Nanoparticles Supported-Methylene Blue Labels and Multiwall Carbon Nanotubes-Based Highly Sensitive Electrochemical Immunosensor

Experimental

Reagents and Apparatus. Chitosan (F.W. 340 g), methylene blue (MB), human immunoglobulin G (hIgG) antigen, monoclonal anti-human immunoglobulin G (anti-hIgG) (produced in mouse), prostate specific antigen (PSA), horseradish peroxidase (HRP), carcinoembryonic antigen (CEA), human α-Thrombin (TB), and gold nanoparticles (AuNPs, 4-5 nm in diameter) were purchased from sigma Co. (USA). The multiwall carbon nanotubes (MWCNTs) were obtained from JEIO Co. Korea and purified and shortened according to our previous report.12 All other chemicals were of extra pure analytical grade and used without further purification. PBS was prepared by mixing Na2HPO4 and NaH2PO4 with appropriate amount and pH 7.4 was adjusted with a pH meter.

The EIS and SWV experiments were performed using CHI 660 (CH Instruments Inc. USA) and ZAHNER ZENNIUM instrument (Serial no. 40282, Germany) electrochemical work stations, respectively. In SWV, the potential was scanned from +0.3 to −0.5 V with 5 mV pulse height, 50 mV amplitude, and 20 Hz frequency. SWV experiments were performed in an oxygen free PBS buffer solution (pH 7.4) with constant purging of N2 gas. The SEM and TEM images were obtained using scanning electron microscope (Model JSM- 7000F JEOL, Japan) and high resolution transmission electron microscope (Model JEM-2100, JEOL, Japan), respectively. UV-vis experiments were carried out with a UV-vis spectrophotometer (model UV-1800, Shimadzu, Japan).

Preparation of the Au/MWCNTs/CS/hIgG Immuno- Sensor Probe. The immunosensor probe was fabricated by sequentially dropping a 3 μL of purified and shortened MWCNTs and 10 μL of CS (7.5 mg/mL CS in the mixture of 1% HCl and Tris (NH2C(CH2OH)3·HCl) buffer, pH 6) solutions on a polished Au electrode. After drying, the Au/ MWCNTs/CS modified electrodes were dipped into a glutaraldehyde solution (25%) for 12 h followed by incubated in a PBS solution containing 1 ng/mL hIgG (pH 7.4) for 12 h. By these steps, hIgG were covalently immobilized on the CS film of Au/MWCNTs/CS modified electrode through the glutaraldehyde cross-linking. The modified electrode was carefully rinsed with a PBS solution (pH 7.4) for removing any unbound free hIgG and was blocked with a 0.1% BSA solution for 1 h.

Preparation of the anti-hIgG/AuNPs/MB Redox Label. The anti-hIgG/AuNPs/MB redox label was prepared by incubating the AuNPs in the PBS solutions of MB (0.1 mM) and anti-hIgG (1000 times diluted) for 12 h at 4 ℃. The resulting conjugate solution was centrifuged. After discarding the supernatant, the conjugate was carefully washed with PBS (pH 7.4) for three times in order to remove unbound anti-hIgG, MB, and AuNPs. The anti-hIgG and MB were attached on the surface of AuNPs through the charge interaction between the positive charges of MB and anti-hIgG and the negative charge of AuNPs. Finally the anti-hIgG/ AuNPs/MB redox label was blocked by using a 0.1% BSA solution for 1 h and was used in the subsequent experiments.