Investigation of DMOAP coated Glasses
To observe the success of DMOAP coatings, 2 µl of 5CB liquid crystal was dropped onto DMOAP- coated microscope glass slide. Figure. 2 a. shows the LC structure can be seen on the non-coated DMOAP glass slide. Figure 2 b, c, d show the LC structure cannot be seen on the DMOAP coated glass slides, since LC molecules have homeotropic orientation through the DMOAP coating.
Determination of Optimum Anti-amyloid beta-42 (Aβ42) Antibody Concentration
In order to determine the optimum antibody concentration, six different samples, which are shown in Table 1, were prepared and then each sample was dropped onto the LC film surface. Before Aβ42 antibodies interacted with Aβ42 peptides, DMSO was dropped onto the Aβ42 antibody surface. No effect of DMSO on the orientation of liquid crystals has been observed. Subsequently, antibodies at these six different concentrations were interacted with three Aβ42 peptide concentrations of 100 pg/ml, 50 pg/ml and 25 pg/ml, respectively.
The formed antibody-antigen immune complex leads to a change in the orientation LCs from homeotropic to a random one. The changes corresponding to the random orientation of the LC molecules were observed by a polarized optical microscope (POM). The POM images showed that a notable change when the samples including the Aβ42 peptide concentrations of 100 pg/ml, 50 pg/ml, 25 pg/ml interacted with 100 µg/ml, 50 µg/ml and 25 µg/ml Aβ42 antibody concentrations as seen in Figure. 3, Figure. 4, Figure 5. The POM images prove that 25 µg/ml Aβ42 antibody concentration can be selected (Sample 3) as the optimum antibody concentration.
Then the selected optimum antibody concentration was interacted with different Aβ42 peptide concentrations as 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, and 1 pg/ml, which are listed in Table 2, to determine the detection limit of the biosensor.
Detection of Amyloid beta-42 (Aβ42) Peptide Concentration
1 µl of 25 μg∕ml anti- Aβ42 antibody was immobilized on the glass with DMOAP alignment layer and then interacted with various concentrations of Aβ42 peptide for 1.5 hours at room temperature. Figure 6 and Figure 7 show that the changes in the orientation of LC direction determined via a POM. In the absence of peptides or antibodies, the images as seen in Figure 6a-e & Figure 7f-k were black because the alignment of nematic LCs was perpendicular to the glass surface with the DMOAP monolayer. Figure. 6a1-e1 & Figure 7f1-k1 show that the immobilized Aβ42 antibodies were insufficient to change the homeotropic alignment of LCs. As the Aβ42 peptide concentration was gradually increased, the number of antigen-antibody immunocomplexes also increased, which caused significant orientational changes in LCs alignment due to their larger size of the immunocomplex (Figure 6a2-e2 & Figure 7f2-k2). Therefore, the optical textures of LCs have changed significantly to reflect the amount of available immunocomplexes. This LC - based optical biosensor was examined to determine the minimum concentration of Aβ42 peptide that could induce an orientational change of LCs. The biosensor was incubated with Aβ42 peptide concentrations in the range of 1000 pg/ml - 1 pg/ml. The change in LC optical textures was considerable when the biosensor was incubated with all the concentrations of Aβ42 peptide as shown in Fig. 6 and Fig. 7. Moreover, these results indicate that a minimum concentration of 1 pg/ml Aβ42 peptide was required to change of LCs orientational on the surface. Therefore, the LOD of this LC-based optical biosensor was determined as 1 pg/ml Aβ42 peptide. Dai and co-workers fabricated that simple in vitro biosensor for the detection of β-amyloid 42 in phosphate-buffered saline (PBS) and undiluted human serum. The concentration range of β-amyloid 42 in this study was from 0.0675 µg/ml to 0.5 µg/ml. Yu and co-workers have been developed gelsolin-bound-Aβ1–40/1–42 detection assay for detecting level variations of Aβ1–40/1–42 associated with AD progress. Compared to other studies, a very low detection limit was achieved in our optical sensor.