We sterilized our glass substrate using plasma treatment and immersed the glass substrate in APTES solution for 1 hour. We washed the excess APTES, and added gold nanoparticles in a 2cm* 2cm marked surface area on the glass using a pipette. We incubated the gold nanoparticles for 9 hours and visualized the glass substrate under Atomic Force Microscope (AFM). Our results indicated that the gold nanoparticles were able to bind to the glass substrate.
Figure 1: a) APTES functionalized glass slide with AuNP solution b) AuNP functionalized surface after washing with MilliQ water.
To further quantitatively characterize the functionalized surface we used Atomic Force Microscope (AFM) (JPK NanoWizard® AFM, Quantitative ImagingTM mode). Figure 2 shows the topographical image of a 3.5 X 3.5 um region on the AuNP functionalized glass slide and the corresponding 3D map. The topographical map obtained from the AFM results indicated that the gold nanoparticles were able to bind to the glass substrate.
Figure 2: AFM image of gold nanoparticle bound glass substrate.
We added various concentrations of aptamer solution (TSO508) to our gold nanoparticle functionalized glass substrate: 2 uM, 25uM, and 50uM and incubated for 30 minutes at room temperature. We used a spectrophotometer (Genesys 10S Vis, Thermo Scientific) to obtain the absorbance spectra of the glass substrates in the visible light range. Our results indicated that the absorbance peaks increased (became higher) as the aptamer concentration increased. The optimum aptamer concentration was selected on the basis of peak intensity. We selected the concentration that resulted in the highest difference in peak absorbance values between AuNPs+ aptamer and bare AuNPs. The highest absorbance among the selected aptamer concentrations was at 50uM, so we decided to use 50uM aptamer for peptide detection.
Figure 3: Absorbance spectra for different aptamer concentrations: only AuNP (blue), AuNP+ 2uM aptamer (red), AuNP+25 uM aptamer (yellow), and AuNP + 50uM aptamer (purple)
We were also able to use AFM to visualize aptamer binding to the gold nanoparticles on the glass substrate. Figure 3 shows the topographical image of a 4 X 4 um region on the AuNP functionalized glass slide obtained using the same parameters as the AFM image previously mentioned. We hypothesize that the increase in the peak height and change in the topographical map is due to the binding of aptamers (50uM) to the AuNP functionalized glass substrate.
Figure 4: 3D AFM image of aptamers bound to gold nanoparticles on the glass substrate.
We prepared a peptide solution (AB-42) of 1000ng/mL, and diluted it to obtain various concentrations of amyloid beta: 500ng/mL, 100ng/mL, 50 ng/mL, 10ng/mL and 1 ng/mL to test the sensitivity of of our aptamer functionalized glass substrate. We added the peptide solutions to the glass substrate functionalized with 50uM aptamers and incubated it for 30 minutes at room temperature. We then measured the absorbance of all glass slides in the spectrophotometer. We used 1000ng/mL as our proof of concept and were able to show that the absorbance of the glass slide with AuNP+ aptamer + peptide changes from that of the glass slide with only AuNP+ aptamer. This indicates that the glass substrate is able to detect the presence of amyloid beta.
Figure 5: Absorbance spectra for bare AuNP (blue), AuNP+ 50uM aptamer (red), and AuNP+50uM aptamer + 1000 ng/mL amyloid beta peptide (yellow)
Once we found that the glass substrate was able to detect the peptide, we determined the limit of detection by measuring the absorbance for each peptide concentration under UV-Vis. We found out that as the concentration of peptide decreases, the absorbance peak becomes shorter. However, after approx. 10ng/mL, decreasing peptide concentration sharply decreases the difference in the peak absorbance value. We can therefore conclude that 10 ng/mL is our limit of detection.
Figure 6: Difference in peak absorbance value for different peptide concentrations.