Antibody Immobilization

The next step in the preparation of the nanobiosensor probes involves the covalent immobilization of receptors onto the nanofiber tip. For antibody binding, several strategies can be used to retain the antibody at the sensing probe.

Figure 5. Scanning electron photograph of a nanofiber having the side-wall coated with silver.

Whatever procedure is involved, one requirement is that the antibody should retain its antigen-binding activity as much as possible. Perhaps the easiest procedure involves enclosure of the antibody in solution, within a semipermeable membrane cap, which fits over the end of the sensor [2]. However, this design is more complicated, and would increase the size of the tip of the nanosensor.

Antibodies can be immobilized onto the nanofiber probes by using a chemical immobilization method. The fiber is derivatized in 10% GOPS in H2O (v/v) at 90 °C for 3 h. The pH of the mixture is maintained below 3 with concentrated HCl (1 M). After derivatization, the fiber is washed in ethanol, and dried overnight in a vacuum oven at 105 °C. The fiber is then coated with silver as described previously. The derivatized fiber is activated in a solution of 100 mg/mL 1,1' carbonyldiimidazole (CDI) in acetonitrile for 20 min, followed by rinsing with acetonitrile and then phosphate-buffered saline (PBS). The fiber tip is then incubated in a 1.2 mg/mL antibody solution (PBS solvent) for four days at 4 °C, and then stored overnight in PBS to hydrolyze any unreacted sites. The fibers are then stored at 4 °C, with the antibody-immobilized tips stored in PBS. This procedure has been shown to maintain over 95% antibody activity [21].

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