Magnetic immunoassay (MIA) is a novel type of diagnostic assay, which utilizes MNPs as labels instead of conventional radioisotopes (radioimmunoassay), fluorescent dyes (fluorescent immunoassay), substrates and enzymes (enzyme-linked immunosorbent assay, ELISA). MNPs are usually conjugated with an antigen or antibody for the recognition of interested molecules. The binding between MNPs and interested molecules causes the clustering of MNPs, which results in an increase of magnetic moment for the detection [22, 23]. For example, Lee et al. [23] developed a handheld diagnostic magnetic resonance (DMR) system, which consisted of planar microcoils, microfluidic channels, and a portable magnet. It used T₂ relaxation time as detection signal, and could be performed in turbid samples (e.g., blood, urine, and sputum) with few or no preparation steps. In a recent report, they determined the accuracy of the DMR system by comparing its performance to a large benchtop NMR relaxometer. In addition to the smaller sample volume required (~5 μL for DMR vs. 300 μL for NMR relaxometer), the DMR system showed a mass detection limit improvement of two orders of magnitude (1 ng vs. 80 ng for avidin detection). This technology could also be extended for the detection of bacteria, cancer cells, and protein biomarkers with a high mass detection sensitivity (more than 800-fold improvement relative to a benchtop relaxometer). The sensitivity could be further improved by preparing new classes of water-soluble MNPs with higher magnetization [23].

Colorimetric immunoassay quantifies the analytes through absorbance generated from a color reaction between substrates and enzymes. One of the most widely used enzymes is peroxidase, which catalyzes the oxidation of organic substrates and produces a color change for detection. Peroxidase enzymes and their mimics contain Fe²⁺ and Fe³⁺ in the reaction centers, which is essential for the catalytic activity. Although MNPs like Fe₃O₄ NPs have been conjugated with horseradish peroxidase (HRP) to introduce peroxidase activity in a number of applications including commercially available magnetic ELISA kits, their peroxidase-like activity has been ignored for a long time. Until recently, Fe₃O₄ NPs have been reported to possess intrinsic peroxidase-like activity. In the presence of H₂O₂, all different sizes (30, 150, and 300 nm) of Fe₃O₄ MNPs catalyzed the reaction and produced a blue color for substrate 3,3,5,5-tetramethylbenzidine (TMB), a brown color for diazoaminobenzene (DAB), and an orange color for o-phenylenediamine (OPD) [24]. If AH represents the substrate, which is a hydrogen donor, the mechanism of the catalytic reaction could be written as follow:

Through a series of studies, the peroxidase-like activity of Fe₃O₄ MNPs was found to be H₂O₂, pH, and temperature dependent. Meanwhile, the reaction followed Michaelis–Menten kinetics. Two formats of immunoassay based on this concept have been performed to detect hepatitis B virus surface antigen and cardiac troponin I (a biomarker for myocardial infarction) [24].

More recently, the peroxidase-like activity of Fe₃O₄ MNPs was used for targeting and visualizing tumor tissues. Magnetoferritin (M-HFn) NPs were prepared by encapsulating iron oxide NPs inside a HFn shell. The schematic process is shown in Fig. 21.2a. TEM images (Fig. 21.2b–d) are shown a core–shell structure with a 4.7-nm core of iron oxide (Fig. 21.2e). The color reactions are shown in Fig. 21.2f, g. The HFn protein shell could specifically bind to tumor cells that overexpressed transferring receptor 1 (TfR1). These M-HFn NPs allowed to the visualization of the tumor tissue through the peroxidase-like activity of iron oxide core without any additional recognition ligands on their surface. This strategy simplified the modification process of conventional NPs and prevented nonspecific binding induced by an excess of ligands on the NP surface; 474 clinical specimens from patients with nine types of cancers were examined. They confirmed that M-HFn NPs could distinguish cancerous cells from normal cells with a sensitivity (positive/cases) of 98 % and a specificity (negative/cases) of 95 % [25].

MRI is a noninvasive imaging modality that has been widely used in clinical diagnosis [26]. This technique is based on the property that the magnetization of hydrogen protons in human body will be aligned around an applied external magnetic field. The presence of MNPs shortens the spin–spin relaxation time and thus produces a negative (decreased) signal in T₂– and T₂*-weighted MR images [27]. Therefore, by labeling the interested cells/tissues with MNPs, we can visualize them noninvasively. So far, MNPs have been applied as MRI contrast agents to improve the sensitivity of detection and diagnosis of major diseases including cancers, cardiovascular diseases, and diabetes which in total account for nearly 2 of every 3 deaths in the USA—close to 1.5 million people in the year 2001.

In cancer prognosis, the status of lymph node is an independent adverse prognostic factor. The enlargement of lymph node usually indicates the potential malignancy or metastasis [28]. Given that intravenously or subcutaneously injected MNPs would be taken up by lymph nodes by means of interstitial-lymphatic fluid transport, we can label lymph nodes with MNPs for cancer diagnosis with MRI. In 2003, 80 patients with presurgical clinical stages T1, T2, or T3 prostate cancer were examined by MRI before and 24 h after the intravenous administration of MNPs. The imaging results were correlated with histological findings; 71.4 % of nodes did not fulfill the usual imaging criteria for malignancy. MNPs have effectively identified small (with a diameter of 5–10 mm) and otherwise undetectable lymph node metastases in patients with prostate cancer [28].

Besides cancer, MNPs are also used in the diagnosis of the cardiovascular diseases such as myocardial injury, atherosclerosis, and vascular disease [29]. The wash-in kinetics of MNPs, which are normally confined to the intravascular space, can be used to demarcate the area of myocardium at risk. The hyperacute stage of myocardial injury increases the capillary permeability and hyperemia, which finally enhances the contrast of the injured areas [30]. To prevent the wash-out of MNPs from acutely injured myocardium, an active ligand binding which achieves specific targeted imaging is also highly promising [31]. Atherosclerosis is another cardiovascular condition in which an artery wall thickens as a result of the accumulation of fatty materials and formation of multiple plaques within the arteries. Long-circulating MNPs are able to penetrate an atherosclerotic plaque and then taken up by the macrophage [32]. Thus, plaque macrophage content could be gauged by MRI for atherosclerosis diagnosis. In addition, MNPs labeled with linear peptides (screened by phage display) can specifically bind to the vascular adhesion molecule-1 (VCAM-1), which is expressed on vascular endothelium. These VCAM-1-targeted MNPs have been demonstrated as sensitive contrast agents for detection of VCAM-1 expression in the aortic roots of statin-treated mice [33].

Diabetes is another major health and development challenge of the twenty-first century. Worldwide, there are already more than 360 million people with diabetes and another 280 million at identifiably high risk of developing diabetes. One major type of diabetes is type 1 diabetes (3–5 % of diabetes globally) is an autoimmune disease that destroys the insulin-producing cells (beta cells) of the pancreas. The initial immune infiltration, termed insulitis, starts many years before the development of type 1 diabetes (T1D) and progresses slowly until a critical mass of beta cells has been annihilated. In the progress of immune infiltration, lymphocytes migrate to the pancreas and destroy beta cells. Thus, pancreatic biopsy is one approach to diagnose insulitis but has been resisted by patients due to its invasiveness. A more attractive alternative to biopsy is provided by noninvasive MRI imaging, in which microvascular leakage was taken as an indicator of inflammation [34]. Specifically, Denis and colleagues developed long-circulating magnetofluorescent NPs with half-lives of over 10 h in blood vessel for both fluorescent and MR imaging, which could follow the microvascular changes accompanying inflammation. Results from mouse showed a positive correlation in magnetofluorescent NPs accumulation in the pancreas and insulitis aggressivity [34]. With this technology, the same group could predict the onset of diabetes (from 6 to 10 weeks) in nonobese diabetic (NOD) mice [35]. In general, MNPs-based imaging holds potential as a useful tool for performing long-term, longitudinal studies of the progression of diabetes in humans.

Targeted drug delivery using MNPs was first proposed in the late 1970s and has been one of the most desirable applications of MNPs for chemotherapy [14]. Following the early studies of Widder and Senyi, the efficacy of this approach was demonstrated in numerous small animal studies and even resulted in a small number of clinical trials. However, despite these efforts and achievements, this technique has yet to develop into a workable clinical application.

One of the reasons is the low payload capacity of existing MNPs because payload (i.e., drugs) can only be attached on the surface or embedded in the double-layer coating around MNPs. To address this issue, a novel platform of engineered Fe₃O₄ porous hollow NPs (HMNPs) was designed for the controlled release of cisplatin. Specifically, cisplatin was encapsulated in the interior cavities, and the targeting agent, Herceptin, was attached on the surface of MNPs. These NPs could then efficiently target and deliver cisplatin to ErbB2-/Neu-positive breast cancer cells (SK-BR-3). The percentage of loaded cisplatin was improved from 4.82 % of Fe₃O₄ MNPs to 24.8 % of Fe₃O₄ HMNPs. Meanwhile, the pores were subject to acid etching in low pH environment and subsequently facilitated cisplatin release. The amount of cisplatin released when pH < 6 was more than 3 times compared to a release in neutral physiological conditions. Once HMNPs were internalized to the endosomes/lysosomes, pH-sensitive pores were further opened up to accelerate the cisplatin release and finally kill the cancer cells [36].

Besides the effort of improving the drug loading efficiency, another focus is to integrate drug delivery and molecular imaging into the same system. For example, dumbbell-shaped Au-Fe₃O₄ NPs were synthesized (Fig. 21.3a, b) and conjugated with cisplatin complexes to serve as a multifunctional platform for targeted cisplatin delivery (Fe₃O₄ part) and optical imaging (Au part) [37, 38]. Herceptin was immobilized onto Fe₃O₄ through PEG3000-CONH-Herceptin to offer specific SK-BR-3 cell targeting ability. While cisplatin complexes were attached onto Au side by reacting with Au-S-CH₂CH₂N(CH₂COOH)₂. Reflection images of SK-BR-3 and MCF-7 cells after incubation with the same concentration of cisplatin-Au-Fe₃O₄-Herceptin NPs (Fig. 21.3d, e) showed the specificity and efficacy of these NPs. Au-Fe₃O₄ NPs did not inhibit cell growth at tested Fe concentration. However, once coupled with cisplatin, these NPs showed remarkable cytotoxicity with a low half-maximal inhibitory concentration (IC₅₀) toward SK-BR-3 cells of 1.76 μg/mL of Pt (Fig. 21.3f) compared to free cisplatin needed (3.5 μg/mL of Pt). This methodology was expected to have a great potential to function as nanovehicles for highly sensitive diagnostic and highly efficient therapeutic applications [38].

Finally, MNPs could also deliver peptide/protein, oligonucleotide, plasmid, etc. One example is magnetofection which uses magnetic field to concentrate MNPs containing nucleic acids into cells. MNPs can be incorporated into viral or nonviral platforms to facilitate gene delivery. Recently, recombinant adeno-associated virus 2 (rAAV) was conjugated onto MNPs to achieve therapeutic levels of transgene expression. This platform enhanced the transfection efficacy. The same level of transfection seen with free vector can be achieved using 1 % of vector conjugated MNPs [39]. For nonviral deliver systems, MNPs can be coated with polyethyleneimine (PEI) or polyethylene glycol-grafted PEI (to reduce cytotoxicity of PEI), which allows the efficient loading and protection of genes and holds a great potential for advanced gene delivery and therapy [40].