The main objective of this project was to create a diagnostic platform to detect Amyloid beta peptides in relevant clinical concentrations. The team hypothesized that by using a shift in the total absorbance and the peak absorbance wavelength of gold nanoparticles using a spectrophotometer (Genesys 10S Vis, Thermo Scientific).
In our project, we were able to use the Atomic Force Microscopy (AFM) to characterize our functionalized glass substrate and visualize the binding of gold nanoparticles as well as aptamers. AFM is a high resolution scanning probe microscopy with a demonstrated resolution in the order of nanometer. In AFM, a sharp tip (a mechanical probe) scans the surface using a feedback loop to adjust parameters needed to image a surface. Hence, atomic forces are used to map the tip-sample interaction and develop an image of the surface.
Image 1 shows the binding of gold nanoparticles to the glass substrate. The shift of color from dark red to yellow indicates an increase in the height of the glass surface. The yellow spots therefore indicate the presence of gold nanoparticles (10nm) that raised the surface height by ~10nm.
Figure 1: AFM image of gold nanoparticle bound glass substrate
Image 2 shows the binding of aptamer (50uM) to the gold nanoparticles on the glass surface. The increase of height of the glass surface by 60 nm indicates that aptamers (with a theoretical height of ~50 nm) were able to bind to gold nanoparticles (~10nm) on the glass surface that together make up 60 nm. Together, these results confirm the functionalization of our glass substrate with gold nanoparticles and aptamers.
Figure 2: 3D AFM image of aptamers bound to gold nanoparticles on the glass substrate.
The initial absorbance (only AuNPs on the slide) was 0.038 at 519 nm but once the aptamers were added, this peak shifted to 533 nm and the total absorbance increased to 0.042. Amyloid beta (the peptide of interest) was added to the slide and incubated for 30 mins. Once this was done, the slide was dried and the new absorbance was measured. The spectrum then shifted upwards again giving an absorbance of 0.077.
As this was done with 1000ng/ml peptide concentration, we needed to establish that this model will work with lower concentrations similar to that in patients affected by Alzheimer’s. We lowered the peptide concentrations till we couldn’t identify a significant change from the initial values (AuNPs and Aptamers without peptides). We concluded that the limit of detection for our method is 10 ng/ml which can further be optimized to detect Alzheimer’s in patients that don’t yet show conclusive clinical symptoms. This is clearly highlighted in the figure below. The y axis values show the difference in absorbance when the peptide binds to the aptamers. The sharp spike around 10 ng/ml indicates that concentrations in this range or above can be detected using this method while those below it will require further optimization.
Figure 3: Graph of change in absorbance against concentration of amyloid-42 peptide. The increase in absorbance from its unbound state is used to determine the presence of the peptide in the sample.