The primary functionalization of iron oxide NPs with organic functionalities is the first step in the covalent binding of biomolecules to their surfaces. The drawbacks of this method stay in the restrictions derived from the biomolecule conformation being imposed by their orientation on the support upon binding. Functionalization of magnetic NPs with carboxyl, amino or hydroxyl groups prior to the covalent binding is traditionally used [6,39,123–138]. Hong et al. [139] functionalized magnetic nanogels through a carbodiimide activation procedure. In this case, polyacrylamide (PAM) coated Fe₃O₄ NPs and Hoffman degradation was used to obtain amino-functionalized magnetic nanogels with a 25 nm diameter. Subsequently, α-chymotrypsin was covalently attached to the nanogel’s surface. The enzymatic activity was largely retained and the reaction temperature and usable pH range were wider after the covalent binding as compared to the free enzyme. In the case of the covalent binding, the enzymatic activity was not dramatically affected by varying the pH between 5.8 and 8.9, while for the free enzyme, the enzymatic activity decreased to less than 50% at pH values between 8 and 8.9. The temperature was varied between 35 and 85 °C and the optimum temperature for the both free and covalently bound enzyme was determined to be at 35 °C, while above this temperature, a decrease in activity was observed in both cases. However, after 60 °C, the free enzyme lost all its activity, while the covalently bound enzyme still retained 60% of its activity. The enzymatic activity was retained after storage at 4 °C for 36 days. The magnetic composite was reusable, with 96.8% of the biological activity being maintained after 12 usage cycles. On the other hand, the affinity between the enzyme and the substrate was lower than that of the free enzyme. An explanation related to sterric effects of diffusion barriers of the substrate to the enzymatic active sites was proposed. More recently [140], the same authors used a hydrophilic polymer with free carboxyl groups to covalently immobilize α-chymotrypsin to the surface of magnetic iron oxide NPs, followed by in-situ polymerization with 1-ethyl-3-(3-dimethylaminepropyl) carbodiimide (EDC) used as a coupling agent. The enzyme retained 80% of its activity at 65 °C, 90% at 25 °C and the full enzymatic activity at 4 °C. The same long term storage stability and reusability as in the case of the amino functionalized nanogels were observed. Other enzymes covalently immobilized on the surface of magnetic NPs include GOx [138] and peroxidase [141,142], as well as cholesterol oxidase, lipase [143], trypsin and chymotrypsin [131,136,137,144–150]. Kuroiwa et al. [151] immobilized chitosanase on amylose-coated Fe₃O₄ NPs. The amylose provided hydroxyl groups on the NPs surface, which were then coupled with the amino groups of chitosanase via covalent binding. Fe₃O₄-chitosan NPs, exposing free amino groups on their surface were used to bind alcohol dehydrogenase via GA coupling, with about 48% retention of enzymatic activity [151]. The authors demonstrated the recovery of the enzyme by magnetic separation.

Core-shell NPs functionalized with polymers were investigated as supports for biomolecule immobilization. Mahmood et al. [152] immobilized lipase on magnetic NPs coated with a oleic acid-Pluronic^® (L-64) block copolymer. In this work, the copolymer was used to improve stability (i.e. reduce agglomeration). Up to 90% enzymatic activity of lipase was retained for seven cycles. This extended usability was attributed to hydrophobic interactions between the preatreated NPs and the copolymer. Shamim et al. [153] synthesized core-shell iron oxide NPs coated with poly-(N-isopropylacrylamide) (PNIPAM) and used them to adsorb bovine serum albumin (BSA) on their surface. Thiodiglycolic and 4-vinylaniline were used as surfactants. The core-shell NPs were thermosensitive. Above 32 °C, the structure of the poly-(NIPAM) changes from hydrophilic to hydrophobic. Due to this effect, at temperatures above 32 °C, the magnetic NPs shrink, and are able to adsorb a larger amount of bovin serum albumin (BSA). Lowering the temperature below 32 °C resulted in desorption of the protein. It has been shown that the protein adsorption takes place a larger extent through hydrogen bonding, and less through hydrophobic interactions. Peng et al. [154] fabricated Fe₃O₄ magnetic NPs with 10 nm in diameter through a chemical precipitation method and further used them for physical adsorption of BSA and lysozyme (LSZ) with partial retention of enzymatic activity. Desorption of both BSA and LSZ was also investigated at the isoelectric points of the enzymes. Kausik et al. [155] used Fe₃O₄ NPs prepared via a co-precipitation method to design a glucose sensor. The NPs dispersed in chitosan formed a film onto an indium-tin oxide (ITO) glass. Dispersion in chitosan helped preventing aggregation, while the NPs facilitated communication with the electrode surface. This biosensor had a rapid response time (5 s), good linearity (10 – 400 mg dL⁻¹), good reproducibility, and high affinity towards glucose. The sensitivity was of 9.3 μA/(mg·dL·cm²) and the sensor was stable for up to eight weeks at 4 °C.

Biomolecules can be entrapped within the different polymeric or silica shells used to form hybrid magnetic composites. For example, spherical silica coated Fe₃O₄ NPs have allowed stable entrapment of horseradish peroxidase (HRP), simultaneously with the formation of the silica layer [96]. This method resulted in biomagnetic catalysts characterized by a long-term stability with temperature up to 85 °C and pH change, as compared to the free enzyme. However, the entrapped enzyme could lose activity due to conformational changes in the silica matrix and also suffers from possible diffusion limitations of the substrate through the silica pores. The same methodology allowed immobilization of an antibody for the development of an immunoassay, for the quantitative determination of gentamicin with a detection limit of 160 ng/mL. More complex systems with entrapped enzymes combining different types of materials were also reported. Such an example is the use of iron oxide NPs with crosslinked enzyme molecules, which were then encapsulated into large pores of mesoporous silica to form a “hierarchically ordered, mesocellular” structure [145]. These nanocomposites were magnetically separable, highly loaded with enzyme, stable under harsh shaking conditions, resistant to different treatment procedures, and reusable.

Site specific immobilization onto magnetic particles is an attractive strategy for attaching biomolecules because it provides a favorable orientation for biorecognition events while avoiding conformational changes, and offering magnetic control of the entire assembly. As opposed to other methods, this strategy involving attachment at a specific pre-determined position eliminate the diffusion barriers or chemical bond formation that could affect the biological activity and, therefore, a lower detection limit and a fast response time could be expected for sensors fabricated based on this method. Johnson et al. [156] developed a method for immobilization of affinity-tagged dehalogenase on iron/iron oxide core-shell NPs through a biomimetic approach. The authors used cloned dehalogenase (DhlA) fusion proteins, with an affinity for either silica or iron oxide surfaces [157,158]. Three different DhLA recombinant enzymes were expressed and immobilized on the NPs surface. The enzymes were able to specifically bind to either iron oxide or silica. The DhLA enzymes tagged with iron oxide or silica affinity peptides showed a higher rate of adsorption onto the magnetic NPs when compared to the His-tagged protein. In another example, pre-activated iron oxide beads carrying Ni- iminodiacetic acid (IDA) complexes were used to immobilize a genetically-modified acetylcholinesterase (AChE), having engineered an hexa(histidine) tag. The 6His-AChE-Ni-NTA-NPs system was deposited onto the surface of a screen-printed electrode surface with a small magnet placed underneath the electrode. This biosensor was used for the detection of two organophosphorous pesticides, via AChE inhibition [159]. The main advantage of this method for inhibition assays is the very high sensitivity with detection limits of 10⁻¹¹ M for chlorpyriphos-oxon and the easy reusability of the same electrode. The system could be easily adapted for integration into an autonomously operated magnetoswitchable device for monitoring AChE inhibiting activities.

Enzymatic sensors based on various magnetic platforms, mainly iron oxides were designed with the advantages of offering high enzyme loading and control of the localization of the sensitive material through the use of a magnet allowing for the enzymatic reactions to occur in the close proximity of the transducer surface [159–163].

The transducing component is easily renewable and reusable. This is due to the ability to control the charging/discharging of its surface by application of a magnetic field, providing reusability of the same electrode for several analyses. Examples include sensors based on tyrosinase for the detection of phenol [162], yeast (YADH/NAD⁺) [164] for the detection of ethanol, GOx for glucose [163], and AChE for organophosphorus pesticides [151]. Enzyme-immobilized magnetic beads can be incorporated in a flow injection analysis (FIA) system as described by Kauffmann et al. [161]. Figure 2 shows the schematic diagram of a FIA assay for detection of glucose using glucose oxidase modified magnetic microparticles. Functionalized magnetic particles were injected into the FIA system and retained near the detector using a pair of two small permanent magnets. An amperometric system for the detection of glucose with glucose oxidase immobilized onto the magnetic particles was developed based on this principle. The porous biomagnetic particles were ferromagnetic spinnel type iron oxide (γ-Fe₂O₃) with the GOx immobilized after silanization with aminopropyltrietoxysilane followed by covalent binding using GA.

Magnetic particles functionalized with specific antibodies (Ab) have been used for the design of immunomagnetic sensors through the immobilization of the Ab-NPs assembly on the surface of an electrochemical transducer [3,165–167]. For these types of immunosensors, the immunomagnetic complex is magnetically attached to the surface of the screen printed electrode. The utilization of Ab-coated magnetic particles is efficient in overcoming the need of regeneration of the sensing surface and makes possible integration into automatic systems, difficult to achieve otherwise due to the obstacles in renewing the sensing surface. The immunocomplex is usually quantified through the use of enzyme labels, with the electrochemical detection of the enzyme reaction product after the complex is exposed to the enzymatic substrate, or through a fluorescent label followed by fluorescence detection. An example of amperometric immunosensing assay obtained by immobilizing the antibody onto a solid carbon paste electrode using core-shell magnetic NPs is shown in Figure 3 [167].

Different analytes were detected using this method, such as: rabbit IgG [168–170], non-pathogenic E. coli O157 [171], polychlorinated biphenyls (PCBs), the herbicide 2,4-D, atrazine [171–175] pesticides and bacterial pathogens [176,177]. For example, streptavidin activated magnetic microbeads were used for the immobilization of an antibody for atrazine, a small pesticide molecule, allowing for the determination of its concentration in biological samples, with a detection limit of 0.027 nmol L⁻¹ [173]. In another work, immunomagnetic separation coupled with differential pulse voltammetry allowed detection of Arochlor 1248 PCB mixture with a detection limit of 0.4 ng/mL using screen-printed three electrode strips [178]. Other immunosensors with renewable electrode surface were reported for the detection of pathogens such as Salmonella Typhimurium [176,177]. Another interesting application of immunomagnetic NPs was for selectively concentrating traces of pathogenic bacteria (Staphyloccocus saprophyticus and Staphyloccocus aureus) via IgGs attached onto the particles surface [179]. It was found that these IgG-NPs conjugates can bind selectively to the cell membrane. Detection limits of as low as 3 × 10⁵ cfu/mL were achieved in aqueous solutions. Other specific examples of monodispersed bio-functional magnetic NPs for protein separation and pathogen detection were discussed recently by Gu et al. [180].

DNA sensors with single-stranded (ss) oligodeoxynucleotides immobilized on electrode surfaces [181–183] via magnetic beads were also fabricated [184–190]. A three layer magnetic NPs structure with a gold surface, silica core and magnetic inner layer was functionalized with DNA and used for quantifying hybridization events [189]. It was found that upon hybridization with complementary oligonucleotides, this structure forms aggregates in the same way as Au NPs. In another work, surfactant-modified oligonucleotides were incorporated into the particle organic shell to create a DNA functionalized surface [188]. For example, monodisperse MnFe₂O₄ magnetic NPs were biofunctionalized with DNA using a combination of alkylphosphonate and ethoxylated fatty alcohols. This method is based on the affinity of alkylphosphonate for metal oxide surfaces. DNA amplification with magnetic primers in combination with electrochemical detection and enzyme labelling was used to develop a genomagnetic assay for detection of food pathogens such as Salmonella spp [190]. The electrode consisted of a graphite epoxy composite and streptavidin modified magnetic beads with immobilized DNA. Loaiza et al. [191] reported a sensitive method for isolation and detection of DNA from bacterial cells using disposable magnetic DNA sensors. Screen-printed gold electrodes with a 4 mm in diameter working electrode surface were used as amperometric transducers in this example.

Figure 4 shows a schematic of the protocol used for enzyme amplification. The magnetic particles were functionalized with streptavidin. A 25-mer capture probe was subsequently attached, followed by the hybridization process. In the next step, streptavidin-peroxidase was attached and the functionalized magnetic particles were attached to the surface of the electrode through a magnet. The sensor was used for the detection of asymmetric DPCR amplified products obtained from E. coli bacterial cultures, with high speed, specificity and sensitivity. Another advantage of this method was elimination of false positive results, a drawback of conventional PCR analysis.