Nanocrystalline Iron Oxides, Composites, and Related Materials as a

Subscriber access provided by Aston University Library & Information Services

Review

Nanocrystalline Iron Oxides, Composites and Related Materials as a Platform for Electrochemical, Magnetic, and Chemical Biosensors Veronika Urbanova, Massimiliano Magro, Aharon Gedanken, Davide Baratella, Fabio Vianello, and Radek Zboril Chem. Mater., Just Accepted Manuscript • DOI: 10.1021/cm500364x • Publication Date (Web): 03 Sep 2014 Downloaded from http://pubs.acs.org on September 6, 2014

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Chemistry of Materials is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Chemistry of Materials

Nanocrystalline Iron Oxides, Composites and Related Materials as a Platform for Electrochemical, Magnetic, and Chemical Biosensors Veronika Urbanova1,$, Massimiliano Magro1,2,$, Aharon Gedanken3, Davide Baratella2, Fabio Vianello1,2,*, and Radek Zboril1,* 1

Regional Centre of Advanced Technologies and Materials, Department of Physical Chemistry, Palacky University, 17. listopadu 1192/12, 771 46 Olomouc, Czech Republic. 2 Department of Comparative Biomedicine and Food Science, University of Padua, Italy. 3 Kanbar Laboratory for Nanomaterials, Bar-Ilan University, Ramat-Gan, Israel. KEYWORDS: Iron oxide, electrochemical sensing, biosensing, nanocomposite materials, fluorescence.

ABSTRACT: This review represents a comprehensive attempt to summarize and discuss various sensing applications of iron oxide nanoparticles (NPs), which have attracted a great deal of attention over recent years because of their easy preparation, biocompatibility, non-toxicity and broad range of biomedical applications. We review the application potential of nanomagnetite based amperometic sensors possessing an intrinsic enzyme mimetic activity similar to that found in natural peroxidases. In addition, we discuss the properties and applications of enzymatic sensors exploiting glucose oxidase, tyrosinase and other enzymes for sensing a variety of important biomedical species. Among iron oxide-based nanocomposites, we highlight the use of Fe3 O4@Au hybrids for designing new electrochemical aptasensors with unique versatility for binding diverse targets, including proteins and peptides. Similarly, sensing applications of composites of iron oxide NPs with graphene derivatives and carbon nanotubes are reviewed. A large part of the review focuses on the development of DNA sensors and iron oxide immunosensors for the detection of biological and chemical pathogens, contaminants and other important analytes. Attention is also given to non-electrochemical sensing, including various types of magnetic, fluorescence and surface plasmon resonance sensors.

INTRODUCTION The development of sensitive analytical methods is of great importance in many different areas, ranging from clinical and environmental studies to the monitoring of biotechnological and industrial production.1 Among the available methods, electroanalytical methods are the most rapidly expanding class of chemical sensors because such devices satisfy many requirements for analytical detection since they are inherently sensitive, selective, fast and inexpensive.2 In general, three types of electrochemical sensors are widely distinguished: potentiometric,3,4 amperometric5 and conductometric.6,7 This classification is related to the type of measured signal during the analysis, i.e., voltage, current or impedance/conductance, that is proportional to the changes caused by different analyte concentration in the samples. Potentiometric sensors or ionselective electrodes are widely used for determination of inorganic or organic ions based on measuring the potential difference between the working and the reference electrode. While amperometric sensors are based on the measurement of the current occurring between working and reference electrodes. Finally, conductometric sensors, which are usually used as gas sensors, detect the changes of electric conductivity generated by presence of analyte. The latter are a very attractive class of analytical devices owing to their high sensitivity. Although conductometric devices have not yet been fully optimized, they offer several advantages over the other types of electroanalytical sensors,

such as low cost and simplicity since no reference electrodes are needed.8 Voltammetric techniques are undoubtedly the most widely applied methods in electrochemical analysis and have been utilized for sensing applications based on different materials, e.g., metals, metal-oxides, semiconductors or composite nanomaterials. Among others, cycling voltammetry (CV), linear sweep voltammetry (LSV), differential pulse voltammetry (DPV) or stripping voltammetry (SV) are regarded as basic tools, which are employed in electroanalysis. Qualitative and/or quantitative information about analyte is related to the current that arises from the potential imposed to an electrode. Contrary, amperometric techniques (e.g., chronoamperometry) are based on application of a constant potential to the working electrode and analyzing steady-state current as a function of time.2 Electrochemical impedance spectroscopy (EIS) constitutes another technique that has been used to study electrochemical systems and its use in the field of the biosensors has been recently reviewed.10 Since the discovery of iron oxide nanoparticles (NPs) about 30 years ago,11 researchers have been exploring their potential in various fields. A number of methods have been developed for synthesizing magnetic NPs with various compositions. Over recent years, enormous advances in the synthesis of various iron oxide NPs have been achieved, and it is now possible to synthesize uniformly sized iron

1 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

oxide NPs ranging from a few nanometers to tens of nanometers.12 Details can be found in several reviews devoted to the synthesis and design of iron oxide NPs.13–20 Briefly, four main crystalline polymorphs with different properties have been described for the iron (III) oxide system: α-Fe2O3 (hematite), β-Fe2O3, γ-Fe2O3 (maghemite) and ε-Fe2 O3.16,17 Hematite (α-Fe2O3) behaves as an antiferromagnet and has been widely used as an effective and selective gas sensor based on the changes in surface resistance in the presence of gas. Similarly, hematite is widely used as a catalyst, photocatalyst and photoanode for direct solar splitting of water.17 β-Fe2O3 is a rare iron (III) oxide phase exhibiting paramagnetic behavior at room temperature. Maghemite (γ-Fe2O3) is the second most common iron (III) oxide polymorph occurring in nature and can be synthetized by a number of solid-state and wet chemical routes. It is typical ferrimagnetic material applicable as magnetic sensor and also in many biomedical fields including cell labelling and separation, targeted drug delivery or MRI contrast enhancement. 17 ε-Fe2O3 is rare polymorph existing only in the form of nanoparticles and can be considered as the intermediate during polymorphous transition of nanocrystalline γ-Fe2O3 to α-Fe2O3. ε-Fe2O3 reveals some unique magnetic properties including giant coercive field, millimeter-wave ferromagnetic resonance, and magnetoelectric coupling.20 Magnetite (Fe3O4) is another important iron oxide phase exhibiting ferrimagnetic or superparamagnetic behavior depending on the particle size. Due to the same spinel structure, the portfolio of applications of magnetite is very similar to that mentioned for its fully oxidized analogue – maghemite. However, it should be noted that uncoated magnetite nanoparticles are susceptible to oxidation when exposed to atmosphere because surface Fe (II) cations react with adsorbed oxygen forming a shell of γ-Fe2O3, which then tends to aggregation. For this reason, several approaches, e.g. formation of a core-shell structure, to protect bare magnetite NPs using polymers or inorganic materials have been demonstrated. Fe3O4 magnetic nanoparticles were used as drug/gene delivery carriers or imaging agents while magnetic NPs with core-shell structure found applications for construction of electrochemical, DNA or enzymatic sensors.13–15 It is worth mentioning that maghemite, magnetite and hematite are most frequently used iron oxides in sensing applications while other phases including also nonstoichiometric wustite (FeO) and iron(III) oxyhydroxides have limited applicability. The popularity of iron oxides as catalysts stems from their interesting characteristics as they are easy to handle, relatively low cost, non-toxic and environmentally friendly. Acceptance of iron oxides is illustrated by the fact that they are routinely employed as catalysts in different processes such as oxidation of carbon monoxide,21 reduction of nitrogen oxides22 and sulfur dioxide,23 photocatalytic splitting of water,24 synthesis of hydrocarbons,25 catalytic decomposition of industrial dyes,26 oxidation of phenolic and other environmentally harmful aromatic compounds,27 dehydrogenation of ethyl-benzene to styrene,28 and catalytic oxidation of various other organic compounds.29 Recently, iron oxide NPs were also proposed for various biomedical applications owing to their superior properties, including

Page 2 of 24

magnetic (e.g., superparamagnetism, high values of saturation magnetization, easy control by small magnetic fields) and biochemical (e.g., non-toxicity, biodegradability, biocompatibility) characteristics. It should be mentioned that magnetic NPs are classified as medical devices and, according to the US-FDA, should conform to ISO 10993 guidelines. Magnetic NPs are usually employed as contrast agents in magnetic resonance imaging (MRI), for radiofrequency induced hyperthermia treatment and magnetically targeted drug delivery.30 Moreover, some magnetic NPs have already been approved for clinical magnetic resonance imaging applications (Feridex by AMAG Pharmaceuticals, Inc., Lexington, MA, USA; Endorem by Guerbet, Villepinte, France).31 Iron oxide NPs also seem to be a promising material for analytical sensing applications.32 In particular, enzymebased electrochemical biosensors, which combine the specificity of enzymes with the analytical power of electrochemistry, are attracting intensive research efforts owing to their potential applications in clinical diagnostics or environmental monitoring. Functionalized NPs have been employed in novel applications for efficiently wiring redox enzymes, and thus have paved the way for novel electrochemical biosensors,33 bioelectronic devices34 or biofuel cells.35 Moreover, the direct immobilization of redox enzymes without diffusible mediators onto nanostructured electrode materials may facilitate the sensitive and selective detection of analytes. Studies on the direct electron transfer of proteins at electrodes may serve as a basis for building innovative electrochemical biosensors, enzymatic bioreactors and biomedical devices. Several immobilization procedures have been used for enzyme immobilization onto an electrode surface.36–38 Besides electrochemical applications, other sensors based on iron oxide NPs with interesting magnetic, colorimetric or optical properties have been described in the literature.39 Important advantages of iron oxide NPs are they are generally unaffected by the measurement process, they can be stored indefinitely and detection techniques based on magnetic labels are often simple to perform and inexpensive. In this paper, we review the current state of art in sensing based on iron oxide NPs. This review is primarily focused on electrochemical sensing. Nevertheless, a brief overview of some non-electrochemical techniques (e.g., fluorescence) is also provided. APPLICATIONS OF IRON OXIDE NANOPARTICLES IN ELECTROCHEMICAL SENSING Interest in magnetic NPs has increased enormously over the past two decades. Fundamental research on NP structure, physical and magnetic properties, and toxicity (among other characteristics) has facilitated the development of magnetic NPs for industrial and biomedical applications.39 Taking advantage of their unique magnetic characteristics and low cost of synthesis, magnetic NPs can be used as an advanced tool for the capture, concentration or separation of many types of analytes from complex matrices.39,40 Among others, iron oxide NPs, such as those of Fe3O4 and γ-Fe2O3, are some of the most promising candidates owing to their large surface area (in the range of 10–50 m2 g–1),


Page 3 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


high mechanical strength, interesting electronic properties, and excellent chemical and thermal stability. Because of their large surface area and biocompatibility, they can be used as nanocarriers for various types of enzymes by means of physical adsorption or covalent immobilization. The most widely used enzyme is glucose oxidase, but immobilization of other enzymes, e.g., hydrolase, horseradish peroxidase (HRP), hemoglobin, creatinase or lactate dehydrogenase, can also be found in the literature.41 Electrochemical sensors based on iron oxide NPs can be divided into two main groups: non-enzymatic sensors, in which bare or functionalized NPs act as the sensing phase, and enzymatic sensors, in which NPs act as mediators.42 In the following text, sensing applications based on different configurations of iron oxide NPs and electrochemical detection will be discussed. Sensing Applications of Iron Oxide Nanoparticles – Non-Enzymatic Sensors. Natural enzymes play an important role in biochemistry and are of significance in chemical industry, medicine or food technology. Wide range of applications in different fields is ascribed to enzyme ability for highly efficient and selective catalysis under mild conditions. However, their use is limited as they can be easily denatured by surrounding environment.43 For this reason, scientific efforts have been devoted to research of material exhibiting a similar catalytic activity to that of natural enzymes. Such materials are called artificial enzyme mimetics.44 Various enzyme mimetics have been proposed based on DNA-organic molecule-enzymes45 or cytochrome P450.46 Among iron oxides, magnetite (Fe3O4) NPs possess an intrinsic enzyme mimetic activity similar to that of natural peroxidases, which are widely used to oxidize organic substrates in order to reduce their toxicity. Thus, magnetite NPs can be used in waste water treatment or as detection tools since Fe2+/Fe3+ ions are known to catalyze the breakdown of hydrogen peroxide.47,48 As an example, Liu and Yu49 monitored colorimetric products of the catalytic oxidation of three different peroxidase substrates, 3,3,5,5tetramethylbenzidine (TMB), di-azo-aminobenzene (DAB) and o-phenylenediamine (OPD), as shown in Figure 1.

Figure 1. Peroxidase-like activity of magnetic nanoparticles. (a) TEM images of Fe3O4 NPs. (b) Different colors generated by the oxidation of various peroxidase substrates catalyzed by Fe3O4 in the presence of H2O2. [Reprinted from ref 47 with permission from Nature Publishing Group].

described in the literature.50–52 The importance of the catalytic decomposition of hydrogen peroxide stems from its vast applicability in waste water treatment because various pollutants can be oxidized and degraded by hydrogen peroxide in the presence of iron oxide.53 Furthermore, the analytical determination of H2O2 is worth considering, since hydrogen peroxide is widely used as oxidizing agent in chemical, food or pharmaceutical industry.54 Up to date, number of methods such as spectrophotometry,55 fluorescence,56 and electrochemical techniques,6,7,57 have been applied for hydrogen peroxide detection. Among these methods, electrochemistry belongs to the most convenient due to its simplicity, low cost and possibility of real-time measurements. In principle, hydrogen peroxide can be either oxidized or reduced directly at the solid electrode surface, but one has to take into account the limitations of sensing performance arising from slow electrode kinetics and high overpotential. For this reason, current research efforts are mainly focused on electrode modifications contributing to the decrease in electrode overpotential and increase the electron transfer kinetics.57 Most of the proposed sensors are based on the electrode surface modification by enzymes. Nevertheless, use of enzymes can have some drawbacks due to the reduce activity of enzyme once it is immobilized, limited lifetime or difficult manipulation during sensor fabrication. Thus, the development of enzyme-free H2 O2 sensors based on different strategies has stimulated scientific attention. In this context, Fe3O4-modified electrodes are usually employed for H2O2 detection, as shown in Figure 2. In initial work, measurement of a peak current that increased with consecutive additions of H2O2 clearly demonstrated the catalytic effect of Fe3O4 toward H2O2 without the need for an enzyme. Moreover, Fe3O4-modified electrodes can provide a cathodic scheme to minimize possible interference in various applications and also exhibits a longer lifetime than other electrochemical schemes, e.g., based on peroxidase or Prussian Blue.50

Figure 2. Typical cyclic voltammetric response of an Fe3O4modified electrode in the absence (curve B) and presence (curves a1-a5, five successive additions) of hydrogen peroxide. Curve a0 shows the voltammetric response in the presence of ambient oxygen. [Reprinted from ref 50 with permission from John Wiley & Sons, Inc.].

Several examples of amperometric sensors employing the electrocatalytic properties of bare iron oxide NPs have been



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Zhang et al.58 have proposed a strategy for H2O2 determination based on layer-by-layer assembly of Fe3O4 NPs and polyelectrolyte – poly(diallyldimethyl)ammonium chloride (PDDA). Morphological characterization by atomic force microscopy (AFM) revealed a coarse film with small NPs and clumps dispersed all over the substrate. With an increasing number of layers, the film became even much coarse with a large number of aggregates. Nevertheless, the modified electrode showed good electrocatalytic behavior toward H2O2, displaying a detection limit of 1.4 µM and 92.3% of its initial response after 50 days. Moreover, no obvious current response was observed after addition of two common interfering species in H2O2 determination, i.e., ascorbic and uric acid. Recently, a carbon paste electrode modified with surface active maghemite NPs59 (SAMN) has been used for hydrogen peroxide detection at low applied potentials.60 To improve the electrocatalytic properties of the modified electrode, an ionic liquid, namely 1-butyl-3methylimidazolium hexafluorophosphate (BMIM-PF6), was introduced. At –0.1 V, the sensitivity of the SAMNBMIM-PF6-CP electrode was 206.51 nA µM–1 cm–2 for H2O2 concentrations in the range 0–1.5 mM, with a detection limit of 0.8 µM. Several attempts have been made to obtain nanostructured NPs since they are highly desired as drugdelivery carriers or medical diagnosis material.61 Porous Fe3O4 NPs (with pore diameter of about 21 nm, see Figure 3) prepared by hydrothermal treatment of ferric ammonium citrate, poly(acrylic acid) and hydrated hydrazine without any template have been used for dopamine (DA) detection.62 A glassy carbon electrode (GCE) was modified with a self-assembled Fe3O4 NPs film using an external magnetic field. The results revealed that the modified electrode could selectively detect DA in the presence of ascorbic acid and showed a linear relationship between the DA concentration and the oxidation peak current, with a detection limit of 10–7 M.

Page 4 of 24

ferrihydrite) have also been tested for H2 O2 sensing.52,63,64 For example, Hrbac et al.63 prepared carbon paste electrodes modified with α-Fe2O3, ß-Fe2O3, γ-Fe2O3, amorphous Fe2O3, and ferrihydrite and employed them for H2O2 reduction as well as compared their responses against a carbon paste electrode modified by Prussian Blue (PB) and Fe3O4. As expected, the best electrocatalyst for hydrogen peroxide reduction was PB.65,66 However, amorphous ferric oxide NPs were much more efficient than magnetite, whereas ferrihydrite was inactive. The authors found that the sensitivity depended firstly on the modifier content and secondly, on the pH. The sensitivity of the Fe2O3-modified carbon paste electrode increased after its onset for a content of between 3 and 5% and reached a maximum at about 15%. If the content was lower than 2.5% or higher than 30%, the electrode was not hydrogen peroxide sensitive. The largest difference in sensitivity between the amorphous ferric oxide and PB electrodes as H2O2 sensors was found at low pH, with the PB electrode being the most sensitive. At pH = 7, the response was almost the same, whereas at pH = 8, PB was less effective than Fe2O3 (Figure 4). These results help to explain the increasing interest in iron oxide NP based sensor development because under some conditions, they can be used as alternatives for PB. Hexagonal α-Fe2O3 nanorods have been shown to exhibit an excellent electrochemical sensing capability toward H2O2 over the concentration range from 40 µM to 4.66 mM.67 Nanorods with exposed high-index facets were synthesized using a double ligand assisted hydrothermal method (involving PO43- and formamide). Scanning electron microscopy (SEM) morphological characterization revealed that homogeneous, well-shaped hexagonal faceted nanorods were generated with a length of around 150 nm and width of about 50 nm.

Figure 3. TEM images of nanostructured Fe3O4. [Reprinted from ref 62 with permission from Elsevier].

Figure 4. Plots of hydrogen peroxide sensitivity versus pH for carbon paste electrodes modified by either Prussian Blue or amorphous Fe2O3. [Reprinted from ref 63 with permission from John Wiley & Sons, Inc.].

Besides Fe3 O4, NPs of various ferric oxides (such as hematite, maghemite, amorphous Fe2 O3, ß-Fe2O3 and

Non-enzymatic iron oxide based sensors have not only been applied for hydrogen peroxide sensing, but other interesting analytes have been reported in the literature. For


Page 5 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


example, α-Fe2O3 dispersed in chitosan (CH) and deposited on a GCE has been used for the electrocatalytic oxidation of nitric oxide.68 Further, α-Fe2O3 nanofibers prepared by a simple electrospinning method have been used for the electrochemical determination of folic acid (FA) in the presence of ascorbic acid.69 Whereas it was not possible to determine the concentration of FA in the presence of ascorbic acid (AA) with a bare GCE, a GCE modified by these nanofibers not only ensured separation of the voltammetric signals of FA and AA but also enhanced the oxidation currents. In addition, the modified electrode was successfully applied for FA determination in human blood serum samples. Fe2O3 nanowire arrays have been proposed as a nonenzymatic glucose sensor, as shown in Figure 5.70 A typical amperometric response of a GCE modified by Fe2 O3 nanowires is also shown in Figure 5. As can be seen, this kind of electrode showed a good electroanalytical performance toward glucose. Further, high selectivity was observed even in the presence of ascorbic acid and dopamine. Fang and co-workers71 used a gold electrode modified by magnetite NPs, for the electrochemical oxidation of dopamine. The modified gold electrode showed a remarkably lower overpotential compared to a bare gold electrode and also provided a well separated and resolvable dopamine peak in the presence of ascorbic acid. The proposed iron oxide modified electrode was characterized by a detection limit of 3×10−8 M and showed good stability and reproducibility. Once again, the unique properties of nanoscale materials enabled a highly sensitive electrochemical tool to be developed, which is of key importance in biological applications. In particular, highly sensitive methodologies are increasingly being demanded for disease diagnoses based on measuring different markers present in ultralow levels during early stages of disease. Another example is the synthesis of hollow hematite (αFe2O3) nano-polyhedrons (Fe-HNPs) by a mild wet chemical method and their use for nitrite detection.72 Electrochemical experiments showed that Fe-HNPs could act as efficient enzyme-like electron mediators for nitrite oxidation. The α-Fe2O3 modified sensor exhibited excellent performance for the determination of nitrite with a response time of less than 10 s, a linear range between 0.009 and 3 mM, and a sensitivity of 70.17 µA mM–1 cm–2. A high selectivity and long-term stability toward nitrite oxidation in the presence of glucose and L-ascorbic acid was also observed at the maximum physiological concentrations tested. Thus, this novel α-Fe2O3 nanomaterial with high indexed facets is a promising material for sensing applications in medicine, biotechnology and environmental chemistry.

Figure 5. SEM, TEM and HRTEM (inset) characterization of Fe2O3 nanowires (up), and amperometric response to successive additions of 1 mM glucose (down) in stirred solution at a glassy carbon electrode modified by Fe2O3 nanowires. The inset shows a differential pulse voltammetric curve measured in the presence of 1 mM glucose. [Reprinted from ref 70 with permission from RSC Publishing].

Moreover, iron oxide NPs prepared by a solution method using reducing agents (urea and NH4OH) in an alkaline phase have been deposited on a GCE and used as a sensor for the detection of an antiemetic drug, namely aprepitant (APPT).73 The studied antiemetic chemical compound belongs to a class of drugs known as substance-Pantagonists, which mediate their effect by blocking the neurokinin 1 (NK1) receptor. In this work, ethyl acetate and butyl carbitol acetate were used as binders for the immobilization of NPs onto the electrode surface (see Figure 6). The modified sensor exhibited good sensitivity and long-term stability as well as an enhanced electrochemical response. The calibration plot obtained was linear over the concentration range from 2.2 nM to 4.1 µM APPT. The sensitivity was 2.53 ± 0.5 µA mM–1 cm–2, the detection limit was 0.38 ± 0.02 nM (S/N = 3), and the response of the sensor was within 10 s. The proposed sensor demonstrated that iron oxide NP modified electrodes could also be used for drug determination.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 6 of 24

Figure 7. Chemical structure of chitosan.

Figure 6. Schematic of a glassy carbon electrode (GCE) modified by iron oxide NPs for aprepitant determination. WE = working electrode. [Reprinted from ref 73 with permission from Elsevier]. Rahman et al.74 also proposed a sensor based on GCE modified by β-Fe2O3 for chloroform detection in a liquid phase. The analytical performance showed that the sensitivity, stability and reproducibility of the sensor were significantly improved by using a thin-film of β-Fe2O3 NPs on the electrode surface. The calibration plot was linear over a large concentration range (12.0 µM – 12.0 mM). The sensitivity was calculated to be 2.18 µA mM–1 cm–2, with a detection limit of 4.4 ± 0.10 µM and short response time (10.0 s). Salek et al.75 have described the insertion of maghemite NPs within the pores of microspheres made of poly(styreneco-divinylbenzene). Sulfo-groups were introduced into the microspheres to prepare an immunomagnetic electrochemical biosensor for protein detection with ovalbumin as a model substance. Based on the above described examples, one can conclude that the emergence of iron NPs has not only opened up new opportunities for highly sensitive bioaffinity and biocatalytic assays based on novel biosensor and chemical sensor protocols but also enabled highly selective bioassays to be designed for various applications, such as the detection of nucleic acids and proteins,76 which will be discussed later. Sensing Applications of Iron Oxide Nanoparticles – Enzymatic Sensors. Since nanostructured semiconducting metal oxide materials exhibit good catalytic properties and higher ionic conductivities, they can be used as interface matrices between analytes and electrodes. In recent years, iron oxide NPs and their composite materials have been used for electrochemical based enzyme biosensor fabrication.42 A variety of biosensors, based on iron oxide NPs, were developed using CH or glutaraldehyde as crosslinking agents suitable for protein immobilization onto the electrode surface. Chitosan (see Figure 7) is a linear polysaccharide composed of randomly distributed β-(1-4)linked D-glucosamine (de-acetylated unit) and N-acetyl-Dglucosamine (acetylated unit), containing a large amount of positive charges at neutral pH. This biopolymer is suitable for film formation and has been widely used as a binder for protein immobilization.77,78

Glucose sensing using immobilized glucose oxidase (GOx) for the conversion of target analytes into electrochemically detectable products (see scheme of Figure 8) is one of the most widely used detection methods for the determination of glucose in blood and food.79 Usually, an electron mediator is used to achieve effective shuttling of electrons from the redox active centre of the enzyme to the electrode surface because a thick protein layer surrounds the flavin adenine dinucleotide (FAD) redox center and introduces an intrinsic barrier to direct electron transfer.80

Figure 8. Schematic showing the principles of glucose sensing using glucose oxidase and a magnetic NP modified electrode.

In this context, iron oxide NPs have been considered as an interesting material for GOx immobilization owing to their biocompatibility and strong electrocatalytic and magnetic properties as well as their large surface-to-volume area. Kaushik et al.81 proposed that for this purpose, magnetite (Fe3O4) NPs dispersed in CH deposited onto an indium-tin oxide (ITO) glass plate could be used. Glucose oxidase (GOx) was then immobilized onto a CH-Fe3O4 modified electrode via physical adsorption. The mechanism for such an immobilization method can be explained by electrostatic interactions; surface charged NPs interact with the –NH2/OH groups in CH, and then because GOx is negatively charged, it can easily be immobilized onto the positively charged CH-Fe3 O4 matrix.82,83 Such a GOx/CHFe3O4/ITO bioelectrode displayed excellent catalytic activity toward glucose with a sensitivity of 9.3 µA mg–1dL cm–2. The Michaelis-Menten (KM) constant of immobilized


Page 7 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


GOx was 0.141 mM, indicating that immobilized GOx displayed a high affinity toward the substrate (glucose). The shelf life of the GOx/CH-Fe3O4/ITO bioelectrode was monitored by measuring absorbance as a function of time and it was observed that about 80% of the GOx activity remained after 8 weeks when stored at 4°C.81 A combination of CH and Nafion for immobilization of magnetite NPs together with GOx onto platinum electrode was described by Yang et al.84 Glucose oxidase (GOx) was simply mixed with magnetite NPs and CH, then crosslinked by glutaraldehyde on a Pt electrode and, finally, covered with a thin Nafion film. The proposed biosensor showed a high sensitivity (11.54 µA mM–1 cm–2), low detection limit (6 µM), and good storage stability. The modified electrode was also successfully applied in human serum samples. A screen-printed electrode modified by Fe3O4 NPs together with ferricyanide and GOx was used for glucose sensing using either cyclic voltammetry or electrochemical impedance spectroscopy.85 Ferricyanide was used to increase the surface concentration of the redox probe because nano-Fe3O4 can adsorb iron-cyanide complexes, and thus improve the current response. As a consequence, the linear response range of the glucose concentration for this biosensor was as high as 600 mg dL– 1 . Tyrosinase is another interesting enzyme that can be considered for immobilization onto iron oxide NPs. Electrochemical tyrosinase biosensors have been shown to be sensitive and convenient tools for the determination of phenolic compounds, which are important contaminants in medical, food and environmental matrices.86 In the presence of oxygen, tyrosinase catalyses the oxidation of phenolic compounds to catechol which is further oxidized to oquinone. O-quinone can be then electrochemically reduced to allow low potential detection87 as shown in the following equation:

(1) Wang et al.51 proposed an amperometric tyrosinase biosensor based on an Fe3O4 NPs/CH modified electrode. This biosensor was shown to exhibit fast and sensitive amperometric responses to various phenolic compounds, and thus offers great promise for the rapid, simple and costeffective analysis of phenolic contaminants in environmental samples. Moreover, the tyrosinase-Fe3O4 NPs/CH based biosensor was applied for the detection of dopamine by electrochemically reducing the biocatalytically liberated dopaquinone.88 A typical electrochemical response for dopamine using the tyrosinase-Fe3O4 NPs/CH modified electrode is shown in Figure 9.

Figure 9. Cyclic voltammetric response to dopamine at (a) Fe3O4/chitosan-GCE, (b) tyrosinase-GCE, and (c) tyrosinase/Fe3O4/chitosan-GCE in phosphate buffer (pH 6.5) at a scan rate of 50 mV/s. [Reprinted from ref 88 with permission from New World Publishing International, Inc.].

Tyrosinase-functionalized Fe3O4 NPs have also been used for the detection of bisphenol A.89 However, the authors found that nickel NPs showed better characteristics in terms of detection limits and sensitivity compared to Fe3O4 NPs. Magnetite (Fe3O4) with a particle size of 25 nm has been deposited onto an ITO electrode together with cholesterol oxidase (ChOx) in order to fabricate an impedimetric cholesterol sensor.90 Electrochemical studies revealed that magnetite NPs (Fe3O4) provide excellent surface properties for ChOx loading, resulting in enhanced electron transfer between ChOx and the electrode. Urease (Ur) and glutamate dehydrogenase (GLDH) have been coimmobilized onto an ITO electrode modified by magnetite NP and CH for the detection of urea.91 Differential pulse voltammetry studies showed that the Ur-GLDH/CHFe3O4/ITO bioelectrode was sensitive in the concentration range of 50 – 1000 mg L–1 urea, with a detection limit of 5 mg L–1. The low value of Michaelis-Menten constant (KM = 0.56 mM) indicated that the enzymes (Ur and GLDH) had a high affinity for urea. A potentiometric urea biosensor has been developed using drop-coating of isopropanol-CH mixture containing Fe3O4 NPs.92 In this case, urease was electrostatically immobilized onto a CH-Fe3O4 nanobiocomposite in order to optimize the sensitivity, specificity, stability and re-usability of the urea biosensor. The biosensor displayed a sensitivity of 42 mV per 10-fold increase in the concentration at room temperature. Thin layer of Nafion was also used in order to enhance the operational stability of the system. The direct electron transfer of hemoglobin (Hb) immobilized onto Fe3O4 NP multilayer films for hydrogen peroxide detection has also been investigated.83 The good sensitivity and long-term stability of the biosensor indicated that direct immobilization of heme proteins on an Fe3O4 matrix may offer a promising approach for hydrogen peroxide sensing. These films were deposited onto several conductive materials (GCE, ITO glass, and aluminium foils) by the electrodeposition of CH/Fe3O4 thin films and then building up a layer-by-layer assembly using phytic



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

acid and CH/Fe3O4 alternately (see Figure 10). The subsequent electrochemical characterization indicated that the constructed magnetic multilayer film was a good matrix for immobilizing Hb and promoting the direct electron transfer of Hb whilst maintaining a high catalytic activity toward many substances.

Page 8 of 24

Owing to the unique properties of gold NPs, extensively applied in optical imaging, hyperthermia, and sensitive biodetection for DNA or proteins, several hybrid nanocomposites based on Au–FexOy have been used to construct enzyme-based electrochemical biosensors.102–104 Different synthetic methods have been proposed for the preparation of monodispersed Au–FexOy nanocomposites with various morphologies, including core/satellite, core/shell, multilayer, dumbbell-like, and flower-like architectures (see Figure 11).97 The size and thickness of both the Au and FexOy components can be controlled to optimize their properties. Further, the surface of the hybrid nanocomposites can be tuned by the attachment of different chemical moieties.

Figure 10. Schematic of hemoglobin-magnetite nanoparticle based biosensor fabrication. [Reprinted from ref 83 with permission from Elsevier].

In addition to the above-mentioned enzymes, superoxide dismutase,93 xanthine oxidase,94 and yeast alcohol dehydrogenase95 have also been used in combination with iron oxide NPs for sensing important analytes in different fields, including food and environmental analysis. Sensing Applications of Composite Materials and Modified Iron Oxide Nanoparticles. Composites of Magnetic Iron Oxide with Noble Metal Nanoparticles as Advanced Immunosensors. Composite NPs and hybrid nanomaterials comprising different functional components and nanoscale functionalities are attracting increasing interest from materials scientists owing to their combined physicochemical properties and great potential for applications in the areas of electronics, photonics, catalysis, biotechnology and nanotechnology.96,97 In particular, bimetallic NPs are particularly attractive because of their peculiar catalytic behavior with respect to monometallic systems. Compared to single metal NPs, bimetallic NPs offer distinct characteristics. Generally, one metal (the core) confers long-term stability and biocompatibility, whereas the other (the shell) shows specific electrical properties. Most studies of electrochemical applications involving bimetallic NPs have been concerned with the combination of Au NPs with other metals, such as Pb, Cu, Pt and Zn.98– 100 Electrodes modified by bimetallic NPs typically exhibit a rapid response and good stability, electrocatalytic response and reproducibility for the selected target detection. Iron oxide based core-shell nanostructures have also shown interesting characteristics.101 Generally, these nanocomposites can be modified with different surface charges, reactive groups or functional moieties to enhance their stability and compatibility. Among such materials, the combination of Au and iron oxides (Fe3O4 or γ-Fe2O3) has been demonstrated to be particularly promising for obtaining hybrid nanocomposites that combine advantageous properties of both Au and iron oxide NPs.

Figure 11. Schematic showing fabrication approaches for different types of Au–FexOy nanocomposites. [Reprinted from ref 97 with permission from RSC Publishing].

As an example, Fe3O4/Au NPs magnetic composites have attracted a particular interest owing to the unique characteristics of magnetite and gold. Combination of superparagnetism105,106 of magnetite and favorable behavior (i.e., electronic and optical) of gold NPs107,108 makes these composites very attractive in the field of biotechnology for cell purification, magnetically controlled transport of anticancer drugs107,109 or for fabrication of immunoassays. Among the reported examples of hybrid synthetic methods, Chin et al. have developed a simple aqueous method at room temperature for the preparation of monodispersed core−shell Fe3O4@Au or Fe3O4@Ag NPs as shown in Figure 12.110 Using this approach, a layer of thin and uniform Au and Ag shells was formed on the surface of Fe3O4 NPs. The strategy involved a “seed-mediated growth” method, whereby pre-formed 2−3 nm Au NPs were attached to −NH2 functionalized Fe3O4 NPs, and then Au3+ or Ag+ was reduced by glucose on the surface of the composite NPs to achieve complete encapsulation in ultrathin shells.


Page 9 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 12. Schematic showing the method for (a) dopamine attachment onto an Fe3O4 NP surface, and (b) Fe3O4@Au or Fe3O4@Ag core shell fabrication. [Reprinted from ref 110 with permission from American Chemical Society].

Fe3O4@Au nanocomposites have been extensively used for immobilization of aptamers, short single-stranded oligonucleotides possessing a high affinity and specificity to diverse target molecules including proteins, toxins or cells.111–113 Zhao et al.114 have developed an ultrasensitive electrochemical aptasensor for thrombin, using Fe3O4@Au cluster/shell nanocomposites for aptamer immobilization. In this electrochemical assay, the Fe3O4@Au cluster/shell nanocomposites served as a capture probe, while a second aptamer dual labeled with AuNPs and HRP served as a detection probe. In the presence of thrombin, a sandwichtype structure was formed, and then HRP-based catalytic reactions could be detected by the electrode. Moreover, the magnetic core helped to concentrate the target protein/sensor NP conjugates and aided immobilization on the electrode surface via application of an external magnetic field, which further lowered the detection limit down to 30 fM. Another example of an electrochemical aptasensor based on a Au3+-assisted core shell iron oxide@poly(ophenylenediamine) nanostructure has been described by Liu et al.115 This novel magneto-controlled electrochemical sensing platform was used for the simple and sensitive detection of adenosine-5´-triphosphate (ATP) as it showed excellent adsorption properties for the selective attachment of aptamers specific to ATP. Figure 10 illustrates the principles behind DNase I-catalyzed target recycling for the signal amplification of Au3+-assisted core shell iron oxide@poly(o-phenylenediamine) nanostructure-assisted aptasensors described by the authors. In this process, aptamers were first immobilized on the sensing platform, followed by addition of the targets in the presence of DNase I and electrochemical measurement, and then immobilization of the whole platform onto a gold electrode via application of an external magnet (see Figure 13). The authors found that the sensitivity of the developed aptasensors was up to 4 orders of magnitude lower than a conventional assay method.

Figure 13. Target-induced release of aptamers from redoxactive Au(III)-assisted core shell iron oxide@poly(ophenylenediamine) nanostructures based on DNase I-catalyzed target recycling. [Reprinted from ref 115 with permission from RSC Publishing].

A polymeric nanocomposite based on Fe3O4@Aupoly(1,6-hexanedithiol)-glucose oxidase has been used for glucose sensing.116 The nanocomposite was synthesized via one-pot chemical oxidation of 1,6-hexanedithiol in an aqueous suspension containing glucose oxidase, Fe3O4@Au core/satellite nanocomposites and 1,4-benzoquinone. The proposed bionanocomposite was efficiently separated and immobilized onto a gold electrode and tested for the amperometric biosensing of glucose, for which it exhibited good sensitivity (110 nA µM–1 cm–2), a rapid response time (5 s) and excellent anti-interference properties and stability. The electrocatalytic behavior of core-shell Fe3O4@Au NPs with immobilized myoglobin has also been studied for the reduction of H2O2.117 In this case, an interconnected threedimensional nanocomposite magnetic film (Fe3 O4@Au NPs) was generated, which provided a large surface area for successful myoglobin immobilization. Sulfite oxidase (SOx) obtained from the leaves of Syzygium cumini has been immobilized onto carboxylated gold coated magnetite NPs (Fe3O4@Au) that were afterwards electrodeposited onto the gold electrodes.118 This amperometric biosensor was applied for detection of sulfite in red wine showing high accuracy when compared to standard DTNB (5,5'-dithio-bis-(2-nitrobenzoic acid)) method. Good long-term stability of biosensor was also shown because it was possible use it around 300 times over a period of 4 months with 30% lost of its initial activity. Hybrid Fe3O4-Ag submicrospheres, containing less than 7% Ag, have been synthesized and used for the development of a hydrogen peroxide sensor.119 The fabrication process for such a hybrid is shown in Figure 14: APTES-functionalized Fe3O4 spheres were first mixed with a Ag NP solution, and then the resulting hybrid spheres were separated after 30 min sonication by application of an external magnet, washed with water several times and, finally, redissolved in water.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 10 of 24

Figure 14. Schematic showing the fabrication process for Fe3O4-Ag hybrid spheres. [Reprinted from ref 119 with permission from Elsevier].

The electrochemical sensor constructed with the hybrid spheres exhibited fast, stable and well-defined electrocatalytic activity toward H2O2 reduction, which was attributed to the synergy between Fe3O4 and Ag. The detection limit was 1.2 µM H2O2, which was lower than some enzyme-based biosensors. The authors concluded that because the sensor combines the properties of both Fe3O4 and Ag, it may be of potential use in many fields, such as magnetic separation and electrochemical detection. Composites of Iron Oxide Nanoparticles with Carbon Nanostructures. Integration of iron oxide NPs and carbon nanomaterials (i.e., graphene oxide - GO, reduced graphene oxide - rGO or carbon nanotubes) into nanocomposite materials has become a hot topic of research owing to their new and/or enhanced functionalities that cannot be achieved by either component alone. Thus, they could potentially be used for wide variety of applications.120,121 Teymourian et al.122 described one-step preparation of rGO decorated by Fe3O4 NPs in which reduction of GO and in-situ generation of Fe3O4 NPs occurred simultaneously. This nanocomposite was used as a platform for electrochemical sensing of various analytes such as NADH, H2O2, nitrite, uric acid, ascorbic acid or dopamine. In all the cases, rGO/Fe3O4 showed a good electrocatalytic activity. In addition, immobilized lactate dehydrogenase (LDH) was used as a model NAD+-dependent dehydrogenase enzyme for lactate sensing. Guanine-bonded graphene and an Fe3O4 NP nanocomposite material has been used for antioxidant amperometric detection.123 The mechanism of the antioxidant sensing (see Figure 15) was explained as follows. During the oxygen reduction process, OH radicals and H2O2 were generated as intermediates. The OH radicals were able to oxidize the guanine probe, thus low currents were obtained during the electrochemical oxidation. When antioxidants (AOs) were present during the oxygen reduction process, the OH radicals and H2O2 could be scavenged by the AOs. Thus, the guanine probe was protected and higher electrochemical oxidation currents were obtained.

Figure 15. Schematic of the antioxidant (AO) sensor working principle. [Reprinted from ref 123 with permission from Elsevier].

Wu et al.124 have used a bifunctional Fe3O4-Pt/r-GO composite, in which Fe3O4 and Pt NPs were loaded onto a rGO surface, for the reduction of methylene blue and aerobic oxidation of benzyl alcohol. An electrochemical sensor based on Fe3 O4 NP-coated poly(diallyldimethylammonium chloride)-functionalized graphene has been proposed for sensitive detection of acetaminophen.125 Carbon nanotubes (CNT) are one of the most studied materials in sensing and biosensing applications.126 They consist of cylindrical graphene sheets of nanometer diameter which uniquely combine high electrical conductivity and high chemical stability. Iron oxide NPs have been used in combination with multi-walled carbon nanotubes (MWCNTs) for interesting biosensing electrochemical applications.127 Cheng et al.128 have reported a magnetite NP-based biosensor for detection of DNA hybridization. First, magnetite NPs (Fe3O4) functionalized with mercaptoacetic acid serve as collector for specific sequence of DNA; the polypyrrole/MWCNT modified electrode was then employed for detection (see Figure 16). The proposed DNA biosensor was successfully applied for the identification of a complementary sequence, a non-complementary sequence and three-base-mismatched sequences with high specificity and good reproducibility.


Page 11 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 16. Schematic showing the procedure for the electrochemical detection of DNA hybridization based on a mercaptoacetic acid (RSH) coated Fe3O4 NP labeled oligonucleotide DNA probe. Step A: preparation of the DNA probe; step B: detection of daunomycin (DNR) attached to the DNA probe; step C: hybridization with target sequences and detection. DPV = differential pulse voltammetric detection; GCE = glassy carbon electrode, MWNT = multi-walled carbon nanotubes; ppy = polypyrrole. [Reprinted from ref 128 with permission from Elsevier].

A system based on MWNTs functionalized with carboxylic acid groups (MWNTs–COOH) combined with an iron oxide (Fe3O4) modified GCE (MWNTs– COOH/Fe3 O4/GCE) has been proposed for the electrochemical determination of rutin (RU).129 Electrochemical characterization by cyclic voltammetry and DPV showed well-defined reversible peaks for RU. Moreover, the redox currents for RU on the MWNTs– COOH/Fe3 O4/GCE were about 20 times higher than on a bare GCE. In addition, this work was extended to a cyclic voltammetric investigation of the interaction of RU with DNA. For this purpose, a MWNTs–COOH/Fe3O4/GCE was immersed in a DNA solution containing 1-ethyl-3-(3dimethylaminopropyl)carbodiimide solution (EDC) and Nhydroxysuccinimide solution (NHS) to ensure immobilization of DNA onto the nanocomposite. A dramatic decrease in the RU peak current was observed without any peak potential shift compared to the previous electrode configuration, while the electron transfer coefficient (α) and the rate constant (ks) remained unchanged. These results indicated that RU and DNA formed a non-electroactive complex. The binding constant and binding ratio were first calculated and then confirmed by UV–visible spectroscopy. Purified acetylcholinesterase (AChE) has been covalently immobilized onto a MWCNTs-COOH/magnetite NP modified gold electrode to obtain an organophosphorus sensor.130 This biosensor was explored for monitoring pesticides in tap water and milk samples spiked with malathion, chlorpyrifos, monocrotophos and endosulfan. The recoveries were found to be between 95 and 109% for the aqueous samples and between 103 and 109% for the milk samples, demonstrating the potential of the device for detection of such analytes. The fabrication process of the biosensor and its principles of electrochemical detection are shown in Figure 17.

Figure 17. Schematic showing the fabrication process for an amperometric biosensor comprising acetylcholinesterase (AChE) immobilized on a MWCNTs-COOH/magnetite NP modified gold electrode along with the principles of immobilized AChE inhibition in pesticide solution. [Reprinted from ref 130 with permission from Elsevier].

Multi-walled carbon nanotubes decorated with magnetite NPs (Fe3O4/MWCNTs) have been used as a biosensor for simultaneous detection of adenine and guanine.131 Electrochemical characterization revealed a higher electron transfer rate of Fe3O4/MWCNTs modified electrode compared to that observed for non-modified one. Relatively large peak potential separation (~300 mV) of adenine and guanine on such modified electrodes enables their simultaneous determination in fish sperm DNA with a very low detection limit. Carbon nanotubes decorated with Fe3O4 have also been used for the construction of an amperometric biosensor for the detection of phenolic compounds.118 This biosensor was based on covalent immobilization of laccase (EC 1.10.3.2) onto a (MWCNTs-COOH)-polyaniline (PANI)-magnetite NPs (Fe3O4) composite that was afterwards electrodeposited onto a gold electrode. Cyclic voltammetry and electrochemical impedance spectroscopy were used for the characterization of modified electrodes. The response of this biosensor was within 3 s when applying + 0.3 V vs. Ag/AgCl under optimal conditions. Two linear concentration ranges (0.1–10 µM and 10-500 µM) were observed, with a detection limit of 0.03 µM. To demonstrate the practical use of this biosensor, the total phenolic content of a tea leaf extract was determined. When stored at 4°C, the enzyme electrode lost 25% of its initial activity after 150 uses over a period of 4 months. Magnetic composite consisting of polymorphous iron oxides (FeyOx), multi-walled carbon nanotubes and chitosan, and decorated with Pt nanoparticles and glucose oxidase, was used as sensitive amperometric sensor for glucose detection.132 The developed biosensor showed, under optimal conditions, detection limits of 2.0 µM and linear dependence of the catalytic current upon glucose concentration over wide range (6.0 µM – 6.2 mM). Another example of a modified iron oxide NP composite that has been used for glucose sensing is Fe3O4@SiO2/MWCNTs.133 Magnetic mesoporous materials, e.g., mesoporous γFe2O3/carbon,134 have also been described in the literature.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Sharma et al.20 have compared the electrochemical sensing behavior toward cholesterol of Fe3O4 coated with carbon (Fe3O4@carbon) and α-Fe2O3 NPs. They found comparable sensitivities for both biosensors, suggesting that encapsulation of Fe3O4 with carbon does not affect the electrocatalytic properties of Fe3O4. Sensing Applications of Organically Modified Iron Oxides Nanoparticles. Surface modification of iron oxide NPs with various organic molecules or polymers has also been extensively studied for electrochemical biosensing applications. Such composite materials are attractive as supports for enzymes and/or biomolecules, offering scope for new types of sensing tools (e.g., electrochemical immunosensors) to be developed. An example of a reagentless electrochemical sensor for H2O2 determination has been described by Miao et al.135 Commercial magnetite NPs (Ocean NanoTech, Springdale, AR, USA) were assembled into a multiple layer-by-layer structure and used as peroxidase mimics. Polythionin was first electrodeposited onto the surface of a GCE to introduce amino groups available for further derivatization. The fabrication process is shown schematically in Figure 18. In this process, MWCNTs-COOH, amino-functionalized magnetite NPs and thionin monomers were alternately anchored onto the surface of polythionin-functionalized GCE by carbodiimide or glutaraldehyde chemistry to generate three layers of both magnetite NPs, and thionin as electron transfer mediator. The multilayer modified electrode exhibited an excellent electrochemical response toward the reduction of H2O2. Yantasee and co-workers136 have functionalized magnetite (Fe3O4) NPs (20 nm diameter) with dimercaptosuccinic acid and prepared a carbon paste and GCE sensor to detect lead in urine and copper, lead, cadmium, and silver in natural water. The proposed sensor exhibited a detection limit lower than 1 ppb and the added advantage that the potential could be fully automated via an electromagnet. The authors also reported the use of colorimetric, optical and MRI as detection tools.

Figure 18. Scheme showing the fabrication process for a polythionin-functionalized GCE modified by iron oxide magnetic nanocrystal (IOMN). [Reprinted from ref 135 with permission from Elsevier].

A glucose biosensor has been successfully developed by simply drop-coating ferricyanide-dextran coated iron oxide (DCIO) NPs onto a screen-printed carbon electrodes and then applying a glucose oxidase overlayer.137 Polyaniline (PANI) coated magnetite NPs have also proposed for

Page 12 of 24

glucose sensing.138 By combining the properties of carbon nanotubes with the high conductivity of PANI, the resulting biosensor (shown schematically in Figure 19) showed improved sensitivity.

Figure 19. Preparation of a polyaniline-Fe3O4-carbon nanotube composite with immobilized glucose oxidase for glucose sensing. [Reprinted from ref 138 with permission from John Wiley & Sons, Inc.].

Yadav et al.139 have been co-immobilized creatininase and sarcosine oxidase onto magnetite NPs coated with CH and grafted with PANI (Fe3O4/CHIT-g-PANI). This composite film was afterwards electrodeposited onto the surface of a platinum electrode by glutaraldehyde coupling. The resulting multi-enzyme electrode (Fe3O4/CHIT-gPANI/Pt) was characterized by cyclic voltammetry, scanning electron microscopy, Fourier transform infrared spectroscopy and electrochemical impedance spectroscopy, and applied for the detection of creatinine. A good sensitivity (3.9 µA µM–1 cm–2) and detection limit (1 µM, S/N = 3) was obtained. The biosensor showed only a 10% loss after 120 uses over 200 days when stored at 4°C. The bioelectrode was tested in human serum samples and the results were in agreement with the standard colorimetric method. Urease covalently attached on poly(glycidylmethacrylate) (PGMA)-grafted magnetite NPs and deposited onto a gold electrode has been used for the fabrication of a potentiometric urea biosensor.140 The potentiometric response was measured as a function of urea concentration in phosphate buffer solution, showing a linear dependence over the range of 0.25–5.0 mM of urea. Putrescine (1,4-diaminobutane), diamine derived from arginine, was shown to be detected using diamine oxidase (DAO) covalently immobilized on iron oxide NPs with subsequent detection of hydrogen peroxide.141 Detection limit was established as 0.65 nM and it was found that common interfering molecules (e.g. ascorbic acid) did not disturb the measurements.


Page 13 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


An example of environmental sensing based on iron oxide NPs has been described by Cevik et al.,142 who proposed a biosensor for the detection of phenol derivatives, such as phenol, catechol, p-cresol, 2aminophenol and pyrogallol. Magnetite NPs were first coated with a telomere of poly(glycidylmethacrylate) (PGMA) in order to obtain a good enzyme immobilization platform. The procedure used for the synthesis of the telomere and coating of the NPs is shown schematically in Figure 20. Horseradish peroxidase (HRP) was afterwards immobilized onto a modified Au electrode by covalent bonding. The results demonstrated that the immobilized HRP retained its activity, suggesting that PGMA coated iron oxide NPs may offer a suitable platform for the immobilization of various types of enzymes.

Figure 20. Schematic showing the synthesis of poly(glycidylmethacrylate)-grafted Fe3O4. [Reprinted from ref 142 with permission from Elsevier]. Iron Oxide Nanoparticles-Biological Macromolecule Composites and Development of Immunosensors and DNA Sensors. The detection of biological and chemical pathogens, contaminants and other important analytes plays a crucial role in the prevention of disease spread, infections and pathologies.143 Enzyme linked immunosorbent assay (ELISA) is the most widely used technique for detection of both antigens and antibodies.144 However, electrochemical methods now offer a promising alternative to this approaches. The possibilities of miniaturization in modern microelectronics has allowed microelectrodes to be developed that can be multiplexed and are effective for detection in very small volumes of samples.145 Electrochemical immunosensors (see Figure 21) are usually obtained through the immobilization of antigen or antibody on the electrode surface, following by injection of a secondary enzyme-labeled antibody and addition of an appropriate enzymatic substrate. The enzymatic reaction generates an electroactive molecule that gives rise to an electrochemical signal.145,146

E

E

enzyme label antigen (target) primary antibody secondary enzymeconjugated antibody

electrode surface Figure 21. Schematic of an electrochemical immunosensor.

The recognition element can be also immobilized onto a separate support, i.e., magnetic- or nano-particles, which is afterwards used for electrode modification to measure the enzymatic product.145 Magnetic NPs (i.e., Fe2O3 or Fe3O4) covered with a thin polymeric shell have most commonly been used for this purpose because they can easily be functionalized with different linkage groups to ensure the fast and specific immobilization of the recognition element.147 Ferrocene-modified magnetite NPs have been used as an electrochemical label in ultrasensitive sandwich-type electrochemical immunosensors for the detection of a cancer biomarker prostate specific antigen (PSA).148 Graphene sheets were used as the sensing platform and magnetite NPs functionalized with ferrocene as the electroactive label (see Figure 22). The label was fabricated as follows. Dopamine (DA) was first anchored onto the magnetite surface, followed by conjugation of ferrocene monocarboxylic acid (FC) and a secondary-antibody (Ab2) onto Fe3O4 through the amino groups of DA (DA-Fe3O4FC-Ab2). The high number of DA molecules anchored to the Fe3O4 surface increased the immobilization of ferrocene and Ab2 onto the magnetite NPs, which in turn increased the sensitivity of the immunosensor. Graphene sheets were used most likely to increase the surface area for capturing a greater amount of primary antibodies (Ab1) as well as for enhancing the detection sensitivity of ferrocene.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 22. Schematic showing the fabrication of DAFe3O4-FC-Ab2 nanoparticles. [Reprinted from ref 148 with permission from Elsevier].

Page 14 of 24

Dumbell-like Au/Fe3 O4 NPs have also been used for the fabrication of an electrochemical immunoassay for PSA detection.149 Core-shell Fe2O3/Au magnetic NPs with covalently immobilized α-fetoprotein antibody have been used in a direct electrochemical immunoassay system.150 Modified NPs were attached to the surface of a carbon paste electrode by means of a permanent magnet to generate a sensor for detecting α-fetoprotein antigen via the change in current or potential measured at the electrode, thereby allowing the antibody-antigen interaction to be studied. In another paper, the same authors proposed a magnetic core/porous shell (CoFe2O4/SiO2) composite system for cancer antigen CA 15-3 detection in clinical immunoassays.151 The principle of such a device is shown in Figure 23. This core/shell nanostructure showed a good capability for the selective attachment of CA 15-3 antibodies. Core-shell NPs based on epoxysilane-modified Fe3O4/SiO2 have been developed by Pan and Yang for carcinoembryonic antigen (CEA) determination in human serum by means of a magneto-controlled immunosensing strategy using flow-injection electrochemical impedance spectroscopy.152 Determination of CEA is particularly important because it was found that CEA appears in the blood of people who have certain kind of cancer, e.g., rectal or colon cancer.153 Thus, the early diagnosis can help in the tumor treatment. Electrochemical impedance spectroscopy (EIS) was used to investigate the immune reaction in the presence of the redox couple. In addition, the process could be switched between “on” and “off” states simply by positioning the external magnet below and above the cell (see scheme in Figure 24).

Figure 24. Schematic showing the regeneration procedure for an immunoassay system based on carcinoembryonic antibodies immobilized on epoxysilane-modified Fe3O4/SiO2 core-shell nanoparticles. CPE = carbon paste electrode. [Reprinted from ref 152 with permission from Springer].

Figure 23. Schematic showing the principles of (a) (bio)magnetic nanoparticle fabrication, and (b,c) their immobilization onto an electrode surface and detection of CA 15-3 antibodies. [Reprinted from ref 151 with permission from John Wiley & Sons, Inc.].

Another system for the detection of CEA and αfetoprotein has been proposed by Zhuo et al.104 Three-layer magnetic NPs comprising an Fe3O4 magnetic core, Prussian Blue interlayer and gold shell, which were functionalized by HRP and glucose oxidase, were developed in order to amplify the signal of the proposed electrochemical immunosensor. The detection limit was found to be of the order of pg/mL, approximately 50-fold more sensitive than an analogous system. A multilayered coating of gold NPs on core-shell Au/Fe3O4 have been used as strategy for increasing the specific surface area for CEA antibody loading.154 The authors found out that the electrochemical immunosensor based on this strategy was 100–1000 times


Page 15 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


more sensitive than achievable with ELISA. This highlights some of the main advantages of using an electrochemical immunoassay based on iron NPs because they are easy to prepare and can be quickly regenerated at low cost. Several other analytes have been studied using iron oxide based electrochemical immunosensors. Determination of ochratoxin A (OTA) has been described using a CH-iron oxide (Fe3O4) nanocomposite as a platform for the immobilization of rabbit immunoglobulin antibodies (IgGs).155 Further, an electrochemical metallo-immunoassay has been suggested for the detection of hepatitis B surface (HBs) antigen based on copper-enhanced gold NPs and magnetic separation.156 The principle of the latter immunosensor is shown schematically in Figure 25. Magnetic NPs modified by hepatitis B surface HBs antibody, HBs antigen solution and an antibody-colloidal gold conjugate were first incubated and then subjected to magnetic separation and a washing step to generate a sandwich-type complex suspension. A copper enhancer solution was then added to this suspension and the magnetic separation and washing procedure was repeated once again. Finally, nitric acid was added to dissolve copper and the resulting solution containing copper ions was transferred to an electrochemical cell where anodic stripping voltammetry was performed. The stripping peak current was proportional to the amount of HBs antigen in the standard solution or sample.

Figure 25. Schematic showing the fabrication process for an electrochemical immunosensor based on copper-enhanced colloidal gold and antibody-immobilized Fe3O4 nanoparticles for hepatitis B surface antigen detection. HBsAb-MNP = hepatitis B surface antibody immobilized magnetic nanoparticles; HBsAg = hepatitis B surface antigen; HBsAbGNP = hepatitis B surface antibody-gold nanoparticles. [Reprinted from ref 156 with permission from Elsevier].

An ultrasensitive electrochemical immunosensor based on CH-magnetite-poly(amino-amine) dendrimers-gold NP (CH-Fe3O4-PAMAM-Au) nanocomposites and HRPMWNTs-antibody (HRP-MWCNTs-Ab) bioconjugates has been developed for the detection of salbutamol (SAL).157 The fabrication process for such an immunosensor is shown schematically in Figure 26. The CH-Fe3O4-PAMAM-Au nanocomposite was used as an immobilization matrix to enhance the electroactivity and stability of the electrode. HRP-MWCNTs-Ab bioconjugates were used as a label to improve the catalytic activity for hydrogen peroxide reduction at the electrode. Under optimized conditions, a calibration plot for SAL was obtained with a linear range of between 0.11 and 1061 µg L–1 and the detection limit was 0.06 µg L–1.

In the last few years, a variety of sensitive DNA or RNA biosensors have been developed for the detection of bacteria, viruses and various chemical substances. Such sensors have displayed attractive features, such as excellent selectivity, high sensitivity, low cost and possibility of onsite measurements.158 Magnetic NPs offer a versatile tool for electrochemical DNA biosensing because, as already mentioned, they can be easily separated from the liquid phase by application of a magnet and then re-disperse immediately after the magnet is removed. An important advantage of DNA electrochemical biosensors over the other types of DNA sensors is their low-cost and simply fabrication.159 Moreover, they can be developed to be compatible with modern microfabrication technologies and independent of sample turbidity or other optical pathways.160,161 Most current electrochemical DNA biosensors rely on the immobilization of single-stranded (ss) oligonucleotides onto an electrode surface labeled with an electrochemical indicator to recognize the complementary target sequences.159,162

Figure 26. Schematic showing the stepwise immunosensor fabrication process. HRP = horseradish peroxidase; SAL = salbutamol; Ab = antibody. [Reprinted from ref 157 with permission from Elsevier].

Figure 27. Schematic showing the main steps of DNA-AuHRP-Fe3O4 biconjugate preparation. PSS = poly(sodium 4styrenesulfonate); PDDA = poly(dimethyldiallylammonium chloride); HRP = horseradish peroxidase. [Reprinted from ref 164 with permission from Elsevier].



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Enzyme-assisted signal amplification strategies have attracted interest in the field of DNA biosensors owing to the outstanding catalytic properties and biocompatible performance of enzymes.163 Horseradish peroxidase modified Fe3O4 NPs have been proposed as a signal amplification platform for the detection of sequence specific DNA.164 HRP was adsorbed onto the surface of NPs via a layer-by-layer technique and then used together with a gold nanofilm as a substrate for anchoring the capture probe. The fabrication strategy for DNA-Au-HRPFe3O4 bioconjugates is shown in Figure 27. A DNA sensor using iron oxide NPs and an enzymatic amplification strategy has also been proposed by Loaiza et al.111 for the detection of specific hybridization processes. In this work, a 19 thiolated 19-mer capture probe was first attached to gold coated Fe3O4 NPs and then streptavidin peroxidase was bonded to the biotinylated target. Afterwards, an external magnetic field was used for iron oxide NP immobilization onto the electrode surface. The hybridization process was detected after the addition of hydrogen peroxide using hydroquinone as mediator. An electrochemical DNA sensor for pyrethroid (artificial pesticides) detection has been fabricated by immobilizing a single strand calf thymus DNA (ssCT-DNA) onto CHmodified magnetite NPs.165 The biocomposite electrode was successfully used for the detection of cypermethrin and permethrin with satisfactory detection limits. Electrochemical impedance spectroscopy together with magnetic NPs (containing about 70% of iron oxide) functionalized by streptavidin and immobilized on a gold electrode has been used for virus detection, namely HIV and HBV.166 In this case, target DNA hybridization induced an increase of the impedance signal, indicating an increase in resistance. Short HIV-1 sequences have also been detected on a CH-coated Fe3O4 based screen-printed electrode.167 An electrochemical DNA biosensor incorporating iron oxides coated with poly-xanthurenic acid has been described by Zhang et al.168 for the specific detection of a PML/RARA fusion gene involved in acute promyelocytic leukemia. An electrochemically fabricated nano-composite film consisting of CH-magnetite (Fe3O4) NPs with immobilized biotinylated probe DNA (BDNA) has been used to detect Neisseria gonorrhoeae, the bacterium responsible for the sexually transmitted disease gonorrhea, using avidin-biotin coupling for rapid and specific DNA hybridization.169 The presence of Fe3O4 NPs increased the electroactive surface area and provided a suitable platform for DNA loading with proper conformation. Differential pulse voltammetric studies were carried out in the presence of methylene blue (MB) as a mediator to facilitate electron transfer. Electrochemical detection of the same bacterium (N. gonorrhoeae) was also performed using a nanocomposite consisting of PANI-coated iron oxide NPs coupled with carbon nanotubes with an immobilized biotinylated nucleic acid probe sequence of 20 bases.170 The biosensor (see Figure 28) was utilized for the detection of DNA extracted from a N. gonorrhoeae culture and infected patient samples. The sensor exhibited good specificity for N. gonorrhoeae species and showed no response toward non-gonorrhoeae Neisseria species and other gram-negative bacteria. Magnetite NPs functionalized by 3glycidoxypropyltrimethoxysilane (GOPS) have been used

Page 16 of 24

for the immobilization of 21-mer peptide nucleic acid (PNA) and subsequent detection of Mycobacterium tuberculosis allowing complementary sequences in its genomic DNA to be detected without the need for PCR amplification.171 On top of that, PNA has been shown to exhibit a higher specificity in the recognition of the DNA sequences and permits the use of shorter probes.172

Figure 28. Schematic showing the fabrication process for polyaniline (PANI) coated Fe3O4 and immobilization of biotinylated DNA using biotin-avidin coupling followed by hybridization for bacterial detection. [Reprinted from ref 170 with permission from Elsevier].

Detection of Bacillus anthracis, a cause of anthrax, has attracted a considerable attention of researcher groups some years ago. Pal and coworkers173 used PANI-coated magnetite NPs for electrochemical detection of these bacterial cells via determination of B. anthracis pag A gene from complex matrices. The detection scheme is shown in Figure 29 and can be described in the following way: the electrochemical sandwich assay consists of a detector DNA probe that is labeled with Fe3O4@PANI NPs, and a capture DNA probe labeled with biotin. After hybridization with DNA targets, Fe3O4@PANI/detector probe-DNA/capture probe-biotin hybrid is formed. This hybrid is then separated from unreacted DNA and non-complementary sequences by magnetic separation stand and used for the electrodes modification. PANI@γ-Fe2O3 NPs have also been applied in a direct-charge transfer biosensor for detecting the anthrax Sterne strain (34F2) of Bacillus anthracis.174 Osaka and coworkers175 have monitored the interaction between biotin and streptavidin using magnetic NPs. They proposed a sensing platform based on chemically driven NP sedimentation. The biomolecular interaction between biotin and streptavidin was successfully detected by using streptavidin-modified magnetite (Fe3O4) NPs and a substrate modified by self-assembled monolayers (SAMs) of biotin. A solution containing streptavidin-modified


Page 17 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


magnetic NPs was loaded into a channel formed on a glass plate. The NPs were then specifically bound to the biotinmodified substrate.

Figure 29. Schematic showing the detection principle of a DNA biosensor based on magnetic nanoparticles. EAM = electrically active magnetic nanoparticles. [Reprinted from ref 173 with permission from Elsevier].

As a result of the swine influenza pandemic, the detection of the influenza A virus (FLUAV) has attracted much attention.176 The pandemic potential relies upon human-tohuman transmissibility, which is dependent upon FLUAV hemagglutinin (HA) specificity for host glycan receptors. Avian FLUAV binds preferentially to alpha 2,3-linked receptors, while human FLUAV binds to alpha 2,6-linked receptors. Iron oxide NPs have been used for detecting the FLUAV H5N1 virus.176 Aniline monomer polymerized around maghemite (γ-Fe2O3) cores served as the basis for a direct electron transfer biosensor developed for the detection of the surface glycoprotein HA from the FLUAV H5N1 (A/Vietnam/1203/04). H5N1 preferentially binds to alpha 2,3-linked host glycan receptors and PANI-modified maghemite NPs (PANI@γ-Fe2O3) were immunofunctionalized with antibodies against the HA target. Glycans pre-incubated with HA in 10% mouse serum were incubated with anti-HA-PANI@γ-Fe2 O3 complexes. The anti-HA-PANI@γ-Fe2O3 complexes effectively enabled the immunomagnetic separation of HA from the mouse serum matrix. Further, the PANI@γ-Fe2O3 NPs served as a transducer for cyclic voltammetry measurements. In this case, the PANI was made electrically conductive by hydrochloric acid doping. The experimental results indicated that the biosensor was able to detect recombinant H5N1 at 1.4 µM in 10% mouse serum, with a high specificity for H5N1 compared to H1 (H1N1 A/South Carolina/1/18). NON-ELECTROCHEMICAL SENSING TECHNIQUES The rapid detection and quantification of low-abundance proteins from biological target samples (body fluids, tissue, etc.) still remains a challenging research topic. The most widely used method of analysis is immunoprecipitation or immunoaffinity separation, followed by measurement of

optical changes. However, immunoassays are time consuming and require relatively large amounts of target protein.177 In contrast, biomolecule-conjugated magnetic NPs can function not only as sensitive biological biosensors but can also be used as a preconcentration or capture probe. An example of such a multiplexed immunoassay is shown in Figure 30.

Figure 30. Schematic showing the fabrication process for polyaniline (PANI) coated Fe3O4 and immobilization of biotinylated DNA using biotin-avidin coupling followed by hybridization for bacterial detection. [Reprinted from ref 177 with permission with permission from John Wiley & Sons, Inc.].

Array-based bioassays based on magnetic NPs are a promising approach for DNA, protein and microbe analysis. Currently, fluorescence and chemiluminescence are used as standard methods for detection on microarrays.175 In the following text, a brief overview of non-electrochemical biosensors based on iron oxide NPs is provided. Magnetic Sensors. There are three main types of magnetic sensors: giant magnetoresistive (GMR), magnetic tunnel junction (MTJ) and SQUID sensors. Compared with the SQUID sensors, the GMR sensors are simpler and more portable. The MTJ sensors have the highest magnetoresistive sensitivity but are still in an early phase of development. Thus, there are relatively few articles in the literature describing their use for analytical purposes.39 Magnetoresistive sensors based on GMR or tunneling magnetoresistance effects have been proposed for the detection of DNA or proteins (see reviews by Freitas et al.178 and Tamanaha et al.179). A common feature of most of these approaches is that magnetic beads are excited by an oscillating applied magnetic field, resulting in a signal detectable by the magnetic field sensor, which, after a washing step, can be compared before and after a sample is introduced. An example of a bioarray using a magnetic label for DNA biorecognition is shown in Figure 31. The principle involves the magnetic labeling of a target molecule, which is then passed over an array on which a complementary molecule (probe) is immobilized. The sensor detects the presence of magnetic labels due to the change in sensor resistance at fixed current. Any unbound target molecules are then washed away and the residual sensor signal depends only on sites where the complementary magnetically labeled target and probe molecule successfully interact.180,181



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 31. Biorecognition assay for the detection of a complementary single strand DNA target biomolecules labeled with magnetic nanoparticles. [Reprinted from ref 180 with permission from MDPI – Open Access Publishing].

A magneto-impedance biosensor for the detection of Fe3O4 magnetic NPs after intracellular uptake has been described by Kumar et al.182 The principle of their experiment was follows. Firstly, magnetic NPs were mixed with a cell sample and then left for uptake inside the cells. After a suitable time, free NPs were removed and then the magneto-impedance in the presence of Fe3 O4 NPs loaded cells was measured. The proposed sensor was tested on human embryo kidney (HEK 293) cells as a model system. Blanc-Beguin et al. have used a giant magneto impedance (GMI) sensor for the magnetic bio-imaging of rat prostate cancer (Mat Ly Lu) cells loaded with maghemite NPs.183 Accumulation of maghemite NPs into the cells was confirmed by optical microscopy, while quantification of the NPs per cell was performed by X-ray fluorescence. Biofunctionalization and detection of manganese ferrite NPs using an MTJ field sensor have been described by Grancharov et al.184 In their work, biotin- or DNAfunctionalized NPs were successfully bound onto an avidin or complementary DNA-functionalized silicon oxide substrate (see Figure 32), showing the potential for introduction of biofunctionalized NPs in both in vitro and in vivo biological environments and sensing capability for proteins and DNA detection.

Page 18 of 24

induced by an aptamer-protein interaction (see schematic illustration in Figure 33). In this work, gold-coated magnetite NPs prepared by reduction of HAuCl4 onto dextran-coated Fe3O4 NPs have been used. Two human alpha-thrombin aptamers, were separately conjugated to two distinct populations of nanostructures, called nanoroses. Once human alpha-thrombin is presented in analyzing solution, the nanoroses start to aggregate and thus cause change in the spin-spin relaxation time (T2) as well as the UV-Vis absorption spectra of the solution. These changes are then used for qualitative and quantitative detection method of human alpha-thrombin. The dual-mode detection was clearly advantageous for obtaining a more reliable result and the detection range was also widened as well. Using this method, a detectable T2 change was observed with 1.0 nM human alpha-thrombin and the detection limit ranged from 1.6 nM to 30.4 nM.

Figure 33. Schematic showing the principles of an aptamerlinked sandwich assay for the detection of thrombin using Fe3O4@Au nanoroses. [Reprinted from ref 185 with permission from Elsevier].

Figure 32. (a) Epifluorescence microscopy image of FITClabeled avidin patterned onto a SiO2 substrate. (b) SEM image of biotinPEG(2000)-PE conjugated MnFe2O4 nanoparticles bound to an avidin patterned SiO2 substrate. (c) SEM image of ssDNA-conjugated MnFe2O4 nanoparticles bound to a SiO2 substrate patterned with complementary ssDNA. [Reprinted from ref 184 with permission from American Chemical Society].

Liang and coworkers185 have described a sensitive biosensing system that is based on magnetic relaxation switch diagnosis with colorimetric detection of human alpha-thrombin based on the aggregation of Fe3O4@Au NPs

Joshi and co-workers have developed a multifunctional implantable system for biosensing, drug delivery and magnetic resonance imaging (MRI) monitoring incorporating an anti-inflammatory drug.186 A glucose biosensor, sodium diclofenac and magnetic NPs were used as biosensor components. The final system, comprising the biosensor, the drug and magnetic NPs loaded inside alginate microspheres had a final size of 10–60 µm. Biosensing studies indicated an excellent glucose response curve, with a regression coefficient of 0.974, over the concentration range of 0–10 mM glucose, and a response time of 4 min. In vitro diclofenac release showed that the magnetic NPs loaded in alginate microspheres increased the burst release percentage by 11–12% in both 60 and 10 µm particles. However, the duration of drug release decreased with the


Page 19 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


size of the magnetic NPs from 7 to 6 days for 60 and 10 µm particles, respectively. Magnetic detection of the cholera toxin B subunit (CTB) has also been reported.187 In this case, galactose conjugated magnetite NPs interacted with CTB resulting in the detectable change in spin-spin relaxation times (T2), and thus demonstrating the interaction between them which was also confirmed by surface plasmon resonance. Fluorecence and Magneto-Fluorescence Sensors. Design of fluorescent biosensors is very complex process involving selection of appropriate macromolecular receptor that has affinity and selectivity to the target and transducing the recognition process into detectable fluorescent signal. The receptor has rarely such transduction function and thus a foreign fluorescent probe, i.e., synthetic fluorophore or fluorescent protein, has to be introduced into the system. Fluorescent biosensors can serve for very sensitive detection of important protein biomarkers and metabolites as well as they can help in drug development because they offer real-time monitoring of dynamic process. For this reason, such biosensors are usually employed in fundamental studies to investigate gene expression, protein localization and activity, function and conformation in a wide variety of fields. Moreover, they are found useful as a sensitive method for detecting the characteristic biomarkers of several pathological factors related to arthritis, viral infection, cancer and metastasis and also to inflammatory, cardiovascular or neurodegenerative diseases.188

liquid sample iron loading.189 Diluted suspensions of colloidal magnetite NPs were supplied in the culture medium to simulate magnetic loading with iron oxides of either environmental waters or human body fluids. Electromagnetic exposure to radiofrequency waves of bacterial samples grown in the presence of magnetic NPs was also carried out (see Figure 34). The inhibitory influence of magnetite NP addition combined with radiofrequency exposure was evidenced from fluorescence data. In a recent study,190 magnetite NPs coated with fluorescent labeled antibodies (chicken IgG labeled with fluorescent dye AlexaFluor647) have been used for target pre-concentration through magnetic separation and also as tracer of signal in an array biosensor. Such arrangement led to improvement of detection limits compared to those reported for conventional array biosensors. In addition, the authors carried out experiments regarding the functionalization of the NPs-IgG- AlexaFluor647 surface with PEG in order to prevent aggregation.

Figure 35. Schematic showing the principles of the fluorescent magnetically derivable nanocatalyst based on rhodamine B isothiocyanate. [Reprinted from ref 191 with permission from Elsevier].

Figure 34. (a) Principles of the proposed biosensor. (b) Electromagnetic exposure cell to subject samples to radiofrequency waves. [Reprinted from ref 189 with permission from European Optical Society].

Fluorescence emission of pyoverdine (the siderophore synthesized by iron scavenging bacteria) has been studied using in vitro cultures of Pseudomonas aeruginosa in initial work toward the development of a biosensor system for

Maghemite NPs (γ-Fe2O3) stabilized with OH- groups can enable the simple electrostatic immobilization of positively charged organic molecules and thus can be used as universal nanocarriers for the construction of magnetofluorescent biosensors). Rhodamine B isothiocyanate has been used to generate a fluorescent magnetic nanocarrier whilst at the same time serving as a spacer molecule for the covalent immobilization of glucose oxidase (see Figure 35).191 Surface Plasmon Resonance Sensing. Biosensors based on surface plasmon resonance (SPR) have become most advanced and developed label-free optical biosensor technology useful for powerful detection and analysis of kinetic properties of biomolecules and their interactions. SPR sensing is based on the measurement of changes in the refractive index caused by biomolecular interactions occurring at the biosensor surface.192,193 On the other hand, it is well known that this technique is not suitable for the detection of small molecules present in a low concentration in the sample as their interactions do not



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

give detectable refractive index changes. For this reason, amplification based on magnetic NPs have been proposed in some works. Wang et al.194 have demonstrated that magnetite (Fe3O4) NPs can enhance the signal of SPR spectroscopy and developed Fe3O4 NPs-aptamer conjugates that can be used as powerful reagent for immunoassays. This sensor was employed for thrombine detection based on its capturing by antithrombin aptamer immobilized on SPR gold film while Fe3O4 NPs-aptamer conjugates were used for amplification. Magnetic NPs with a magnetite (Fe3O4) core have been used for the construction of a highly sensitive SPR biosensor.195 In this work, magnetic NPs served partially for fast delivery of analyte from sample to the sensor surface and also for enhancing refractive index changes. Experimental set-up combined grating-coupled surface plasmon resonance (GC-SPR) and magnetic field gradient (see Figure 36) in order to facilitate manipulation with magnetic NPS on the SPR chip surface. Performance of the biosensor was investigated on immunoassay-based detection of β-human chorionic gonadotropin (β-hCG) and the results revealed that the developed system improved the sensitivity of detection by 4 orders of magnitude with respect to the conventional SPR sensors; in addition, the limit of detection was found in the range of pM. An amplification technique using core-shell Fe3O4@Au NPs for an SPR bioassay has been proposed by Liang et al.196 Their sandwich-type immunoassay was used for the detection of alpha-fetoprotein (AFP) based on the immobilization of primary AFP antibody (Ab1) on the surface of a 3-mercapto-1-propanesulfonate/CHferrocene/Au nanoparticles with secondary antibody conjugate with Fe3O4@Au-AFP for amplification.

Figure 36. Schematic showing different methods of detection at an SPR chip: (a) direct detection, (b) sandwich assay with amplification by detection antibody, (c) and (d) magnetic NPs-antigen without (c) or with (d) applied magnetic field, (e) preincubation of magnetic NPs-antigen with β-hCG followed by sandwich assay in the presence of an applied magnetic field gradient. [Reprinted from ref 195 with permission from American Chemical Society]. CONCLUSIONS AND CHALLENGES The goal of the review was to provide an overview of recent trends in the field of electrochemical and/or chemical sensing based on iron oxide NPs, including details on their surface modification and composites with noble metals or carbon nanostructures. The exploitation of nanomaterials in electroanalysis has resulted in the enhancement of sensor

Page 20 of 24

performance owing to (i) the increased electrode surface area and (ii) enhanced electron transfer related to the catalytic ability of iron oxide NPs. The unique properties of magnetic NPs and the ability to control their magnetization and localization by application of an external magnetic field has stimulated evolution of a number of medical applications, such as molecular sensing, detection of biological and chemical pathogens, drug delivery and cell purification. Moreover, magnetic NP labeling processes are relatively simple and the biochemical activity of the labeled compound remains unaffected. Future developments are likely to focus on hybrid materials, e.g., iron oxide composites with graphene, carbon dots or core-shell structures with noble metals, because such engineered materials hold particular promise for the fabrication of many biomedical devices, nanocarrier drug delivery systems, immunoassays or DNA biosensors. Besides, the use of magnetic NPs as labels for biological molecules offers some advantages compared to the traditional fluorescent markers. They have almost unlimited life-time while fluorescent dyes undergo to photobleaching. Alongside, the sensitivity of sensors based on magnetic NPs or fluorescent tagging is nowadays comparable. The promising way is also related to the development of dual magneto-fluorescent sensors based on combination of iron oxide NPs with quantum dots including carbon dots with excellent biocompatibility.197 Similarly, the hybrids composed of iron oxide NPs with attached noble nanometals (gold, silver) seem to be selective sensors of various biomolecules exploiting the primary magnetic separation and secondary SERS detection of the biomolecule.198 Nevertheless, there are still several challenges in the development of biomagnetic sensors. For example, magnetic nanoparticle labels work well with big molecules such as DNA but they are less efficient in detection of the smaller molecules (such as proteins). Similarly, the applications of nano-magnetite require the suitable surface stabilization to prevent its possible oxidation during the sensing process. In addition, attention should be devoted to the miniaturization of such devices and in vivo biomedical applications.

AUTHOR INFORMATION Corresponding authors Phone: +39-049-8276863, Fax: 0039-049-8073310, E-mail address: [email protected] (Fabio Vianello); Phone: +420-58-563-4337, Fax: +420-58-563-4761, E-mail address: [email protected] (Radek Zboril). Notes $ These authors contributed equally. The authors declare no competing financial interest. ACKNOWLEDGMENTS The authors acknowledge the support by the Operational Program Research and Development for Innovations – European Regional Development Fund (CZ.1.05/2.1.00/03.0058) and Operational Program Education for Competitiveness – European Social Fund (CZ.1.07/2.3.00/20.0017, CZ.1.07/2.3.00/20.0170, CZ.1.07/2.3.00/20.0155, and CZ.1.07/2.3.00/20.0058) of


Page 21 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


the Ministry of Education, Youth and Sports of the Czech Republic. The authors deeply thank to Prof. Dr. Michal Otyepka (Regional Centre of Advanced Technologies and Materials, Faculty of Science, Palacky University, Olomouc, Czech Republic) for the artwork help.

REFERENCES (1) Janata, J.; Josowicz, M.; Vanysek, P.; DeVancy D. M. Anal. Chem. 1998, 70, 179R–208R. (2) Wang, J. Analytical Electrochemistry; VCH Publishers: New York, U.S.A., 1994. (3) Bratov, A.; Abramova, N.; Ipatov, A. Anal. Chim. Acta 2010, 678, 149–159. (4) Bakker, E.; Bühlmann, P.; Pretsch, E. Chem. Rev. 1997, 97, 3083–3132. (5) Walcarius, A. In Chemical and Biological Microsensors: Apllication in Liquid Media, Fouletier, J.; Fabry, P., Eds.; John Wiley & Sons, Inc.: Hoboken, New Jersey, U.S.A., 2013. (6) Vianello, F.; Boscolo-Chio, R.; Signorini, S.; Rigo, A. Biosens. Bioelectron. 2007, 22, 920–925. (7) Vianello, F.; Zennaro, L.; Rigo, A. Biosens. Bioelectron. 2007, 22, 2694–2699. (8) Eggins, B. R. Analytical Techniques in the Science: Chemical Sensors and Biosensors; John Wiley & Sons, West Sussex, 2002. (9) Grieshaber, D.; MacKenzie, R.; Voros, J.; Reimhult, E. Sensors 2008, 8, 1400–1458. (10) Katz, E.; Willner, I. Electroanalysis 2003, 15, 913–947. (11) Glavee, G. N.; Klabunde, K. J.; Sorensen, C. M.; Hadjipanayis, G. C. Inorg. Chem. 1995, 34, 28–35. (12) Park, J.; An, K.; Hwang, Y.; Park, J.-G.; Noh, H.-J.; Kim, J.-Y.; Park, J.-H.; Hwang, N.-M.; Hyeon, T. Nat. Mater. 2004, 3, 891–895. (13) Gupta, A. K.; Gupta, M. Biomaterials 2005, 26, 3995–4021. (14) Mornet, S.; Vasseur, S.; Grasset, F.; Duguet, E. J. Mater. Chem. 2004, 14, 2161–2175. (15) Laurent, S.; Forge, D.; Port, M.; Roch, A.; Robic, C.; Elst, L. V.; Muller, R. N. Chem. Rev. 2008, 108, 2064–2110. (16) Machala, L.; Zboril, R.; Gedanken, A. Chem. Mater. 2007, 111, 4003–4018. (17) Machala, L.; Tucek, J.; Zboril, R. Chem. Mater. 2011, 23, 3255– 3272. (18) Lee, N.; Hyeon, T. Chem. Soc. Rev. 2012, 41, 2575–2589. (19) Zhang, L.; Dong, W.-F.; Sun, H.-B. Nanoscale 2013, 5, 7664– 7684. (20) Tucek, J.; Zboril, R.; Namai, A.; Ohkoshi, S. Chem. Mater. 2010, 22, 6483–6505. (21) Li, P.; Miser. D. E.; Rabiei, S.; Yadav, R. T.; Hajaligol, M. R. Appl. Catal. B 2003, 43, 151–162. (22) Randall, H.; Doepper, R.; Renken, A. Appl. Catal. B 1998, 17, 357–369. (23) Shi, Y.; Fan, M. Ind. Eng. Chem. Res. 2007, 46, 80–86. (24) Lin, Y.; Zhou, S.; Sheehan, S. W.; Wang, D. J. Am. Chem. Soc. 2011, 133, 2398–2401. (25) Li, S.; Meitzner, G. D.; Iglesia, E. J. Phys. Chem. B 2001, 105, 5743–5750. (26) Huang, C. P.; Huang, Y. F.; Cheng, H. P.; Huang, Y. H. Catal. Commun. 2009, 10, 561–566. (27) Pham, A. L.-T.; Lee, C.; Doyle, F. M.; Sedlak, D. L. Environ. Sci. Technol. 2009, 43, 8930–8935. (28) Castro, A. J. R.; Soares, J. M.; Filho, J. M.; Oliveira, A. C.; Campos, A.; Milet, E. R. C. Fuel 2013, 108, 740–748. (29) Alonso, D. A.; Naja, C.; Pastor, I. M.; Yus, M. Chem. Eur. J. 2010, 16, 5274–5284. (30) Ahmed, N.; Fessi, H.; Elaissari, A. Drug Discov. Today 2012, 17928–17934. (31) Huang, J.; Zhong, X.; Wang, L.; Yang, L.; Mao, H. Theranostics 2012, 2, 86–102. (32) Ho, D.; Sun, X.; Sun, S. Acc. Chem. Res. 2011, 44, 875–882. (33) Nöll, T.; Nöll, G. Chem. Soc. Rev. 2011, 40, 3564–3576. (34) Turner, A. P. Chem. Soc. Rev. 2013, 42, 3184–3196. (35) Sleutels, T. H.; Ter Heijne, A.; Buisman, C. J.; Hamelers, H. V. ChemSusChem 2012, 5, 1012–1019. (36) Tischer, W.; Wedekind, F. Top. Curr. Chem. 1999, 200, 95–126.

(37) Zelinski, T.; Waldmann, H. Angew. Chem. Int. Ed. 1997, 36, 722–724. (38) Urbanova, V.; Kohring, G.-W.; Klein, T.; Wang, Z.; Mert, O.; Emrullahoglu, M.; Buran, K.; Demir, A. S.; Etienne, M.; Walcarius, A. Z. Phys. Chem. 2013, 227, 667–689. (39) Beveridge, J. S.; Stephens, J. R.; Williams, M. E. Annu. Rev. Anal. Chem. 2011, 4, 251–273. (40) Hsing, I.-M.; Xu, Y.; Zhao, W. Electroanalysis 2007, 19, 755– 768. (41) Shi, X.; Gu, W.; Li, B.; Chen, N.; Zhao, K.; Xian, Y.; Microchim. Acta 2013, DOI: 10.1007/s00604-013-1069-5. (42) Hernández-Santos, D.; González-García, M. B.; García, A. C. Electroanalysis 2002, 14, 1225–1235. (43) Breslow, R. Acc. Chem. Res. 1995, 28, 146–153. (44) Wei, H.; Wang, E. Anal. Chem. 2008, 80, 2250–2254. (45) Guenter, W. Chem. Rev. 2002, 102, 1–27. (46) Aissaoui, H.; Bachmann, R.; Schweiger, A.; Woggon, W. D. Angew. Chem. Int. Ed. 1998, 37, 2998–3002. (47) Gao, L.; Zhuang, J.; Nie, L.; Zhang, J.; Zhang, Y.; Gu, N.; Wang, T.; Feng, J.; Yang, D.; Perrett, S.; Yan, X. Nat. Nanotechnol. 2007, 2, 577–583. (48) Hermanek, M.; Zboril, R.; Medrik, I.; Pechousek, J.; Gregor, C. J. Am. Chem. Soc. 2007, 129, 10929–10936. (49) Liu, Y.; Yu, F. Nanotechnology 2011, 22, 145704. (50) Lin, M. S.; Leu, H. L. Electroanalysis 2005, 17, 2068–2073. (51) Wang, S.; Tan, Z.; Zhao, D.; Liu, G. Biosens. Bioelectron. 2008, 23, 1781–1787. (52) Magro, M.; Baratella, D.; Pianca, N.; Toninello, A.; Grancara, S.; Zboril, R.; Vianello, F. Sens. Actuat. B-Chem. 2013, 176, 315–322. (53) Xu, P.; Zeng, G. M.; Huang, D. L.; Feng, C. L.; Hu, S.; Zhao, M. H.; Lai, C.; Wei, Z.; Huang, C.; Xie, G. X.; Liu, Z. F. Sci. Total Environ. 2012, 424, 1–10. (54) Sanderson, W. R. Pure Appl. Chem. 2000, 72, 1289–1304. (55) Sunil, K.; Narayana, B. Contam. Toxicol. 2008, 81, 422–426. (56) Towne, V.; Will, M.; Oswald, B.; Zhao Q. Anal. Biochem. 2004, 334, 290–296. (57) Chen, W.; Cai, S.; Ren, Q. Q.; Wen, W.; Zhao, Y. D. Analyst 2012, 137, 49–58. (58) Zhang, L. H.; Zhai, Y. M.; Gao, D.; Wen, D., Dong S. J. Electrochem. Commun. 2008, 10, 1524–1526. (59) Magro, M.; Valle, G.; Russo, U.; Nodari, L.; Vianello, F. Maghemite Nanoparticles and Method for Preparing Thereof. Patent No. PCT/EP2010/060486, 2010. (60) Baratella, D.; Magro, M.; Sinigaglia, G.; Zboril, R.; Salviulo, G.; Vianello, F. Biosens. Bioelectron. 2013, 45, 13–18. (61) Cheng, K.; Peng, S.; Xu, C.; Sun, S. J. Am. Chem. Soc. 2009, 131, 10637–10644. (62) Xu, H.; Shao, M.; Chen, T.; Zhuo, S.; Weu, C.; Peng, M. Micropor. Mesoporous. Mater. 2012, 153, 35–40. (63) Hrbac, J.; Halouzka, V.; Zboril, R.; Papadopoulos, K.; Triantis T. Electroanalysis 2007, 19, 1850–1854. (64) Li, J.; Gao, H.; Chen, Z.; Wei, X.; Yang, C. F. Anal. Chim. Acta 2010, 665, 98–104. (65) Gorton, L. Electroanalysis 1995, 7, 23–45. (66) Ricci, F.; Amine, A.; Palleschi, G.; Moscone, D. Biosen. Bioelectron. 2003, 18, 165–174. (67) Liu, Z.; Lv, B.; Xu, Y.; Wu, D. J. Mater. Chem. A 2013, 1, 3040–3046. (68) Zhang, L.; Ni, Y.; Wang, X.; Zhao, G. Talanta 2010, 82, 196– 201. (69) Maiyalagan, T.; Sundaramurthy, J.; Kumar, P. S.; Kannan, P.; Opallo, M.; Ramakrishna, S. Analyst 2013, 138, 1779–1786. (70) Cao, X.; Wang, N. Analyst 2011, 136, 4241–4246. (71) Fang, B.; Wang, G.; Zhang, W.; Li, M.; Kan X. Electroanalysis 2005, 17, 744–748. (72) Cao, X.; Xu, Y. J.; Wang, N. Electrochim. Acta 2012, 59, 81–85. (73) Rahman, M. M.; Khan, S. B.; Faisal, M.; Asiri, A. M.; Abu Tariq, M. Electrochim. Acta 2012, 75, 164–170. (74) Rahman, M. M.; Jamal, A.; Khan, S. B.; Faisal, M. Superlatt. Microstuct. 2011, 50, 369–376. (75) Salek, P.; Korecka, L.; Horak, D.; Petrovsky, E.; Kovarova, J.; Metelka, R.; Cadkova, M.; Bilkova, Z. J. Mater. Chem. 2011, 21, 14783–14792. (76) Zhang, Y.; Zhou, D. Expert Rev. Mol. Diagn. 2012, 12, 565– 571.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(77) Nagarale, R. K.; Lee, J. M.; Shin, W. Electrochim. Acta 2009, 54, 6508–6514. (78) Kang, X. H.; Wang, J.; Wu, H.; Aksay, I. A.; Liu, J.; Lin, Y. H. Biosens. Bioelectron. 2009, 25, 901–905. (79) Bakker, E. Anal. Chem. 2004, 76, 3285–3292. (80) Wang, J. Chem. Rev. 2008, 108, 814–825. (81) Kaushik, A.; Khan, R.; Solanki, P. R.; Pandey, P.; Alam, J.; Ahmad, S.; Malhotra, B. D. Biosens. Bioelectron. 2008, 24, 676– 683. (82) Pandey, P.; Singh, S. P.; Arya, S. K.; Gupta, V.; Datta, M.; Singh, S.; Manhotra, B. D. Langmuir 2007, 23, 3333–3337. (83) Zhao, G.; Xu, J. J.; Chen, H. Y. Electrochem. Commun. 2006, 8, 148–154. (84) Yang, L. Q.; Ren, X. L.; Tang, F. Q.; Zhang, L. Biosens. Bioelectron. 2009, 25, 889–895. (85) Lu, B.-W.; Chen, W.-C. J. Magn. Magn. Mater. 2006, 304, e400–e402. (86) Besombes, J.-L.; Cosnier, S.; Labbe, P.; Reverdy, G. Anal. Chim. Acta 1995, 311, 255–263. (87) Burestedt, E.; Ruzgas, A.; Gorton, T.; Emneus, J.; Dominquez, E.; Marko-Varga, G. Anal. Chem. 1996, 68, 1605–1611. (88) Wang, Y.; Zhang, X.; Chen, Y.; Xu, H.; Tan, Y.; Wang, S. Am. J. Biomed. Sci. 2010, 2, 209–216. (89) Alkasir, R. S. J.; Ganesana, M.; Won, Y. H.; Stanciu, L.; Andreescu, S. Biosens. Bioelectron. 2010, 26, 43–49. (90) Kaushik, A.; Solanki, P. R.; Kaneto, K.; Kim, C. G.; Ahmad, S.; Malhotra, B. D. Electroanalalysis 2010, 22, 1045–1055. (91) Kaushik, A.; Solanki, P. R.; Ansari, A. A.; Sumana, G.; Ahmad, S.; Malhotra, B. D. Sens. Actuat. B-Chem. 2009, 138, 572–580. (92) Ali, A.; Al Salhi, M. S.; Atif, M.; Ansari, A. A.; Israr, M. Q.; Sadaf, J. R.; Ahmed, E.; Nur, O.; Willander, M. J. Phys.: Confer. Series 2013, 414, 012024. (93) Thandavan, K.; Gandhi, S.; Sethuraman, S.; Rayappan, J. B. B.; Krishman, U. M. Sens. Actuat. B-Chem. 2013, 176, 884–892. (94) Thandavan, K.; Gandhi, S.; Sethuraman, S.; Rayappan, J. B. B.; Krishman, U. M. Food Chem. 2013, 139, 963–969. (95) Liao, M.-H.; Guo, J.-C.; Chen, W.-C. J. Magn. Magn. Mater. 2006, 304, 421–423. (96) Sanchez, C.; Julian, B.; Belleville, P.; Popall, M. J. Mater. Chem. 2005, 15, 3559–3592. (97) Leung, K. C.; Xuan, S.; Zhu, X.; Wang, D.; Chak, C. P.; Lee, S. F.; Ho, W. K.; Chung, B. C. Chem. Soc. Rev. 2012, 41, 1911–1928. (98) Zhong, C. J.; Maye, M. M. Adv. Mat. 2001, 13, 1507–1511. (99) Sanchez, S. I.; Small, M. W.; Bozin, E. S.; Wen, J. G.; Zuo, J. M.; Nuzzo, R. G. ACS Nano 2013, 7, 1542–1557. (100) Mallat, T.; Baiker, A. Annu. Rev. Chem. Biomol. Eng. 2012, 3, 11–28. (101) Peng, H. P.; Liang, R. P.; Qiu, J. D. Biosens. Bioelectron. 2011, 26, 3005–3011. (102) Liu, Y.; Han, T.; Chen, C.; Bao, N.; Yu, C.-M.; Gu, H.-Y. Electrochim. Acta 2011, 56, 3238–3247. (103) Kang, L.; Lai, Y.; Zhang, W.; Jin, L. Talanta 2011, 84, 607– 613. (104) Zhuo, Y.; Yuan, P.-X.; Yuan, R.; Chai, Y.-Q.; Hong, C.-L. Biomaterials 2009, 30, 2284–2290. (105) Kim, D. K.; Zhang, Y.; Voit, W.; Rao, K. V.; Kehr, J.; Bjelke, B.; Muhammed, M. Scripta Mater. 2001, 44, 1713–1717. (106) Pankhurst, Q. A.; Connolly, J.; Jones, S. K.; Dobson, J. J. Phys. D: Appl. Phys. 2003, 36, R167–R181. (107) Qiu, J. D.; Xiong, M.; Liang, R.-P.; Peng, H.-P.; Liu, F. Biosens. Bioelectron. 2009, 24, 2649–2653. (108) Gan, N.; Jin, H.; Li, T.; Zheng, L. Int. J. Nanomed. 2011, 6, 3259–3269. (109) Park, H. Y.; Schadt, M. J.; Wang, L.; Lim, I.; Im, S.; Njoki, P. N.; Kim, S.-H.; Jang, M.-H.; Luo J.; Zhong, C.-J. Langmuir 2007, 23, 9050–9056. (110) Chin, S. F.; Iyer, K. S.; Raston, C. L. Cryst. Growth Des. 2009, 9, 2685–2689. (111) Loaiza, O. A.; Jubete, E.; Ochoteco, E.; Estibalitz, C.; German, G. H.; Rodriguez, J. Biosens. Bioelectron. 2011, 26, 2194–2200. (112) Palchetti, I.; Mascini, M. Anal. Bioanal.Chem. 2012, 402, 3103–3114. (113) Lee, J. H.; Yigit, M. V.; Mazumdar, D.; Lu, Y. Adv. Drug. Deliv. Rev. 2010, 62, 592–605. (114) Zhao, J.; Zhang, Y.; Li, H.; Wen, Y.; Fan, X.; Lin, F.; Tan, L.; Yao S. Biosens. Bioelectron. 2011, 26, 2297–2303.

Page 22 of 24

(115) Liu, B.; Cui, Y.; Tang, D.; Yang, H.; Chen, G. Chem. Commun. 2012, 48, 2624–2626. (116) Zou, C.; Fu, Y.; Xie, Q.; Yao, S. Biosens. Bioelectron. 2010, 25, 1277–1282. (117) Qiu, J. D.; Peng, H. P.; Liang, R. P.; Xia, X. H. Biosens. Bioelectron. 2010, 25, 1447–1453. (118) Rawal, R.; Chawla, S.; Pundir, Ch.S. Biosens. Bioelectron. 2012, 31, 144–150. (119) Liu, Z. L.; Zhao, B.; Shi, Y.; Guo, C. L.; Yang, H. B.; Li, Z. A. Talanta 2010, 81, 1650–1654. (120) Si, Y.; Samulski, E. T. Chem. Mater. 2008, 20, 6792–6797. (121) Cong, H.-P.; He, J.-J.; Lu, Y.; Yu, S.-H. Small 2010, 6, 169– 173. (122) Teymourian, H.; Salimi, A.; Khezrian, S. Biosens. Bioelectron. 2013, 49, 1–8. (123) Li, P.; Zhang, W.; Zhao, J.; Meng, F.; Yue, Q.; Wang, L.; Li, H.; Gu, X.; Zhang, S.; Liu, J. Analyst 2012, 137, 4318–4326. (124) Wu, S.; He, Q.; Zhou, C.; Qi, X.; Huang, X.; Yin, Z.; Yang, Y.; Zhang, H. Nanoscale 2012, 4, 2478–2483. (125) Lu, D.; Zhang, Y.; Wang, L.; Lin, S.; Wang, C.; Chen, X. Talanta 2012, 88, 181–186. (126) Merkoςi, A.; Pumera, M.; Llopis, X.; Pérez, B.; del Valle, M.; Alegret, S. Trends Anal. Chem. 2005, 24, 826–838. (127) Cao, H.; Zhu, M.; Li, Y. J. Solid. State Chem. 2006, 179, 1208–1213. (128) Cheng, G.; Zhao, J.; Tu, Y.; He, P.; Fang, Y. Anal. Chim. Acta 2005, 533, 11–16. (129) Bian, C. L.; Zeng, Q. X.; Yang, L. J.; Xiong, H. Y.; Zhang, X. H.; Wang, S. F. Sens. Actuat. B-Chem. 2011, 156, 615–620. (130) Chauhan, N.; Pundir, C. S. Anal. Chim. Acta 2011, 701, 66–74. (131) Shahrokhian, S.; Rastgar, S.; Amini, M. K.; Adeli, M. Bioelectrochem. 2012, 86, 78–86. (132) Li, J.; Yuan, R.; Chai, Y. Q.; Che, X. J. Mol. Catal. B-Enzym. 2010, 66, 8–14. (133) Baby, T. T.; Ramaprabhu, S. Talanta 2010, 80, 2016–2022. (134) Yu, J.; Tu, J. X.; Zhao, F. Q.; Zeng, B. Z. J. Sol. State Electrochem. 2010, 14, 1595–1600. (135) Miao, Y. Q.; Wang, H.; Shao, Y. Y.; Tang, Z. W.; Wang, J.; Lin, Y. H. Sens. Actuat. B-Chem. 2009, 138, 182–188. (136) Yantasee, W.; Hongsirikarn, K.; Warner, C. L.; Choi, D.; Sangvanich, T. Analyst 2008, 133, 348–355. (137) Chen, W.-C.; Lin, Y.-Y. Biomed. Eng. Appl. Basis Commun. 2009, 21, 437–441. (138) Liu, Z.; Wang, J.; Xie, D.; Chen, G. Small 2008, 4, 462–466. (139) Yadav, S.; Devi, R.; Bhar, P.; Singhla, S.; Pundir, C. S. Enzyme Microb. Technol. 2012, 50, 247–254. (140) Cevik, E.; Senel, M.; Baykal, A. Curr. Appl. Phys. 2013, 13, 280–286. (141) Shanmugam, S.; Thandava, K.; Gandhi, S.; Sethuraman, S.; Rayappan, J. B. B.; Krishnan, U. M. Analyst 2011, 136, 5234–5240. (142) Cevik, E.; Senel, M.; Baykal, A.; Abasiyanik, M. F. Sens. Actuat. B-Chem. 2012, 173, 396–405. (143) Borrebaeck, C. A. K. Immunology Today 2000, 21, 379–382. (144) Lequin, R. M. Clin. Chem. 2005, 51, 2415–2418. (145) Ricci, F.; Adornetto, G.; Palleschi, G. Electrochim. Acta 2012, 84, 74–83. (146) Marquette, C. A.; Blum, L. J. Biosens. Bioelectron. 2006, 21, 1424–1433. (147) Moreno-Guzmán, M.; Gonzáles-Cortés, A.; Yánez-Sedeno, P.; Pingarrón, J. M. Anal. Chim. Acta 2011, 692, 125–130. (148) Li, H.; Wei, Q.; He, J.; Li, T.; Zhao, Y. F.; Cai, Y. Y.; Du, B.; Qian, Z. Y.; Yang, M. H. Biosens. Bioelectron. 2001, 26, 3590–3595. (149) Wei, Q.; Xiang, Z.; He, J.; Wang, G.; Li, H.; Qian, Z.; Yang, M. Biosens. Bioelectron. 2010, 26, 627–631. (150) Tang, D.; Yuan, R.; Chai, Y. Biotechnol. Lett. 2006, 28, 559– 565. (151) Tang, D.; Yuan, R.; Chai, Y.; An, H. Adv. Funct. Mater. 2007, 17, 976–982. (152) Pan, J.; Yang, Q. Anal. Bioanal. Chem. 2007, 388, 279–286. (153) Chen, C.; Yang, S.; Lin, J.; Lin, T.; Chen, W.; Jiang, J.; Wang, H.; Chang, S. J. Surg. Res. 2005, 124, 169–174. (154) Li, Z.; Ni, Y.; Wang, X.; Zhao, G. Talanta 2010, 82, 196–201. (155) Kaushik, A.; Solanki, P. R.; Ansari, A. A.; Ahmad, S.; Malhotra, B. D. Electrochem. Commun. 2008, 10, 1364–1368. (156) Shen, G.; Zhang, Y. Anal. Chim. Acta 2010, 674, 27–31.


Page 23 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(157) Liu, S.; Lin, Q.; Zhang, X. M.; He, X. R.; Xing, X. R.; Lian, W. J.; Huang, J. D. Sens. Actuat. B-Chem. 2011, 156, 71–78. (158) Merkoςi, A. FEBS Journal 2007, 274, 310–316. (159) Palecek, E.; Fojta, M. Anal. Chem. 2001, 73, 74A–83A. (160) Palecek, E.; Fojta, M.; Tomschik, M.; Wang, J. Biosens. Bioelectron. 1998, 13, 621–628. (161) Marrazza, G.; Chianella, I.; Mascini, M. Biosens. Bioelectron. 1999, 14, 43–51. (162) Azek, F.; Grossiord, C.; Joannes, M.; Limoges, B.; Brossier, P. Anal. Biochem. 2003, 284, 107–113. (163) Mao, X.; Jiang, J.; Xu, X.; Chu, X.; Luo, Y.; Shen, G.; Yu, R. Biosens. Bioelectron. 2008, 23, 1555–1561. (164) Dong, X.-Y.; Mi, X.-N.; Wang, B.; Xu, J.-J.; Chen, H.-Y. Talanta 2011, 84, 531–537. (165) Kaushik, A.; Solanki, P. R.; Ansari, A. A.; Malhotra, B. D.; Ahmad, S. Biochem. Eng. J. 2009, 46, 132–140. (166) Hassen, W. M.; Chaix, C.; Abdelghani, A.; Bessueille, F.; Leonard, D.; Jaffrezic-Renault, N. Sens. Actuat. B-Chem. 2008, 134, 755–760. (167) Tran, L. D.; Nguyen, B. H.; Hieu, N. V.; Tran, H. V.; Nguyen, H. L.; Nguyen, P. X. Mater. Sci. Eng. C 2011, 31, 477–485. (168) Zhang, W.; Yang, T.; Jiao, K. Biosens. Bioelectron. 2012, 31, 182–189. (169) Singh, R.; Verma, R.; Kaushik, A.; Sumana, G.; Sood, S.; Gupta, R. K.; Malhotra, B. D. Biosens. Bioelectron. 2011, 26, 2967– 2974. (170) Singh, R.; Verma, R.; Sumana, G.; Srivastava, A. K.; Sood, S.; Gupta, R. K.; Malhotra, B. D. Bioelectrochem. 2012, 86, 30–37. (171) Prabhakar, N.; Solanki, P. R.; Kaushik, A.; Pandey, M. K.; Malhotra, B. D. Electroanalysis 2010, 22, 2672–2682. (172) Wang, J.; Palecek, E.; Nielsen, P. E.; Rivas, G.; Cai, X.; Shiraishi, H.; Dontha, N.; Luo, D.; Farias, P. A. M. J. Am. Chem. Soc. 1996, 118, 7667–7670. (173) Pal, S.; Alocilja, E. C. Biosens. Bioelectron. 2010, 26, 1624– 1630. (174) Pal, S.; Setterington, E. B.; Alocilja, E. C. IEEE Sens. J. 2008, 8, 647–654. (175) Osaka, T.; Matsunaga, T.; Nakanishi, T.; Arakaki, A.; Niwa, D.; Iida, H. Anal. Bioanal. Chem. 2006, 384, 593–600. (176) Kamikawa, T. L.; Mikolajczyk, M. G.; Kennedy, M.; Zhong, L. L.; Zhang, P.; Setterington, E. B.; Scott, D. E.; Alocilja, E. C. IEEE Trans. Nanotechnol. 2012, 11, 88–96. (177) Lin, P.-C.; Chou, P.-H.; Chen, S.-H.; Liao, H.-K.; Wang, K.-Y.; Chen, Y.-J.; Chen, Y.-J.; Lin C.-C. Small 2006, 2, 485–489. (178) Freitas, P. P.; Ferreira, R.; Cardoso, S.; Cardoso, F. J. Phys.: Condens. Mater. 2007, 19, 165221. (179) Tamanaha, C. R.; Mulvaney, S. P.; Rife, J. C.; Whitman, L. J. Biosens. Bioelectron. 2008, 24, 1–13. (180) Germano, J.; Martins, V. C.; Cardoso, F. A.; Almeida, T. M.; Sousa, L.; Freitas, P. P.; Piedada, M. S. Sensors 2009, 9, 4119–4137. (181) Graham, D. L.; Ferreira, H. A.; Freitas, P. P. Trends Biotechnol. 2004, 22, 455–462. (182) Kumar, A.; Mohapatra, S.; Fal-Miyar, V.; Cerdeira, A.; Garcia, J. A.; Srikanth, H.; Gass, J.; Kurlyandskaya, G. V. Appl. Phys. Lett. 2007, 91, 143902. (183) Blanc-Beguin, F.; Nabily, S.; Gieraltowski, J.; Turzo, A.; Querellou, S.; Salaun, P. Y. J. Magn. Magn. Mater. 2009, 321, 192– 197. (184) Grancharov, S. G.; Zeng, H.; Sun, S. H.; Wang, S. X.; O'Brien, S.; Murray, C. B.; Kirtley, J. R.; Held, G. A. J. Phys. Chem. B 2005, 109, 13030–13035. (185) Liang, G. H.; Cai, S. Y.; Zhang, P.; Peng, Y. Y.; Chen, H.; Zhang, S.; Kong, J. L. Anal. Chim. Acta 2011, 689, 243–249. (186) Joshi, A.; Solanki, S.; Chaudhari, R.; Bahadur, D.; Aslam, M.; Srivastava, R. Acta Biomater. 2011, 7, 3955–3963. (187) Kaittanis, C.; Banerjee, T.; Santra, S.; Santiesteban, O. J.; Teter, K.; Perez, J. M. Bioconj. Chem. 2011, 22, 307–314. (188) Morris, M. C. Biochim. Biophys. Acta 2013, 1834, 1387–1395. (189) Poiata, A.; Creanga, D. E.; Airinei, A.; Tupu, P.; Goiceanu, C.; Avadanei, O. J. Eur. Opt. Soc.-Rapid Publ. 2009, 4, 09024. (190) Smith, J. E.; Sapsford, K. E.; Tan, W. H.; Ligler, F. S. Anal. Biochem. 2011, 410, 124–132. (191) Magro, M.; Sinigaglia, G.; Nodari, L.; Tucek, J.; Polakova, K.; Marusak, Z.; Cardillo, S.; Salviulo G.; Russo, U.; Stevanato, R.; Zboril, R.; Vianello, F. Acta Biomater. 2012, 8, 2068–2076. (192) Sipova, H.; Homola, J. Anal. Chim. Acta 2013, 773, 9–23.

(193) Homola, J. Chem. Rev. 2008, 108, 462–493. (194) Wang, J. L., Zhu, Z. Z.; Munir, A.; Zhou, H. S. Talanta 2011, 84, 783–788. (195) Wang, Y.; Dostalek, J.; Knoll, W. Anal. Chem. 2011, 83, 6202– 6207. (196) Liang, R. P.; Yao, G. H.; Fan, L. X.; Qiu, J. D. Anal. Chim. Acta 2012, 737, 22–28. (197) Markova, Z.; Bourlinos, A. B.; Safarova, K.; Polakova, K.; Tucek, J.; Medrik, I.; Siskova, K.; Petr, J.; Krysmann, M.; Giannelis, E. P.; Zboril, R. J. Mater. Chem. 2012, 22, 16219–16223. (198) Ranc, V.; Markova, Z.; Hajduch, M.; Prucek, R.; Kvitek, L.; Kaslik, J.; Safarova, K.; Zboril, R. Anal. Chem. 2014, 86, 2939–2946.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 24 of 24

Table of Contents


Nanocrystalline Iron Oxides, Composites, and Related Materials as a

Recommend Documents