## 4 Applications of Fe\({}_{3}\)O\({}_{4}\) Nanoparticles
### Biosensing  
Magnetic nanoparticle-based solutions for biosensing have been widely studied, and significant progress has been achieved [7]. In general, magnetic NPs have been used for labeling magnetic signals for various biological sensing purposes such as biomolecular detections based on ligand-receptor binding of biological substances. In these systems, the magnetic NPs are functionalized for facile conjugation with biomarkers. The magnetic signals can be detected by different magnetic detection techniques including spintronic sensors, nuclear magnetic resonance sensors, atomic magnetometer-based sensors, and superconducting quantum interference devices. Various types of magnetic responses can be used for detection depending on the sensing purposes and detection techniques such as relaxation, remnant magnetization, susceptibility, induced \({}^{1}\)H NMR, and frequency mixing. In-depth methods for functionalization of magnetic particles, strategies for bioconjugation, and designs of magnetic detection for magnetic particle-based biosensing platforms were summarized in the reviews of [PERSON] et al. [7] and [PERSON] et al. [134]. Due to their strong magnetic properties, biocompatibility, and facile functionalized surface, Fe\({}_{3}\)O\({}_{4}\) nanoparticles have been utilized in various biosensor platforms [135; 136; 137; 138]. Various biosensing platforms utilizing Fe\({}_{3}\)O\({}_{4}\) NPs were summarized in the review by [PERSON] et al. [139]. The magnetic properties, sizes, morphologies, and crystallinity of NPs are important factors defining the performance of Fe\({}_{3}\)O\({}_{4}\) nanoparticle-based sensing systems. [PERSON] and colleagues examined the biosensing performance of Fe\({}_{3}\)O\({}_{4}\) nanoparticles from two aspects: the sizes and geometries of NPs (cubes and spheres) [42; 43]. As an approach to study the size effect of NPs in biosensing, three different sizes of 120, 440, and 700 nm Fe\({}_{3}\)O\({}_{4}\) nanospheres with comparable degrees of crystallinity (grain size ~17-18 nm) were synthesized, coated with SiO\({}_{2}\), functionalized with poly(acrylic) acid (PAA) before conjugation with streptavidin, and utilized to detect specific proteins. As shown in Figure 11a, larger spherical NPs exhibit better magnetic signals and surprisingly better performance than commercial magnetic beads (2.8 \(\upmu\)m size) in an experiment detecting the well-known streptavidin-biotin interaction. In addition, the streptavidin-conjugated magnetic NPs were further combined with an exchange-induced remnant magnetization (EXIRM) platform for the specific detection of two immunoglobulins G (IgG1 and IgG2a) bound to Protein A. Figure 11b shows a schematic illustrating protein-immunoglobulin exchange detection using EXIRM measurements. Protein A has a stronger affinity for IgG2a than IgG1. Therefore, Protein A is immobilized covalently on the glass surface and then attaches to biotinylated IgG1, followed by the attachment of streptavidin-coated NPs to bound biotinylated IgG1. With stronger affinity for Protein A, IgG2a is then introduced, causes an exchange reaction, and influences the magnetization signal. Figure 11c shows that larger MPs exhibit larger slopes, and magnetic NPs with a size of 120 nm show equivalent signals to commercial magnetic beads. The combination of the larger size and strong magnetic properties of functionalized NPs increases the sensitivity by up to 9-fold compared with commercial magnetic beads and achieves a high detection specificity. In addition to the importance of appropriate sizes in biosensing, the morphology of NPs has been identified as an important parameter for efficient biosensing performance. [PERSON] et al. synthesized spherical and cubic Fe\({}_{3}\)O\({}_{4}\) NPs with multiple domains and sizes ranging from 100 to 225 nm and studied their crystallinity, magnetic properties, and biosensing performance using force-induced remnant magnetization force spectroscopy (FIRMS).