Electronic Supplementary Material

Electrochemical immunoassay for subgroup J of avian leukosis viruses using a glassy carbon electrode modified with a film of poly(3-thiophene boronic acid), gold nanoparticles, graphene and immobilized antibody

Zhenzhen Wangb,1, Kun Shanga,1, Jing Donga, Ziqiang Chengb*, Shiyun Aia*

a College of Chemistry and Material Science, Shandong Agricultural University, Taian, Shandong 271018, P.R. China

b College of Animal Science and Technology, Shandong Agricultural University, Taian, Shandong 271018, P.R. China

* Corresponding authors: Tel: +86 538 8247660, Fax: +86 538 8242251, E-mail address: (S.Y. Ai), (Z.Q. Cheng).

1 These authors contributed equally to this work.

Preparation of graphene (GR)

Briefly, graphite powder was reacted with concentrated H2SO4, K2S2O8, and P2O5 at 80 C for 6 h. After that, the mixture was diluted with deionized water, filtered with 0.2 m Nylon film and dried naturally. The product was reoxidized with concentrated H2SO4 and KMnO4 and graphite oxide (GO) was obtained. In order to exfoliate GO, 0.25 mg·mL-1 GO dispersion was sonicated for 1 h. And then, appropriate hydrazine was added into the above solution and kept stirring for 1 h at 95 C. Finally, black GR was obtained by filtration and dried in vacuum.

Affinity interaction of Ab with the PTBA-AuNPs-GR film

Previous studies have demonstrated that the glycosyl group can be effectively covalently bound to the boronic acid group, and the specific binding is reversible and can be removed by sorbitol solution [1-3]. Therefore, to determine the covalent binding of Ab to the PTBA-AuNPs-GR film, the effects of sorbitol on the binding was studied. Briefly, after immersing the Ab/PTBA-AuNPs-GR/GCE in pH 7.0 PBS containing 10 mM sorbitol for 60 min (Fig. 1S, curve c), the peak current increased and the Ret decreased significantly, which was due to the release of the specifically bound Ab. However, when this electrode was re-incubated in Ab solution in 0.1 M PBS (pH 7.0) for 60 min, the re-incubated electrode showed similar peak current and Ret with its initial value (Fig. 1S, curve d), demonstrating that Ab was re-immoblized on the PTBA-AuNPs-GR film. These results showed that the glycosylation sites of Ab specifically bound to the PTBA-AuNPs-GR nanocomposite as shown in Fig. 1, and the specific binding was reversible and the bound Ab could be split by sorbitol.

Fig. 1S. Cycle voltammograms (A) and Nyquist plots (B): (a) PTBA-AuNPs-GR/GCE, (b) Ab/PTBA-AuNPs-GR/GCE, (c) was (b) incubated in 10 mM sorbitol solution + 0.1M PBS (pH 7.0) for 60 min, (d) was (c) re-incubated in Ab solution for 60 min.

Optimization of immunoassay conditions

The experimental parameters, including incubation temperature and time which could affect performance of the immunosensor, were optimized by electrochemical impedance spectroscopy (EIS) measurement. The effect of the incubation temperature was examined from 20 to 55 C (Fig. 2S A). It was found that the Ret increased with the increasing temperature of up to 37 C and then decreased with the temperature going up. This result implied 37 C was the optimal incubation temperature as it was a favor of the physiological temperature about the immunoreaction. Since the incubation time would influence the degree of the specific recognition reaction between the antigen and antibody, the incubation time on Ret was also investigated (Fig. 2S B). The proposed immunosensor was first incubated in a standard ALV-J solution with 527 TCID50/mL for different times, and then tested by EIS. It was found that the Ret increased with the incubation time up to 60 min and after that it tended to level off. This result indicated the time of saturated binding of antibodies and antigens was 60 min. Therefore, the incubation time of 60 min was adopted in the subsequent work.

Fig. 2S. Effect of incubation temperature (A) and immuno-reaction time (B).

Specificity of the immunosensor

Fig. 3S. Investigation of the immunosensor specificity. The concentration of ALV-J and REV was 2635 TCID50/mL.

References

1. Zhang X, Wu Y, Tu Y, Liu S (2008) A reusable electrochemical immunosensor for carcinoembryonic antigenvia molecular recognition of glycoproteinantibody by phenylboronic acid self-assembly layer on gold. Analyst 133:485

2. De Guzman JM, Soper SA, McCarley RL (2010) Assessment of glycoprotein interactions with 4-[(2-aminoethyl)carbamoyl]phenylboronic acid surfaces using surface plasmon resonance spectroscopy. Anal Chem 82:8970

3. Liu S, Miller B, Chen A (2005) Phenylboronic acid self-assembled layer on glassy carbon electrode for recognition of glycoprotein peroxidase. Electrochem Commun 7:1232

1