## 2 Materials and Methods
### Experimental Procedures  
#### 2.1.1 Immobilization of BBSA on Gold Chips  
After reviewing the literature [22], the incubation times and concentrations related to PDMS stamping were optimized by trial and error. The PDMS stamp that contains 20 \(\upmu\)m alternating lines was washed using an ultrasonic cleaner using 70% ethanol and rinsed using 70% ethanol followed by deionized (DI) water. The stamp was gently dried by using a stream of nitrogen gas. The gold chips of size 8.3 mm \(\times\) 3.8 mm were prepared by first cutting the gold-coated glass slides, cleaning  
Figure 2: Schematic of the complete biosensing experiments: streptavidin was used as the target molecule, biotin-coated beads captured and separated the target, signal amplification occurred in the microfluidic chamber using iron nano particles. with ethanol and DI water, followed by drying with nitrogen gas. Then, the PDMS stamp was inked with 2 mg/mL of BBSA, and BBSA solution was left on the stamp for 15 min before removing the excess solution from the stamp using a wick of tissue paper. The stamp was again dried using a gentle stream of nitrogen gas. The stamp was brought into contact with the gold chip, and a small force was applied to make a contact between the two surfaces. After waiting for 1 min, the stamp was removed. The gold chip was rinsed gently with PBS to remove the unbound BBSA. In order to passivate the rest of the surface that had no BBSA, the gold chips were covered with a solution of BSA (1 mg/mL) and incubated for 2 min before washing with DI water to remove the unbound BSA. 1.2 First Set of Experiments: Magnetic Accumulation of Iron Nano Particles around the Immobilized Streptavidin-Coated Magnetic Beads  
The purpose of the first experiments was to test the magnetic accumulation of iron nano particles around the immobilized streptavidin-coated magnetic beads, and to determine the quantification method for the measurement of the signal amplification. The streptavidin-coated beads were bound to the BBSA-coated gold chips. Under the applied external magnetic field, the introduced iron nano particles accumulate around the streptavidin-coated beads through magnetic dipole-dipole interaction. Then, images were recorded for quantification of the signal amplification. The concentration of streptavidin-coated magnetic beads was changed, and a fixed amount of iron nano particles was exposed to show that when more base beads were present on the surface, a higher number of iron nano particles accumulate. Figure 1 illustrates the steps followed for the first experiments. The gold chip containing the BBSA pattern was incubated with various concentrations (0.0335 mg/mL, 0.067 mg/mL, 0.67 mg/mL and 6.7 mg/mL) of streptavidin-coated magnetic beads. After 20 min of waiting, the gold chip was washed with DI water and dried. The gold chips with immobilized streptavidin-coated magnetic beads (base beads) were then placed in the fluidic chamber for signal amplification. Two permanent magnets were placed at both sides of the fluidic chamber. The gold chip was placed at exactly the center of the magnetic field where the base beads were magnetized. The fluidic chamber was placed under the microscope, as shown in Figure 1b. Iron nano particles were used as the added beads for signal amplification. After the optimization experiments, a concentration of 0.25 mg/mL of iron nano particles were placed in a syringe. The syringe was connected to the fluidic chamber through the inlet tube, and the iron nano particles were pumped to the fluidic chamber at a rate of 100 \(\upmu\)L /min for 3 min. The interaction of the iron nano particles with base beads was observed, and images both before and after amplification were recorded using the optical microscope.