## 4 Discussion and Conclusions  
In this paper, we described a new method of integrating magnetic particles and image processing for the detection of biomolecules. The micrometer-sized magnetic beads used in this study had two important functions: (1) as agents of immunomagnetic capture and separation; and (2) acting as base beads on a chip to attract added magnetic particles under an applied magnetic field. The PDMS stamp formed alternating lines of probe molecules on the surface, and these alternating lines allowed differential measurements on the same chip. It is known that immunomagnetic diffractometry for the detection of various important biomolecules related to diseases has shown potential as a simple and sensitive method [22; 25]. In this paper, we demonstrated that the magnetic accumulation-based signal amplification method can be easily integrated with the existing immunomagnetic diffractometry methods. Simply applying an external magnetic field and introducing magnetic particles as an additional step would result in six-fold signal enhancement. As a comparison, in our previous work [16], in order to reach four-to-five-fold signal amplification on a chip, temperature control, extra biomolecules, and extra time were needed. Figure 5: (**A**) Optical micrographs of some of the gold chips for streptavidin detection before and after magnetic signal amplification; (**B**) The calculated \(CNR\) and \(\Delta\)PN using the images in (**A**). Values are mean \(\pm\) standard error of the mean. The magnetic accumulation-based signal amplification method does not require expensive fluorescent and radioactive labels or time-consuming multiple signal enhancement steps. It is rapid, easy to implement, and inexpensive. There are several signal transduction methods used in biosensors. In this manuscript, we showed that image processing could be considered as a signal readout method for point-of-care devices. We calculated pixel areas and intensity values (_CNR_) to quantify the amount of micro and nano particle accumulation on the chip. This signal readout method can be considered as an alternative to laser-based methods, where a laser beam illuminates the diffraction gratings and spots of varying intensities are formed from the reflected light. An optical setup including photo detectors and lenses together with a voltage filter/amplifier are needed to measure the intensity of the spots [26]. Such a laser based measurement method is sensitive but bulky. Cell phone cameras and processing powers are improving from day-to-day, and as a result, cellphone microscopes are becoming popular to acquire images [27]. New strategies have been developed to utilize the improved hardware and software features of cell phones to use them for microscopy [28; 29]. Using images to calculate _CNR_ for quantification shows that it is possible to use mobile devices as signal read out devices for the chips having diffraction gratings. The _CNR_ measurements reveal that added iron nano particles accumulate around the base beads and act as contrast agents to enhance the signal. This signal enhancement on the chip has the potential to be integrated with simpler signal readout systems such as cellphone microscopy [30], and thus can be used in devices for point-of-care diagnostics. Figure 5 shows an image of the control experiment where 0 nM streptavidin was used, and biotin-coated magnetic beads were washed away from the surface. However, even though there was not a significant amount of base beads, some of the added iron nano particles for signal amplification were present on the surface due to the applied external magnetic field, resulting in an offset _CNR_ value of 24. Few iron nano particles were distributed randomly on the surface along the magnetic field lines. In order to show the impact of base beads on the distribution of iron nano particles, a differential measurement was performed by calculating the \(\Delta\)PN (Figure 5B bottom).