## 1 Introduction  
The advances in micro/nano fabrication methods have led to the development of various biosensing platforms using magnetic particles. Micro/nano-cantilevers [1, 2], stripe/wires [3, 4], and pores [5, 6] are some of the platforms. There are also magnetic particle-based Food and Drug Admisnistration (FDA) approved isolation technologies available in the market [7], and in the literature, many microfluidic devices have been developed for the separation of various biomolecules [4, 8, 9]. After the separation step, there are various techniques to read out signal from magnetic particles; electrochemical detection [10], magnetometer [11], magnetic remanence [12], magnetoresistance [13, 14], hall sensors [15], optical methods such as laser diffraction [16], optical light microscope [17], and fluorescent detection [18]. Common approaches to amplifying the signal from magnetic particle-based biosensors are the use of additional magnetic [16] or nonmagnetic particles, such as labels coated with biomolecules to either bind to a target molecule [19] or bind to magnetic beads [18, 20]. In a companion paper [21] we introduced an alternative signal amplification method based on magnetic bead accumulation. Briefly, in this method, immunomagnetic beads are first used to capture and separate the target molecules; then, under an external magnetic field, added uncoated magnetic particles form flocs around the immunomagnetic beads due to magnetic dipole-dipole interactions. These accumulations increase the total volume and area of the magnetic particles, thus resulting in increased pixel area in the recorded images. This method does not require biomolecular interactions for accumulation/signal amplification, and this feature brings some advantages, such as time and cost effectiveness and independence from temperature and pH. In the previous work, we experimentally investigated accumulation dynamics in detail; in this manuscript, we demonstrate step-by-step the application of this amplification method to a complete biosensing scenario, including target capture, separation, and specific binding to surface receptors. We also explore various quantification methods in this manuscript, such as calculating pixel numbers, pixel intensity changes, and differential pixel numbers from the recorded images to measure the signal amplification. The first set of experiments was performed to investigate the accumulation of iron nano particles around the magnetic particles already immobilized on the gold chip in a microfluidic chamber, and to investigate the different quantification methods using the recorded images. For this purpose, periodic patterns of biotin-coupled bovine serum albumin (BBSA) were micro contact-printed on gold chips using a polydimethylsiloxane (PDMS) stamp. The uncoated parts of the gold surface were passivated by using bovine serum albumin (BSA). Then, the suspension of streptavidin-coated immunomagnetic particles was transferred onto the gold chip containing BBSA micropatterns for the in situ assembly of gratings. Streptavidin-coated beads were observed to specifically attach to the BBSA micropatterns, while unbound beads were removed by rinsing with phosphate-buffered saline (PBS). Then, the gold chip was placed in a microfluidic chamber made of polymethyl methacrylate (PMMA) to implement magnetic accumulation-based signal amplification. The iron nano particles were injected in the microfluidic channel using a syringe pump, while the external magnetic field was applied perpendicular to fluid flow using permanent magnets (Figure 1). In the first set of experiments, the streptavidin-coated magnetic beads were immobilized on the printed pattern and acted as base beads to attract iron nanoparticles under an applied magnetic field for signal amplification. The second set of experiments was performed to demonstrate a complete biosensing; streptavidin was chosen as the model target molecule, and biotin-coated magnetic beads were used as the capture and separation agents.