## 3 Results  
The purpose of the first set of experiments was to compare the _CNR_ and _number of pixel_ calculations to determine which method to use for the complete biosensing experiments. For that reason, the gold chips were examined, and images were recorded using an optical microscope (Nikon). Figure 4 shows some examples of the gold chips before and after the signal amplification was applied. The different concentrations of streptavidin-coated beads (base beads) were added on the gold chips, and the same amount of iron nano particles was injected into the microfluidic chamber (Figure 4). The number of pixels and _CNR_ values were calculated from the images recorded before and after the injection of iron nano particles. These experiments reveal that the amount of iron nano particles accumulated around the base beads depends on the number of base beads present on the gold surface. As the concentration of base beads was increased, the amount of nano particles that accumulated around the base beads also increased.  
By optimizing the flow parameters, concentrations, and magnetic field strength, the magnetic accumulation can be controlled and used to amplify the _CNR_. Minimum six-fold, and maximum nine-fold _CNR_ amplifications were calculated from the recorded images (Figure 4A). However, calculating the number of pixels for before and after the amplification step revealed only one or two-fold amplification (Figure 4B).  
Figure 4: (**A**) Optical micrographs of some of the gold surface before and after magnetic signal amplification; (**B**) The contrast-to-noise ratio (_CNR_) and calculated number of pixels of the images in (**A**) for the corresponding concentrations in the range of 0.0335–6.7 mg/mL. Values are mean \(\pm\) standard error of the mean.  
Figure 3: Impact of iron nano particles. (**A**) Optical micrographs of the gold surface before and after magnetic signal amplification; (**B**) Pixel intensity changes along the red reference line for red, green, and blue colors.  
Calculating \(CNR\) as a quantification method resulted in higher amplification ratios compared to calculating the pixel area of the beads on the surface. In light of the first set of experiments, calculating the \(CNR\) was the preferred quantification method for the streptavidin detection experiments.  
For the second set of experiments, streptavidin was chosen as the target molecule, and biotin-coated magnetic particles were used as the capture agents. Figure 5 shows some examples of the gold chips for the streptavidin detection experiments. After the images were recorded, they were processed to calculate the \(CNR\). Figure 5 demonstrates the dependence of \(CNR\) on the target concentration. As a result of streptavidin detection experiments based on magnetic dipole-dipole interaction and accumulation, a minimum eight-fold amplification in \(CNR\) was recorded.  
The exact same procedure was followed for the control experiments, except no streptavidin was added to the mixture tubes. Biotin-coated magnetic beads did not significantly bind to BBSA-functionalized gold chips, and patterns were not formed as expected (Figure 5A). After adding the same amount of iron nano particles, due to the magnetic field, some iron nano particles accumulated around each other and were not washed away. For the 0 streptavidin case, \(CNR\) was measured as 24, and the distribution of iron nano particles on the gold surface was random. In order to show the impact of base beads on the distribution of iron nano particles, the number of iron nano particles on the printed lines and non-printed lines were counted using image-processing tools (Matlab code and Image). For the 0 nM streptavidin case, the average difference in the number of pixels between the printed and non-printed lines was \(\Delta\)PN = 69, whereas the average difference \(\Delta\)PN was above 1500 for the 100 nM concentration (Figure 5B).