MATERIALS AND METHODS
Materials. Microscope glass slides (15 mm × 20 mm) were received from Mesostate (Taiwan). Dimethyloctadecyl[3-(trimethoxysilyl)propyl]ammonium chloride (DMOAP) as the aligning agent dissolved in methanol was purchased from Sigma–Aldrich. Dimethyl sulfoxide (DMSO) and 4-Cyano-4′-pentylbiphenyl (5CB) were received from Merck. Anti-beta Amyloid - 42 antibody (ab10148) was obtained from Abcam. Amyloid β Protein Fragment 1-42 (A9810) was purchased from Sigma-Aldrich.
Instruments. Optical observations were performed on Nikon CIPOL-50X-1000X EPI-DIA Polarized Optical Microscope (USA). Reflection analysis were performed on Ocean Optics Spectrometer USB2000+(UK).
Preparation of DMOAP-Coated Microscope Glass Slide and Formation of the LC Film. Glass slides were cleaned in distilled water (deionized), IPA (Isopropyl alcohol ) and ethanol by ultrasonication for 15 min at room temperature. The glass slides were dried under the stream nitrogen and then baked at 80°C for 15 min. After the cleaning process was completed, the slides were immersed in an aqueous solution of 1% (v/v) DMOAP for 15, 30 and 60 minutes followed by rinsing with DI water for removing the excess DMOAP. Subsequently, the glass slides were dried again with nitrogen and baked at 80°C for 15 min. The slides left in the oven during the day to cool down to room temperature. The purpose of this step is to provide the homeotropic alignment of LC molecules on the glass slide. The next step is the formation of LC films. Prior to the addition of the Aβ42 antibody on the DMOAP-coated glass slides, 2 µl 5CB was dropped on these glass slides to observe the alignment of LC molecules which are intended to provide a homeotropic alignment on the glass surface with the help of DMOAP. Thus, a LC film was successfully formed on DMOAP coated glass slide.
Immobilization of Amyloid beta-42(Aβ42) Antibodies and Formation of Amyloid beta-42 (Aβ42) Antibody-Antigen Immunocomplex. Aβ42 antibody and Aβ42 peptide of specific concentrations were prepared. In the first step, aqueous solutions of the Aβ42 antibody were diluted with DI water. The diluted Aβ42 antibody solution was immobilized by dispensing 1 µl droplet of Aβ42 antibody (at 100 µg/ml, 50 µg/ml, 25 µg/ml, 20 µg/ml, 10 µg/ml, 1 µg/ml) on the formed LC films. These antibodies were incubated overnight at room temperature. Afterward, Aβ42 peptide was firstly dissolved in dimethyl sulfur oxide (DMSO) at a concentration of 1mg/ml. The particular Aβ42 peptide concentration was prepared by diluting in 10 mM sodium phosphate buffer (Na-PB)(pH 7.5). Then, the diluted Aβ42 peptide solution (25 pg/ml, 50 pg/ml, 100 pg/ml) was reacted with the determined antibody concentrations for 30 minutes, 60 minutes and 90 minutes in order to determine the optimum antibody concentration. The optimum time was determined as 1.5 hours. As a result of these steps, an Aβ42 antibody-antigen immunocomplex was formed as seen in Figure 1. After the optimum antibody concentration was selected, different Aβ42 peptide concentrations of 1000 pg/ml, 500 pg/ml, 200 pg/ml, 100 pg/ml, 50 pg/ml, 25 pg/ml, 20 pg/ml, 15 pg/ml, 10 pg/ml, 5 pg/ml, 1 pg/ml were reacted with the optimum antibody concentrations, respectively.
Optical Analysis. Optical textures were determined via a polarizing optical microscope and changes in LC orientation resulting from antigen-antibody binding were observed using an optically polarizing light microscope. All changes in the structure of LC were captured by a CMOS (complementary metal-oxide-semiconductor) camera attached to the microscope using a 10X objective lens (Nikon). The reflectance spectra of all samples were determined using an Ocean Optics spectrometer. Spectral experiments were carried out at room temperature in the range of 200-900 nm of wavelength. The experimental results showed that all the samples have a maximum reflectance peak at 547 nm.
RESULTS AND DISCUSSIONMicroscopic Analysis