The key feature of this protocol, it can be applied on a wide range of iron-iron oxide nanomaterial. Also, it can be applied on multi-segmented nanomaterial, such as iron-gold nanowires. This protocol can be performed safely in any standard biological lab.
The main advantages of this biofunctionalization technique is the ability to produce a specifically targeted nanomaterial based on a strong covalent bond between the nanowire and the antibodies. Begin by cutting the aluminum discs into small pieces with a single-edge blade and a small hammer. Place the cut pieces into a two-milliliter tube and fill it with one milliliter of one-molar sodium hydroxide, making sure all aluminum pieces are covered.
Leave the tube at room temperature in a chemical fume hood for 30 minutes. After the incubation, remove the aluminum pieces with a tweezer and keep the released nanowires in sodium hydroxide for an additional 30 minutes. Place the tube in a magnetic dry rack, wait two minutes, and replace the solution with fresh sodium hydroxide.
Sonicate the two-milliliter tube with the nanowires for 30 seconds and leave it for one hour inside a chemical fume hood. Repeat this step at least four times. When finished, place the two-milliliter tube in the magnetic rack, discard the sodium hydroxide, and replace it with one milliliter of absolute ethanol.
Sonicate the nanowires for 30 seconds, place the tube in the magnetic rack, and replace the ethanol. Repeat the ethanol wash at least four times. After washing the nanowires, transfer them to a five-milliliter glass tube with a one-milliliter pipette.
Add 100 microliters of APTES to the nanowire solution and vortex the glass tube for 20 seconds. Place the tube in an ultrasonic bath and sonicate it for one hour. Then take the tube out of the bath and add 400 microliters of deionized water and 20 microliters of one-molar sodium hydroxide.
Repeat the one-hour sonication. After taking the glass tube out of the ultrasonic bath, place it next to a magnet to collect the nanowires. Replace the supernatant with one milliliter of fresh absolute ethanol and sonicate the tube for 10 seconds.
Repeat the ethanol wash and sonication twice. Dissolve 0.4 milligrams of EDC and 1.1 milligrams of Sulfo-NHS and one milliliter of MES in a two-milliliter tube. In another two-milliliter tube, add 960 microliters of 0.1-molar PBS and add 30 microliters of anti-CD44 antibody to it.
Next, add 10 microliters of the previously prepared EDC/Sulfo-NHS mixture to this tube. Place the antibody tube in a shaker and shake it at 10 times G for 15 minutes at room temperature. Meanwhile, place a magnet next to the tube with the APTES-coated nanowires for two minutes, discard the ethanol, and replace it with PBS.
Sonicate the nanowires for 10 seconds. After the last wash with PBS, collect the nanowires and transfer the activated antibody solution to the nanowires'tube. Then sonicate it for 10 seconds.
Replace the tube in the rotator overnight at four degree Celsius. On the next day, collect the nanowires with the magnet and discard the supernatant. Add one milliliter of 2%BSA and incubate the tube for one hour at four degrees Celsius to block the reaction.
Electron energy loss spectroscopy or EELS maps can be used to visualize the silicon atoms on the nanowire surface and confirm the presence of the APTES coating. On the non-coated nanowires, the iron atoms appear dark blue and the iron and oxygen mixed atoms are light blue. The corresponding EELS mapping of non-coated nanowires shows a higher intensity of iron versus oxygen in the center than on the surface, indicating an iron-iron oxide structure.
The mapping demonstrated that the iron oxide layer consists of more iron(II, III)oxide than iron(III)oxide. The antigenicity of the attached antibodies was confirmed using the immunoprecipitation and western blot assays. The BCA assay was used to quantify the antibodies'number on the nanowires.
Measurement of zeta potential was used to elucidate surface functionalization. The terminal amine group on APTES reduced the negative charge compared to non-coated nanowires. Antibody functionalization further reduced the charge.
The specific cell targeting of the antibody-functionalized nanowires was confirmed using confocal microscopy. Figures A and B represent the brightfield microscopy images of specimen C and D respectively. The color red corresponds to the depiction of the cell membrane, blue represents the nucleus, and the green clusters depict the presence of attached, targeted CD44 nanowires.
Furthermore, the cell viability assay was used to verify that the non-coated, APTES-coated, and antibody-coated nanowires were biocompatible. In the APTES process coating, it's very important to add the deionized water before the sodium hydroxide solution to enhance the bonding between the APTES molecule and the nanowires. Also, during the biofunctionalization process, it's necessary to wash the nanowire with PBS buffer a couple of times to enhance the covalent bond between the antibody and the nanowires.
