Carbon Nanotubes
CNTs used in the experiment is as-grown-horizontally aligned, thread-like, and double-walled (T-DW). It was obtained from Lijie Ci during his post-doc fellow at Ajayan research group, Rice University, Houston, TX USA with permission from Prof. Pulickel M. Ajayan. The T-DWNT was synthesized and characterized as described in Ci et al. (2002) [27]. The peeled T-DWNT strand diameter was approximately 1–2 μm as shown in Figure 8.
Chemicals and microorganisms
All chemicals used in the experiment were analytical grade. N-hydroxysuccinimide (NHS), bovine serum albumin (BSA) and 2-morpholineethanesulfonic acid monohydrate (MES) were purchased from Sigma-Aldrich (USA). N-(−3-Dimethylaminopropyl)-N-ethyl-carbodiimide hydrochloride (EDC) was purchased from Fluka (USA). All microbiological media were obtained from Difco (USA). Rabbit anti-Salmonella spp. monoclonal antibody was purchased from Biodesign International (USA). Glycine, all buffer reagents, and other chemicals were obtained from Merck (USA). Milli-Q water and double-distilled water were used throughout the experiment. Salmonella typhimurium and Escherichia coli were obtained from BioSensor Lab, King Mongkut’s University of Technology Thonburi, Bangkok, Thailand.
Bacterial suspension preparation
The active S. typhimurium cells were grown in nutrient broth (NB) and incubated at 30 C and at 150 rpm until an approximately cell number of 109 cells/mL was obtained. The cell suspension was then harvested, centrifuged, washed with phosphate buffer saline (PBS) solution (0.10 M pH 7.4), and re-suspended with the same volume of the original solution to obtain the required cell concentrations for the measurement.
Optimization of the anti-Salmonellaspp antibody immunoassay
Antibody immobilization and microbial incorporation
A CNT strand was peeled out from the CNT forest and the electrode fabrication was performed as stated in the literature [13, 14]. The anti-Salmonella monoclonal antibody (MAb) was covalently immobilized onto the T-DW electrode by using the EDC crosslink method with some modifications. Briefly, 10 μL of a mixture of 400 mM EDC and 100 mM NHS in MES buffer solution (0.1 M, pH 6.0) was dropped onto the T-DWNT electrode surface and incubated for 1 hr at room temperature. Then, the electrode was washed with PBS buffer solution (0.1 M, pH 7.2) three times and dried at room temperature. Next, 10 μL of the MAb solution (10 μg/mL in deionized water) was dropped to cover the whole area of the electrode, and then it was incubated for another 2 hrs at room temperature. The electrode was then passed three times through washing steps using first a PBS (0.1 M, pH 7.2) containing 0.05% (v/v), and then a Tween 20 (PBS-T), followed by deionized water. Then, 10 μL of phosphate buffer saline (PBS) solution (0.1 M, pH 7.2) containing 2% BSA (w/v) was dropped onto the electrode to block the area without the MAb attachment and the same washing steps were conducted after 30 min of incubation at room temperature. The MAb-DW electrode was stored at 4 C in a humid condition until it was used. For the microbial detection, the bacterial cell suspension was dropped on to the electrode, and it was incubated at room temperature for 2 hrs before being put through the washing steps. The electrode was then used for electrochemical characterization.
Electrochemical characterization of the MAb-DW electrode
Cyclic voltammetry (CV) was employed in a standard three-electrode system. with the MAb-DW electrode, Ag/AgCl, and Pt disk as working, reference and counter electrodes, respectively. The experiment was conducted on PGSTAT12 by using the GPES software acquisition data. This was done to characterize the electrochemical properties of the MAb-DW electrode. The potential was cycled from −1.0 to 1.0 V vs. Ag/AgCl at a scan rate of 20 mV/sec in citrate phosphate buffer (0.05 M, pH 5.5) working solution at a fixed MAb concentration (10 μg/mL in deionized water). This was performed in order to obtain an optimum potential range for further amperometric sensing investigations. The CV experiments were sequentially conducted at the same electrode before and after MAb modification, after blocking with BSA (MAg-DW electrode), and also after the S. typhimurium cells (109 CFU/mL) were attached to the electrode (cells-MAg-DW electrode).
Immunoassay at the MAb-DW electrode optimization
Figure 9 shows the schematic of the electrode platform for S. typhimurium assay and how the signal was obtained.
Working potential optimization
Chronoamperometry (CA) was conducted at the MAb-DW electrode, without the cells incorporated, at a fixed concentration of MAb 10 μg/mL with various working potentials obtained from the CV measurements. These measurements were used to obtain the optimum working potential for cell detection. In the CA measurement, the potential was fixed at 0 V for 100 sec, then stepped to the working potential.for another 250 sec, in which the current response was observed.
MAb concentration optimization
The signal at the electrode assembled with various MAb concentrations (10, 25, 50 and 100 μg/mL) with and without the attached S. typhimurium cells (109 CFU/mL) was observed at the electrode in the citrate phosphate buffer (0.05 M, pH 5.5) working solution. The MAb concentration provided the higher signal and was subsequently further used for sensitivity testing of S. typhimurium cells.
The MAb-DW electrode sensitivity testing
Different Salmonella cell concentrations (10-109 CFU/mL) were immobilized on to the MAb-DW electrode and the CA measurements were conducted to obtain the detection range at the electrode. Non-specific adsorption of the Salmonella cells on the BSA-incorporated DWNT electrode was also tested. Escherichia coli (107 CFU/mL) was also used for specificity testing at the electrode.