. Hallaj, Biosens. Bioelectron. 42 (2013) 439.
[4]
V.
Escamilla-Gómez, D.
Hernández-Santos,
M.
B. González-García,
J.
M. Pingarrón-Carrazón, A. Costa-García, Biosens. Bioelectron. 24 (2009) 2678.
[5] J. Okuno, K. Maehashi, K. Kerman, Y. Takamura, K. Matsumoto, E. Tamiya, Biosens.
Bioelectron. 22 (2007) 2377.