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