Magnetic‐Nanoparticle‐Based Immunoassays‐on‐Chip...

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versatile techniques available for their sur-face conjugation using antibodies, MNPs have been actively studied for applications in both in vitro and in vivo cancer diag-nostic imaging and therapy. [ 5,6 ]

As cancer is a highly heterogeneous dis-ease, [ 7 ] the idea of “personalized medicine” has been put forward for the diagnosis and therapy of cancers, where the cancer patient is given the medical care based on his/her individual molecular phenotype. [ 8 ] This concept requires the isolation and analysis of cells of interest from the indi-vidual patients. However, these cells, such as circulating tumor cell (CTC), [ 9 ] cancer stem cell, [ 10 ] disseminated tumor cell, [ 11 ] and dormant cancer cell, [ 12 ] are often rare in a mixture of heterogeneous cells (usu-ally less than 1% of the total cell number). The low number of target cells in blood sample largely limits the detection capa-bility and reduces effectiveness of the

time-sensitive cancer treatment. Therefore, the isolation and detection of the rare cells for early diagnosis and prognosis of cancers have drawn increasing attention in recent years. Accu-rate enumeration of enriched cancer cells may provide insights for metastasis, tumor growth, and cancer development for risk assessment, response monitoring, and development of new therapies. [ 13–15 ]

To facilitate the effective monitoring and therapies for cancer, various types of separation methods have been devel-oped to capture carcinoma cells as well as to analyze the cells at the molecular scale. [ 16 ] The separation methods for rare cancer cells can be divided into two categories. The fi rst category is “label-free” isolation which is based on differences in physical properties, such as size and density. These methods include density gradient centrifugation, size-based fi ltration methods, and dielectrophoretic methods. These label-free methods have been reviewed comprehensively in recent years and are beyond the scope of this review. [ 17 ] Microfl uidic technologies are usually employed to achieve high-effi ciency cell capture for label-free isolation. The second category is based on specifi c biomedical properties, such as the affi nity mediated immunoassay using antigen-modifi ed particles or chips. [ 18–20 ] Compared to physical parameter-based methods, various affi nity immunoassays usu-ally display both better specifi city and selectivity. Based on the

Magnetic-Nanoparticle-Based Immunoassays-on-Chip: Materials Synthesis, Surface Functionalization, and Cancer Cell Screening

Ying Zhu , Katsiaryna Kekalo , Christian NDong , Yu-Yen Huang , Fridon Shubitidze , Karl E. Griswold , Ian Baker , and John X. J. Zhang *

The unique properties of magnetic nanoparticles (MNPs), coupled with versatile surface engineering techniques, have led to a rising class of screening methods that enable separation of specifi c cell populations from complex biological samples. The growing sophistication and effi ciency of these methods have far reaching implications for both fundamental research and clinical applications. In this study, the synthesis and surface engineering of MNPs is reviewed. Here, a model is introduced to illustrate how MNP morphology and particle–particle interactions infl uence magnetization, which is a key consideration in designing and selecting MNPs for effi cient cell separations. Building upon these themes, immunomagnetic assays for capturing, isolating, and characterizing rare cell types from complex biolog-ical mixtures are reviewed. Although the focus of this study is on circulating tumor cells, these same techniques can be applied in screening for other rare cells of interest, such as various stem cell populations. In conclusion, current challenges and future directions for magnetic -nanomaterial-based cell screening systems are discussed.

DOI: 10.1002/adfm.201504176

Dr. Y. Zhu, Dr. K. Kekalo, Dr. C. NDong, Dr. Y. Y. Huang, Prof. F. Shubitidze, Prof. K. E. Griswold, Prof. I. Baker Thayer School of Engineering Dartmouth College Hanover , NH 03755 , USA Prof. J. X. J. Zhang Thayer School of Engineering Dartmouth-Hitchcock Norris Cotton Cancer Center Dartmouth College Hanover , NH 03755 , USA E-mail: [email protected]

1. Introduction

Emerging multifunctional nanomaterials plays an increasingly signifi cant role in fundamental biomedical research as well as clinical diagnostics and therapy, particularly for addressing the complicated diseases such as cancer. [ 1–3 ] Cancer is the second leading cause of death in the US according to statistics reported by the Centers of Disease Control and Prevention. In 2015, it is estimated that there will be about 1.7 million new cancer cases diagnosed and 589 430 cancer deaths in the US. [ 4 ] Due to the unique properties of magnetic nanoparticles (MNPs) and the

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difference in sorting techniques, various affi nity ligands can be employed for (1) cell-affi nity chromatography or chip, [ 18,21–23 ] in which a ligand is chemically immobilized to a solid sup-port to capture specifi c cell type in a suspension. Nanostruc-tured surfaces have been applied to chip-based systems to enhance the capture effi ciency [ 23–25 ] ; (2) fl uorescence-activated cell sorting, [ 26–28 ] which sorts a heterogeneous mixture of cells using fl uorescent signals; 3) magnet-based cell separation, [ 29–31 ] in which an external magnetic fi eld is applied to separate the magnetic-material-labeled cells from a heterogeneous popula-tion of cells. Compared with other enrichment technologies, an immunomagnetic assay has several advantages that make it especially suitable for rare cell screening. First, it has high sen-sitivity due to specifi c antibody-antigen binding. Increased sen-sitivity and specifi city can be achieved by expanding the types of antigens which can be multiplexed for recognition. Second, as magnetic force has been shown to be effective to work as the retaining force across multiple length scales, an immunomag-netic assay implemented in microfl uidic chips increases the specifi city between the target and nontarget cells. [ 32 ] In addition, high-throughput and hybrid integration with other separation or analysis platform can be achieved with an immunomagnetic assay. We have discussed the advantages of multiscale immu-nomagnetic assay in a previous paper which interested readers can refer to for more details. [ 32 ] Since magnetic nanomaterials such as MNPs can be manipulated using magnets located at a close distance, magnetic separation based cell assays using MNPs is shown to be an effective process for both fundamental studies in cancer biology and clinical implementations.

In this review, we will discuss each of the key steps leading to an effective development of an MNP-based immunomag-netic assay, which are as follows: 1) synthesis of MNPs, 2) sur-face engineering of MNPs, 3) assays for capture and isolation of cells, and 4) molecular characterization of the separated cells, ideally at single cell level. Most of the discussion will focus on CTCs, the mostly studied type of rare cells so far, which have been shown to be good indicators in the bloodstream for the presence of a primary tumor and metastasis. These techniques can be directly used for the screening of other rare cancer cells such as cancer stem cells and disseminated tumor cells.

2. Design and Synthesis of MNPs

The major components of cells, such as water, phospholipid bilayers, proteins, and DNA, are diamagnetic, which means the material displays a weak repulsion to a magnetic fi eld. [ 33,34 ] The diamagnetism of the cell is important for magnetic cell separa-tion in that the cells do not substantially interact with the mag-netic fi eld unless artifi cially modifi ed. [ 29,35–38 ] Cell separation can be driven by a difference in magnetic susceptibility between different cell subsets, which is typically achieved by selective binding of the magnetizable micrometer- or submicrometer-sized particles to target cells. MNPs can be utilized to label the target cells and separate them from those unlabeled ones, as the applied magnetic fi eld attracts the labeled cells while uni-versally repulses un-labeled ones from the magnetic source. [ 39 ]

Current magnetic materials for cell separation are antibody-targeted micrometer-sized magnetic beads and magnetic fl uids

which consist of nanoparticles. The fi rst magnetic particles used for clinical cell separation applications, Dynabeads produced by Invitrogen Inc., were large and uniform superparamagnetic polystyrene beads (4.5 µm diameter) coated with a monoclonal human IgG antibody. [ 35,40,41 ] Currently, Invitrogen Inc. provides

Karl Griswold is an Associate Professor of Bioengineering at Dartmouth College, NH, USA. As a DOW Foundation Scholar, he earned a B.S. in chemistry from Texas State University in San Marco, TX, USA in 1995, after which he worked for two years at Thermo Electron Corporation in Austin, TX, USA and two years with Huntsman

Corporation in Austin, TX, USA. He then returned to academia, receiving a PhD from the University of Texas at Austin, TX, USA in 2005, and he joined the Thayer School of Engineering faculty in 2007. His research interests are biotechnology and biotherapeutics.

Ian Baker is the Sherman Fairchild Professor of Engineering and Senior Associate Dean for Academic Affairs in the Thayer School of Engineering at Dartmouth College, NH, USA and Director, Dartmouth Center for Cancer Nanotechnology Excellence. He is a Chartered Engineer (UK) and a Fellow of ASM international, TMS,

IOM, 3 and the MRS. His research interests include: metallic materials for high temperature applications, novel magnetic materials, the mechanical properties and micro-structural evolution of snow and ice, nanoparticle-based magnetic hyperthermia of cancer.

John X. J. Zhang is a Professor at the Thayer School of Engineering at Dartmouth, NH, USA, and a Fellow of AIMBE. He received his PhD from Stanford University, CA, USA. His research is on devel-oping miniature medical systems to improve global health, through innovations in bio-inspired nanomate-

rials, lab-on-chip design, and advanced nanofabrication technologies.

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a wide range of magnetic beads for cell separation. The other main producers and products in this area are: R&D Systems’ Immunicon MagCellect Ferrofl uid which contains 200 nm superparamagnetic particles with a relatively uniform particle size; Miltenyi Biotec GmbH’s magnetic colloid called MACS [ 29 ] ; StemCell Technologies Inc.’s magnetic dextran colloid and BD Immunosciences’ magnetic colloid. Some of these companies also produce the columns used to separate magnetic particles.

2.1. Design Principles of Core MNPs

MNPs, because of their high surface area per volume, express properties different from bulk materials (such as their behavior under a magnetic fi eld, lower melting and boiling points, lower sintering temperature, and reduced fl ow resistance, etc.) and have a wide range of biomedical applications, such as spe-cifi c cell labeling and separation, diagnostics, drug delivery including controlled release, contrast agents for MRI, and mag-netic hyperthermia. [ 33,42–48 ] Each application requires particles of different physical and chemical properties for optimum use. The chemical composition, shape, size and method of synthesis of the magnetic core ensure the desired behavior of MNPs under an applied static or alternating magnetic fi eld.

For magnetic cell separation, the MNP core (1) has to be chemically stable in both aqueous solutions and biological fl uids; (2) has narrow size distribution and ideally a spherical shape, so as to avoid undesired excessive core-core interac-tion and aggregation (which will be discussed in Section 2.4); (3) has to be biocompatible; (4) has to be superparamagnetic; and (5) has a high saturation magnetization. Size reduction in multidomain magnetic materials results in the formation of single-domain particles and gives rise to the phenomenon of superparamagnetism. Superparamagnetic materials exhibit a large magnetization response in the presence of a magnetic fi eld but have zero response in the absence of a fi eld. This prop-erty is very useful in the applications that require strong force which can be “turned on and off” by a magnetic fi eld, such as cell separation. The maximum induced magnetization can be characterized by saturation magnetization. Increasing satura-tion magnetization can lead to increased cell capture effi ciency. The saturation magnetization depends on both the material ( Figure 1 ) and the size of particles ( Figure 2 ). The saturation magnetization of MNPs increases with the increase of par-ticle size, whereas at a critical point the MNPs transform from single-domain to multidomain structures (see Table 1 ) and their magnetic behavior changes from superparamagnetic to ferrimagnetic- or ferromagnetic, which is undesirable for cell screening application. Therefore, MNPs for the application of magnetic cell labels are comprised either of individual nano-particles (10–50 nm) or of microbeads consisting of individual superparamagnetic nanoparticles embedded in a large body. [ 49 ]

Coercivity ( H c ), which is the fi eld required to bring magneti-zation to zero, is another parameter for evaluation the magnetic properties of MNPs. In the zero-fi eld condition, superparamag-netic nanoparticles exhibit zero coercivity, i.e., zero resistance to magnetic reversal, which makes them highly manipulatable in magnetic fi eld. The shape anisotropy, which is the departure from sphericity of single-domain particles, can also have a very

large infl uence on the coercivity. Table 2 shows the infl uence of aspect ratio on coercivity for Fe nanoparticles. [ 53 ]

The sum total of these properties, e.g., superparamagnetic, high saturation magnetization, and low coercivity, makes MNPs the best candidate for cell-screening applications in magnet-based platforms.

As noted earlier, when selecting the core magnetic material and size for cell separation, it is critical that the core should be chemically stable in water and biological fl uids. Naked metallic nanoparticles are chemically active and easily oxidized in air, which results in loss of magnetism and dispersibility. [ 42 ] For example, it was shown that 8–16 nm Fe/Fe oxide core–shell particles show extraordinary saturation magnetization com-pared to other MNPs, but oxidize rapidly in water and espe-cially in saline solution. [ 54 ] Nanoparticles of Co or Co ferrites could be a good alternative to iron MNPs since they show better chemical stability in aquatic solutions. For most applications it is crucial to develop protection strategies to chemically stabilize the naked MNPs by surface coating, which will be discussed in Section 50 .

2.2. Synthesis Methods of MNPs

A large number of methods have been described for prepara-tion of various nano- and micrometer-sized magnetic particles for biomedical applications. The widely known and employed methods for the synthesis of MNPs can be categorized as fol-lows: (1) Physical methods, such as gas-phase deposition, [ 55 ] and fl ame spray synthesis. [ 56 ] (2) Wet chemical methods, such as chemical coprecipitation, [ 57,58 ] thermal decomposition, [ 59 ]

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Figure 1. Magnetization curves of nine ferromagnetic materials, showing saturation: 1) Sheet steel; 2) silicon steel; 3) cast steel; 4) tungsten steel; 5) magnet steel; 6) cast iron; 7) nickel; 8) cobalt; 9) magnetite. Data from ref. [ 50 ]

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microemulsion techniques, [ 60 ] and hydrothermal reaction. [ 61,62 ] (3) Microbial methods, in which Fe(III)-reducing bacteria are usually employed for large-scale production of MNPs under anaerobic conditions. [ 63 ] A complete list of all the methods has been given in recent reviews. [ 64,65 ] Wet chemical processes have been investigated more extensively than physical methods, as they can provide higher level of controls over the size, compo-sition, magnetic properties and shape of MNPs. This is par-ticularly important toward the rare cell screening in a liquid environment, which is the central focus in terms of applications for this review. Therefore, we specifi cally describe several pop-ular and representative wet-chemical routes for the fabrication of shape-controlled, highly stable and monodisperse MNPs.

The general principle of wet-chemical synthesis (i.e., liquid-phase synthesis) is based on the classic LaMer mechanism. [ 66 ]

As the solution concentration passes its saturation, nucleation and growth of monodisperse particles occur. The chemical syn-thesis of coprecipitation is a very facile and convenient way to synthesize iron oxides Fe x O y (including Fe 3 O 4 and γ-Fe 2 O 3 ), which was developed in the early 1980s by Massart who used ferrous and ferric salts in alkaline and acidic aqueous solutions to produce Fe 3 O 4 nanoparticles in the size range 10–20 nm. [ 67 ]

Briefl y, alkaline salt solutions containing a mixture of Fe 2+ /Fe 3+ are precipitated under an inert atmosphere and at either room or elevated temperatures. The size, shape, and composition of the resultant MNPs can be tuned via a wide range of param-eters including: the type of salts used (e.g., chloride, sulfates, nitrates), the Fe 2+ /Fe 3+ ratio, the reaction temperature, the pH value and ionic strength of the media. Controlling of the nucle-ation and growth is essential for the synthesis of monodisperse MNPs using coprecipitation method. Several stabilizer and/or reducing agents have been employed to improve the size disper-sity and prevent aggregation, such as polyvinyl alcohol, [ 68 ] citric acid [ 69 ] and oleic acid. [ 68,70 ] This method is usually employed to obtain MNPs of magnetite and other ferrites in the size range 2–80 nm. [ 71 ] With fi xed synthesis conditions, the quality of the magnetite nanoparticles is highly reproducible.

Another important chemical synthesis method is thermal decomposition. In this method, organometallic compounds are undergone decomposition in high-boiling organic solvents con-taining stabilizing surfactants. [ 72,73 ] Control of the size (from 4 to 16 nm) and morphology of MNPs can be managed by a careful control of the molar ratio of the metal precursor, surfactant, and solvent. For example, monodisperse γ-Fe 2 O 3 MNPs has been synthesized by decomposition of Fe(CO) 5 . [ 74 ] As an alternative to using highly fl ammable Fe(CO) 5 , iron (III) acetylacetonate can be used. Decomposition of iron (III) acetylacetonate during high-temperature (265 °C) treatment of phenyl ether in the pres-ence of alcohol, oleic acid, and oleylamine has been reported to produce 4 nm Fe 3 O 4. [ 75 ] Larger MNPs, up to 20 nm in diameter, can be further obtained through a seed-mediated growth method by adding additional precursors to the 4 nm magnetite particles.

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Table 1. Critical diameter (at room temperature) above which a single-domain spherical particle with axial magnetic anisotropy changes to a multidomain particle. [ 52 ]

Material Co Ni Fe Fe 3 O 4 γ-Fe 2 O 3

D [nm] 70 55 14 128 166

Figure 2. Saturation magnetization normalized to the saturation magnetization of bulk material versus particle diameter for various oxide nanoparticles. Reproduced with permission. [ 51 ] Copyright 2007, IOP Publishing.

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In the method of water-in-oil microemulsion, or reverse micelle, nanoparticles are formed by the isotropic dispersion of two immiscible liquids, which form microdomains that are stabilized by an interfacial surfactant fi lm. This method can be considered as a derivative of precipitation or reduction tech-nique with the difference that the reaction occurs in small water droplets stabilized in an organic solvent. By mixing two microe-mulsions containing the desired reactants, microdroplets collide and break up again under agitation, and fi nally form a precipi-tate containing nanoparticle. [ 76 ] The size of the resulting MNPs can be successfully controlled by the size of the micro reactor by varying the ratios between the water, organic solvent and sur-factant. [ 60 ] However, the yield of nanoparticles is low compared to the other methods, and large amount of solvent are necessary, which compromises effi ciency and manufacturing scale-up.

The hydrothermal synthesis allows a wide variety of nano-structured shapes and compositions. [ 77,78 ] This synthesis method is based on a phase transfer and separation that occurs at the interfaces of liquid, solid and solution phases. For example, Deng et al. [ 79 ] utilized hydrothermal synthesis to fabri-cate monodispersed MNPs in the size range of 200–800 nm. In their work, a mixture of iron salt (e.g., FeCl 3) , high-boiling-point reducing agent (e.g., ethelyne glycol), electrostatic stabilizer (e.g., sodium acetate), and a surfactant (e.g., polyethylene glycol) are sealed in a Tefl on-lined stainless-steel autoclave, heated to 200 °C, and maintained at that temperature for 8–72 h.

Examples of MNPs synthesized by different methods are pre-sented in Figure 3 , and the comparison of most commonly used techniques is presented in Table 3 . [ 42 ] Coprecipitation is the general preferred method for ease of use and high throughput. However, thermal decomposition is the best method for a good control over the size and morphology. Due to the large amount of solvent, the microemulsion technique is not scalable or cost-effective yet. Finally, the hydrothermal synthesis is still in the stage of experimental validation. Overall, most common meth-odologies for MNP production are coprecipitation and thermal decomposition, and they are commercially scalable.

2.3. Surface Coating of Magnetic Cores

The hydrodynamic size, surface charge, and biological proper-ties in most cases are functions of MNP’s surface modifi cation and/or coating. Besides the tailoring of surface properties, the surface coating also acts as a barrier against the interactions between particles, thus constitutes a barrier to prevent aggrega-tion and improve the stability of MNPs in biological environ-ment. The use of a variety of surface functional groups such as hydroxyl, carboxyl, pyridine, amide, aldehyde, phenyl chlo-romethyl, and others has been described in the literature. [ 80–83 ] Most common coating materials for MNPs include polystyrene, polysaccharides, polyethylene glycol, peptides, and phospho-lipids. [ 84–93 ] Besides polymeric materials, inorganic materials such as silica, porous carbon, and graphene-based materials have also been employed as coating materials. [ 94–98 ] However, as the target application is cell screening, the main perspec-tive of the surface coating discussed here will be focused on carbohydrate-related polymers, so as to assist the discussion of

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Table 2. Infl uence of aspect ratio of Fe nanoparticles on coercivity. [ 53 ]

Aspect ratio (c/a) 1.1 1.5 2.0 5.0 10

H c [Oe] 820 3300 5200 9000 10 100

Figure 3. Transmission electron microscopy images of MNPs made by: a) thermal decomposition (Ocean Nanotech); b) a combination of synthesis in a matrix with hydrothermal growth (Dartmouth college); c) coprecipitation followed by high-temperature homogenization (Micromod GmBH); d) hydrothermal growth (Dartmouth college); e) coprecipitation (Dartmouth college); f) microemulsion synthesis (Dartmouth college).

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surface engineering of MNPs in Section 70 . Surface modifi ca-tion and coatings are usually performed through one of four ways: (1) physical encapsulation, [ 86–93 ] (2) physical sorption, [ 99 ] (3) chemical binding [ 84,85,92,93,99–103 ] or 4) synthesis in a polymer matrix. [ 104–106 ]

Physical encapsulation is a method for encapsulating MNPs in a polymer shell without physical or chemical binding. This method is convenient to make microcapsules loaded with MNPs and other substances (for example, drugs). [ 89–93 ] Usu-ally a mixture of components is squeezed through the nozzle of certain size (related to the desired size of microcapsules) into an environment that produces rapid polymerization of the cell material (solvent, air, radiation, environment at polymerization temperature, etc.). [ 86,87 ] Using standard nozzles of different sizes it is possible to produce narrowly size-distributed micro-spheres. When a smaller size of spheres is desired, a piezoe-lectric vibrating nozzle method as a droplet ultrafi ne generator can be used. [ 88 ] For magnetic liposome formation, a mixture of magnetic material and lipids is produced by slowly evaporating a magnetic lipid fi lm formation followed by re-hydration and suspension. [ 93 ]

Physical sorption can be performed in two ways: high-pressure homogenization techniques [ 107 ] and electrostatic layer-by-layer self-assembly. [ 99 ] In high-pressure homogeniza-tion techniques, a coating material is mixed together with the MNPs and treated with high pressure (100–1000 Bar). The layer-by-layer self-assembly method is based on electrostatic interaction of positive and negative charged absorbed layers of polyelectrolytes.

Chemical binding is the most commonly used technique. Modifi cation of the MNP surface with desired functional groups allows numerous kinds of surface engineering. [ 80–83 ] The easiest and most widely used way to start MNP function-alization is coating the MNPs with amorphous silica. After the silica coating, surface-reactive groups such as tetraethyl ortho-silicate (TEOS) and aminopropyltriethoxysilane (APTMES) are added to MNPs and kept for 2–72 h at 100–120 °C either in a sonic bath or mechanically stirred/shaken. [ 84,100–103 ] The sur-face-reactive groups facilitate the stability and allow the design of multifunctional MNPs. The surface engineering of MNPs for conjugation of biomarkers will be discussed in more details in Section 70 .

Synthesis in a polymer matrix is a one-pot method of pro-ducing coated MNPs. [ 104–106 ] Polymers like saccharides, polysac-charides, polysaccharides derivatives, and other polymers could

be used for this purpose. The sizes of the magnetic crystals formed using this process is normally 1–3 nm. When larger sizes of crystals and/or aggregation are desired, hydrothermal growth can be used. [ 106 ]

2.4. Theory and Modeling of MNP Interactions

An immuno-magnetic assay (IMA) combines an external direct current (DC) magnetic fi eld source, MNPs, and bio-markers for recognizing cancers cells. A key characteristic of an IMA is the use of a high magnetic force, which depends on the applied external DC magnetic fi eld gradient, and the shape, size, and properties of the MNPs. There are a number of MNP types available for use in IMAs; however, there are many limitations that must be taken into consideration when selecting MNPs for this use. First, if the MNP cores are too small then the exerted magnetic force on the MNPs will be insuffi cient to interact with them. On the other hand, if the MNP cores are too large then they will have high gravitational masses/forces, resulting in insuffi cient external applied DC magnetic fi eld for interacting with the MNPs in solution, which may compromise the mag-netic separation in the cell screening application. In addition, if the concentration of MNPs present in the fl uid is too low, there will be insuffi cient force per unit volume of samples to capture cancer cells and achieve high accuracy IMA analyses. MNP aggregation produced by an external DC magnetic fi eld gradient leads to particle-particle interactions, which in turn affects the magnetic fi eld and force distributions. Finally, due to fast distance decay, the external DC magnetic fi eld force is only able to capture nearby MNPs, and the recognition of deep cells is limited by the external exerted magnetic forces and bio-markers. As a result, current IMA is limited to surface MNP assays. Therefore, high accuracy and effective IMA requires selection of MNPs which can optimize magnetic force.

The magnetic force depends on both the external magnet geometry and the MNPs properties. The magnetic moment ( M ) of a superparamagnetic MNP is always aligned along the easy axis of magnetization. Under an externally applied DC magnetic fi eld ( H ), the MNP experiences translational ( )tF V MM BB= ⋅ ∇ and rotational ( )rF V MM HH= × forces, where V is the volume of the MNP and B is the magnetic induction. Assuming there is no remnant magnetization of the MNP, the analytical expression for the translational force exerted on a superparamagnetic MNP in an inhomogeneous magnetic B

� fi eld is well known [ 108,109 ] and

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Table 3. Comparison of MNP synthesis methods. [ 42 ]

Synthesis method Coprecipitation Thermal decomposition Microemulsion Hydrothermal

Synthesis route Very simple, ambient conditions Complicated, inert atmosphere Complicated, ambient conditions Simple, high pressure

Reaction temp [°C] 20–90 100–320 20–50 220

Reaction time Minutes Hours–days Hours Hours–days

Solvent Water Organic compound Organic compound Water–ethanol

Surface-capping agents Needed, added during or after reaction Needed, added during reaction Needed, added during reaction Needed, added during reaction

Size distribution Relatively narrow Very narrow Relatively narrow Very narrow

Shape control Not good Very good Good Very good

Yield High/scalable High/scalable Low Medium

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it is proportional to the applied magnetic fi eld gradient and the induced dipole moment in the MNP. Therefore, to achieve the high magnetic force and accurate IMA analysis, it is necessary to optimize the shape, size and magnetic properties of both the external applied DC magnet and the MNPs. The highest pulling forces arise in the vicinity of the sharp edges of the external DC magnets, a feature which has allowed the design and building of microscale magnetic array to enhance IMA. [ 20 ] To further improve the effi ciency of IMA one must account for MNP remanent magnetization, which is due to strong particle-particle interactions. Figure 4 shows comparisons between the modeled magnetic susceptibilities for noncoupling ( p = 0), medium coupling ( p < 100) and strong coupling ( p > 100) MNPs (where p is an interaction coeffi cient). [ 47 ] These results indicate that as the interaction increases the remanent magneti-zation increases as well.

Similar results were reported for two 5 nm spheres oriented in the same vertical direction. [ 47 ] However, numerical studies for horizontally oriented spheres (not shown here) demonstrate the opposite trend, i.e., the fi eld between the spheres decreases as they approach each other. Thus, in both cases there are sig-nifi cant magnetic interactions between MNPs; namely, MNPs oriented along the applied AMF produce local fi eld enhance-ments, while MNPs oriented horizontally to the AMF decrease the effective local fi eld around the MNP. The comparisons between interacting fi eld values for spheroids and spheres (see Figure 5 ) show that the coupling between spheroids is

stronger than between spheres, indicating that the morphology of MNPs has an infl uence on the coupling. To further under-stand how the coupling changes depending on the shape of the interacting parts, we modeled the electromagnetic interaction between two identical isotropic fl ower-like MNPs with 100 nm cross-sections. For these studies we considered two cases (1) when the sharp tips of the fl ower-like MNPs approach each other ( Figure 6 a), and (2) when one MNP sharp tip approaches the fl at smooth surface of the other MNP (Figure 6 b). The fi eld distributions inside two interacting fl ower-shaped MNPs are more nonlinear (see fi elds between −100 and 100 nm on Figure 6 ) than fi elds distributions inside prolate spheroids ( Figure 5: fi elds between –300 and 300 nm) and spheres ( Figure 5: fi elds between –100 and 100 nm). Since the induced magnetic forces on MNPs are proportional to the magnetic fi eld gradient, these results show that the fl ower-like shaped MNPs experience stronger forces than spherical or spheroidal particles. The reason is that the fl ower-like MNPs provide more translational and rotational movement. In addition, our mod-eled data for fl ower-shaped MNPs in Figure 6 illustrate that the magnetic fi eld coupling between the MNPs strongly depends on the shapes of the interacting parts. Namely, the coupling fi elds between sharp tips (Figure 6 a) is stronger than between the sharp tip and fl at smooth edge (Figure 6 b). Overall, the interaction effects depend strongly on the MNPs size, shape and the distance between them. However, these effects become negligible for separations of more than three particle dia-meters. [ 47 ] The interactions between MNPs described here and other physical properties (such as magnetization, size, shape, etc.) play signifi cant roles and must be taken into account for biomedical applications. For instance, from the simulations described above, it is suggested that spherical shape, non-coupling MNPs are recommended for applications such as IMA, cells separation, and drug targeting in which a precise control by the external magnetic fi eld is required; while non-regular shape, strong-coupling MNPs are recommended for

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Figure 4. Modeled MNP magnetizations for different interactions between MNPs. To further understand how the interactions between MNPs affect their magnetization, we present the magnetic fi eld distri-bution between identical magnetic isotropic MNPs. We consider three situations: a) prolate spheroids with a 150 nm major axis and a 50 nm minor axis; b) spheres of 100 nm diameter; and c) two fl ower-like MNPs with 100 nm cross-sections, all with equal relative permeability µ r = 100, for detailed analysis of magnetic fi eld couplings. The MNPs were exposed to a DC magnetic fi eld and calculations were performed for three separa-tions: h = 1, 5, and 10 nm. The calculated magnetic fi eld distributions are shown in Figure 5 (for sphere and spheroid MNPs) and Figure 6 (for fl ower-like MNPs). The numerical data in Figure 5 show that the magnetic fi eld between two vertically oriented spheroids (solid lines) and spheres (dashed) increases by an order of magnitude when the distance between spheres decreases from 10 to 1 nm.

Figure 5. Total magnetic fi eld versus distance along the z -axis for two identical isotropic targets separated by h = 1, 5, and 10 nm. Solid lines are for two vertically oriented spheroids, with a = 150 nm major axis and b = 50 nm minor axis, respectively, and dashed lines are for two spheres, with a = b = 50 nm radius. The insert shows a schematic representation of the Tx loop and spheroids in a coordinate system. The y -axis is in plane. The Tx loop is placed at x = y = 0, z = 3 cm.

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applications that require generation of hysteresis, frictional and other losses such as hyperthermia.

3. Surface Engineering of MNPs

In addition to the key roles played by MNP’s material proper-ties, structures, and interactions, surface functionalization of MNPs is critical to enabling high-performance immunoassays. In particular, MNPs must effi ciently associate with target cancer cells, and this goal is best achieved through active molecular targeting. There currently exists a wide array of bioconjuga-tion strategies and targeting ligands that have proven useful in specifi c targeting of MNPs to cancer cells, the most prominent of which will be reviewed here.

3.1. Surface Biomarkers for Targeting Cancer Cells

It bears noting that, beyond the properties of the MNPs them-selves, the nature of the target cancer cells are central to the design of effective immunoassays. In particular, detailed knowl-edge of cancer associated biomarkers is key to enabling mole-cular targeting. A thorough consideration of this important topic is beyond the scope of the current review, but we pro-vide an abbreviated list of cancer cell surface markers and/or secreted tumor antigens that have been targeted using func-tionalized MNPs ( Table 4 ). More detailed reviews of cancer biomarker discovery and exploitation are available elsewhere, including recent articles discussing technical innovations in single cell analyses, [ 110 ] genomics, [ 111 ] proteomics, [ 112 ] glyco-nomics, [ 113 ] and systems biology. [ 114 ] As a general comment, it

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Figure 6. Total magnetic fi eld versus distance along the z -axis for two vertically orientated isotropic fl ower-like shaped MNPs, with 100 nm cross-section size, separated by h = 1, 5, or 10 nm. The insert shows a schematic representation of the Tx loop and MNPs in a coordinate system. The y -axis is in plane. The Tx loop is placed at x = y = 0, z = 3 cm. a) Sharp tips are close to each other; b) a sharp tip is close to a fl at smooth edge.

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has been well established that the effi ciency of molecular tar-geting correlates directly with cellular expression levels of the cognate receptor. See, for example, rigorous quantitative studies on targeting epidermal growth factor receptor (EGFR) [ 115 ] and human epidermal growth factor receptor 2 (HER2), [ 116 ] as well as a seminal article on the effi ciency of MNP targeting to the α-folate receptor (α-FR). [ 117 ]

3.2. Affi nity Ligands for MNPs-Based Molecular Targeting

Molecular targeting of MNPs has been realized using a wide variety of ligands ( Table 5 ), but antibodies and engineered antibody fragments continue to be the most widely employed targeting moieties. As affi nity reagents, antibodies exhibit numerous desirable properties including good stability, high

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Table 4. Cancer biomarkers targeted with MNPs.

Biomarker Type (surface or soluble) Cancer type Objective (diagnostic, in vivo Imaging, therapeutic)

Ref.

Human protein tyrosine kinase-7

(PTK-7)

Surface Various Diagnostic [118]

IgM B cell receptor Surface B cell lymphoma Diagnostic [118]

Prostate-specifi c membrane

antigen (PSMA)

Surface Prostate Imaging/therapeutic [119,120]

Alpha-fetoprotein (AFP) Surface Liver Imaging [121]

Integral membrane glycoprotein Surface Various Imaging/therapy [122]

α v β 3 integrin Surface Various Imaging [123,124]

Matrix metalloproteinase-2

(MMP-2)

Surface Various Imaging [123]

Underglycosylated mucin-1

(uMUC-1)

Surface Various Imaging/therapy [125]

Stage specifi c embryonic

antigen-3 (SSEA-3)


Gastrin-releasing peptide (GRP)

receptor

Surface Prostate Imaging [127]

Human epidermal growth factor

receptor 2 (HER2)

Surface Breast, various Imaging/therapy [128–130]

α-folate receptor (α-FR) Surface Ovarian, various Diagnostic/Imaging/therapy [48,117,129,131,132]

Glypican-3 (GPC3) Surface Liver Imaging [133]

Endothelin B receptor Surface Brain Imaging [134]

CD22 Surface Leukemia Therapy [135]

Epidermal growth factor receptor

(EGFR)

Surface Various Imaging/therapy [136]

CD146 Surface Gastric Imaging [137]

Phosphatidylserine Surface Various Imaging [138]

Placental alkaline phosphatase

(ALPP)


Transferrin receptor 1 (TfR1) Surface Various Imaging diagnostic [140]

Secreted protein, acidic and rich

in cysteine (SPARC)

Secreted matricellular protein Various Imaging [141]

Tenascin-C (Tnc) Surface Various Imaging [124]

Nucleolin Surface Various Imaging [124]

Clotted plasma proteins Extracellular environment Various Imaging [142]

Glucose transporter (GLUT) Surface Various Imaging [143]

Epithelial cell adhesion molecule

(EpCAM)

Surface Various Diagnostics [20]

Urokinase plasminogen activator

receptor (uPAR)

Surface Various Proof-of-concept [144,145]

Carcinoembryonic antigen (CEA) Surface Various Diagnostics [146]

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affi nity, exquisite specifi city, and the inherent capacity to target virtually any antigen of interest. Additionally, antibody dis-covery and engineering technologies are mature and robust, which further facilitate their general applicability as targeting ligands.

Numerous studies have shown that nanoparticle targeting, particularly in vivo, is critically dependent on size. [ 128,147,170–172 ] As a class of targeting ligands, full length IgG antibodies are large, and their coupling to nanoparticle surfaces can result in disadvantageously large conjugates. However, antibodies exhibit a modular architecture that enables reformatting as engineered fragments of various sizes, e.g., from single domain “nano-bodies” (≈15 kDa) to full length IgGs (≈150 kDa). There exist comparative analyses of various antibody formats for MNP tar-geting, [ 130 ] but targeting effi ciency is dictated by a vast number of interacting variables such that there is no single “optimum” design for any given application. In particular, MNP size, and thus antibody size, might be less critical for many in vitro diag-nostic applications. Factors such as selection of an appropriate cancer cell biomarker, the magnetic properties of the inorganic nanoparticle core, successful conjugation with fully functional targeting ligands, and an adequate targeting ligand density are likely to be higher priority concerns for developing in vitro immunoassays.

A second class of prevalent targeting moieties is peptide affi nity ligands. Peptides are signifi cantly smaller than anti-bodies or antibody fragments, although they also exhibit lower monovalent affi nity. This limitation can be mitigated in large part by hypervalent display on nanoparticle scaffolds. Phage display, cell surface display, and other discovery platforms enable identifi cation of peptide affi nity ligands for a wide variety of targets. For example, the SPARC-binding peptide and CREKA peptide, both derived from phage display screens, have been used to target MNPs to cancer cells. [ 141,142 ] Other MNPs have been targeted with the EPPT peptide, [ 125 ] which is derived from the complementary determining region of the ASM2 monoclonal antibody. Several natural peptide sequences have also been used to target MNPs to cancer cells. RGD pep-tides from integrin binding sequences, [ 123,124 ] the U11 peptide derived from urokinase plasminogen activator, [ 144,145 ] the chlo-rotoxin peptide from scorpion venom, [ 123 ] and bombesin pep-tides from amphibians [ 127 ] have all proven capable of selective cancer cell targeting. One key advantage of targeting peptides is that they are amenable to chemical synthesis, which renders them readily accessible and easily modifi ed. In general, their facile production combined with impressive targeting versatility

suggests that peptide affi nity ligands will see increasing use as MNP targeting reagents.

While less common than antibodies or peptide, MNPs have been functionalized with a wide variety of other affi nity ligands. Affi bodies, based on derivative fragments of protein A, repre-sent a nonimmunoglobulin binding scaffold that has been used to target MNP to cancer associated surface receptors, namely the HER2 [ 129,160–163 ] and EGFR [ 164 ] membrane proteins. Lectins represent a class of natural binding proteins that selectively target carbohydrate structures. It has been suggested that lec-tins might offer useful MNP targeting moieties for aberrant gly-coforms associated with cancers, [ 126 ] and more recently lectin-MNP conjugates have been validated in in vivo studies. [ 168,169 ] In a reversal of the lectin strategy, MNPs have also been func-tionalized with carbohydrates so as to target carbohydrate receptors on the surface cancer cells. Examples include arabi-nogalactan and lactobionic acid to target the asialoglycoprotein receptor [ 165,167 ] and 2-deoxy--glucose to target the glucose trans-porter (GLUT). [ 143 ] Somewhat analogous to functionalization with sugars, others have shown that MNPs can be selectivity targeted to cancer and other cells via functionalization with a variety of small chemical entities. [ 166 ] Likewise, MNPs have been successfully targeted to cancers using the cognate ligands of upregulated surface receptors, and in particular folic acid has been used to target cancer cells that overexpress the α-folate receptor. [ 117,129,131,132 ] A more exotic approach to targeting a dif-ferent receptor involves encapsulation of MNPs within human ferritin protein shells. [ 140 ] These iron/protein nanocontainers inherently home to cancer cells that overexpress the transferrin receptor 1 (TfR1). Finally, it should be noted that DNA and RNA aptamers have been widely used to engineer MNPs for specifi c molecular recognition. [ 118,124,148–159 ]

Multiplexed targeting of MNPs to two or more cancer cell surface markers can improve targeting effi ciency and enhance cancer cell detection. For example, compared to use of a single monospecifi c MNP preparation, mixtures of different monospe-cifi c nanoparticle conjugates (e.g., anti-EGFR and anti-EpCAM; anti-HER2 and anti-EpCAM; or anti-MUC1 and anti-EpCAM) have been shown to enable more effi cient capture and detection of cancer cells from blood. [ 173 ] The broad utility of multiplexed targeting with mixtures of monospecifi c MNPs is underscored by the large body of literature describing diverse, monospecifi c MNP constructs; conceptually, simple combinations of this vast array of monospecifi c MNP conjugates should enable develop-ment of high-performance immunoassays.

The success of monospecifi c MNP multiplexing suggests that multispecifi c MNP constructs, in which each individual nanoparticle bears two or more types of targeting ligands, could be of great utility. However, publications describing bispecifi c and multispecifi c MNP targeting are rare. Multispecifi c MNPs that simultaneously target nucleolin, α v β 3 integrin, and Tnc proteins were constructed by cofunctionalization with two distinct aptamers and an RGD targeting peptide. [ 124 ] Relative to their monofunctionalized counterparts, these trispecifi c MNPs were shown to have greater sensitivity in detecting numerous cancer cell lines. More recently, bispecifi c MNP have been con-structed via simultaneous conjugation of commercially avail-able anti-CD45 and anti- myosin light chain IgG antibodies. [ 174 ] While this bispecifi c MNP construct is not relevant to oncology

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Table 5. Example ligands employed in molecular targeting of MNPs.

Ligand Ref.

Antibodies [48,119–122,126,128,130,131,134–138]

Peptides (natural and synthetic) [123–125,127,141,142]

DNA and RNA Aptamers [118,124,147–159]

Folate and folic acid [117,129,131,132]

Affi bodies [129,160–164]

Small chemical entities [143,165–167]

Lectins [126,168,169]

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applications (it was designed for regeneration of cardiac tissue), the work demonstrates proof of concept for antibody mediated bispecifi c targeting of MNP. We have developed anti-body targeted bispecifi c MNP platforms for cancer diagnostics, imaging, and therapy (Griswold and Ndong, unpublished data). Specifi cally, we have shown that during incubation with SKBR3 breast cancer cells, MNPs targeted to both HER2 and EpCAM exhibit higher cellular accumulation than their monospecifi c counterparts ( Figure 7 ). Given the impressive preliminary results generated with multispecifi c MNP constructs, we antici-pate that bi- and multispecifi c MNPs will play an increasingly important role in magnetic immunoassays and other cancer applications. Looking to the future, the performance of mul-tispecifi c MNP platforms will be enhanced considerably by advances in conjugation strategies. In particular, the multi-specifi c MNPs described to date relied on stochastic coupling of distinct targeting moieties, which were all coupled to the MNP surface using the same chemistry. Thus, the ratio of one targeting ligand to another could be controlled only through adjusting reaction stoichiometry, and the reaction products exhibit a distribution of targeting ligand ratios. Implementa-tion of more sophisticated orthogonal site-specifi c chemistries should enable fi ner control over the density of complementary targeting ligands, and this in turn is expected to improve the performance of multispecifi c constructs.

3.3. Conjugation and Functionalization Strategies

As indicated above, conjugation strategies are a key aspect of designing and fabricating high-performance MNPs for cancer cell targeting and detection. Perhaps the simplest strategy is biophysical adsorption, but this approach is reversible in nature and can ultimately lead to loss of surface adsorbed targeting ligand. More robust coupling strategies are based on covalent chemical linkages. To date, the dominant reaction chemistry for MNP functionalization has been carbodimide and/or N -hydrox-ysuccinimide (NHS) ester-mediated amide bond formation between carboxylates on the MNP and amines on the targeting ligand, or vice versa (e.g., refs. [124,165]). While these covalent amide bonds provide highly stable linkages, they offer little to no control over molecular orientation of the targeting ligand on the MNP surface. Particularly in the case of targeting proteins, there exist a multitude of free reactive amines and carboxylates distributed across the protein surface, and inevitably some proportion of coupled ligands is oriented with its binding site occluded.

To improve MNP conjugation and targeting effi ciency, a variety of site-specifi c conjugation strategies have been devel-oped and implemented. These methods seek to uniformly orient ligands with their binding sites freely accessible. One example is engineering unpaired cysteines into proteins or peptides, which can then be site specifi cally conjugated using iodoacetyl [ 123 ] or maleimide [ 48,128,160 ] functionalized MNPs. An alternative site-specifi c reaction chemistry is copper-free and copper-catalyzed click conjugation, [ 127,129 ] which can be accom-plished via site specifi cally incorporated azide or alkyne reactive groups within the targeting reagent. A third biorthogonal reac-tion chemistry is the tetrazine and trans-cyclooctene mediated “BOND” strategy. [ 175 ] While BOND chemistry itself is highly specifi c in nature, to date it has been used only after NHS-mediated preactivation of targeting antibodies (e.g., randomly activating lysine residues), which fails to capitalize on the site-specifi c potential of this strategy. An additional approach to site-specifi c conjugation leverages selective oxidation of glycans on IgG antibodies followed by coupling of the resulting aldehydes to hydrazide functionalized MNPs. [ 173 ] Finally, it bears noting that engineered antibodies containing biorthogonal non-natural amino acids are becoming more prevalent, particularly in the fi eld of antibody drug conjugates. [ 176,177 ] Although non-natural amino acids have yet to be leveraged in producing targeted MNP conjugates, the exquisite control and selectivity offered by these customized reaction chemistries is sure to impact future development of high-performance hybrid nanomaterials for in vitro diagnostics.

Other approaches to site-specifi c MNP functionalization rely on noncovalent but high affi nity interactions. For example, NHS ester reactions have been used to covalently couple pro-tein A or protein G to MNPs, followed by site-specifi c capture of IgG antibodies. [ 178,179 ] Similarly, MNP can be functional-ized with streptavidin via NHS ester coupling, followed by capture of site specifi cally biotinylated proteins, peptides, or nucleic acids. [ 118,148,180 ] As an aside, site-specifi c biotinylation of targeting peptides and nucleic acid aptamers is readily accom-plished during chemical synthesis, while site-specifi c bioti-nylation of antibodies and other proteins requires alternative

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Figure 7. Bispecifi c MNPs exhibit enhanced cancer cell association. Maleimide functionalized SPIO MNPs, purchased from Micromod Gmb, was site-specifi cally functionalized with genetically engineered Fab anti-body fragments that bore a free C-terminal cysteine. Three Fab antibody fragments were studied: Bfab, targeting the negative control botulinum toxin; Tfab, targeting HER2; and Epfab, targeting EpCAM. The monospe-cifi c Tfab and Epfab MNPs show signifi cant cellular accumulation relative to the negative Bfab control MNPs, and the bispecifi c Tfab-Epfab MNPs accumulated ≈60% more MNPs than their monospecifi c counterparts.

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strategies, such as fusion with a short biotin ligase recogni-tion peptide. [ 181 ] A relatively recent innovation in site-specifi c attachment of targeting peptides and antibodies is the use of enzyme fusion proteins and MNPs that are surface functional-ized with corresponding enzyme inhibitors. Incubation of the MNPs and enzyme fusion proteins leads to covalent linkage of the enzyme active site to the nanoparticle surface, and as a result the cargo fusion peptide or antibody can be precisely oriented on the MNP surface. To date, this strategy has been implemented with several enzyme fusion partners including O6 -alkylguanine-DNA transferase, cutinase, and haloalkane dehalogenase. [ 130,144,182 ] Another innovative site-specifi c tar-geting strategy integrates MNP nucleation and synthesis with an intact phage display platform. Specifi cally, M13 phage dis-playing the SPARC-binding peptide was engineered to bear a triglutamate motif in their multicopy P8 coat protein. The triglutamate motif was leveraged to nucleate formation of multiple iron oxide nanoparticles along the length of the phage, providing for signifi cant signal enhancement following homing of the phage to prostate cancer cells via the SPARC-binding peptide. [ 141 ] As a whole, the ongoing advances in site-specifi c conjugation strategies should continue to improve the perfor-mance of MNPs in immunoassays.

3.4. Characterization of Functionalized MNPs

There exists a suite of relatively standardized assays by which targeted MNPs are functionally validated. Nanoparticle size is frequently determined by electron microscopy (for iron core) or dynamic light scattering (for hydrodynamic dia-meter). [ 48,117,123,128,183 ] Nanoparticle concentration can be deter-mined by UV–vis spectroscopy or inductively coupled mass spectrometry, and the concentration of attached biomolecules (e.g., antibodies, affi bodies, etc.) is often determined with Brad-ford or bicinchoninic acid assays, typically with an appropriate correction for interference by the MNPs themselves. [ 48,123,128 ] In other cases, the extent of conjugation is determined by quan-tifying MNP reactive surface groups followed by subtractive analysis, [ 166,184 ] and capillary electrophoresis with laser-induced fl uorescence detection has also been leveraged to assess con-jugation effi ciency. [ 185 ] More qualitative assessments of pro-tein or chemical functionalization can be obtained by dynamic light scattering size analysis [ 128,184 ] or infrared spectroscopy. [ 123 ] Although less common, nuclear magnetic resonance spectros-copy has also been used to assess ligands conjugated to the sur-face of MNPs. [ 117,186 ]

Once fabricated, the binding activity of targeted MNP con-jugates is often determined by ELISA assay, where the bound fraction of MNPs is readily determined using a ferrozine assay. Such ELISA assays can be performed with either purifi ed pro-tein target [ 48,128,187 ] or live cancer cells. [ 48,123,128 ] Alternatively, protein binding affi nity can be determined by surface plasmon resonance, [ 117,188 ] whereas measurement of magnetic relaxa-tion times [ 184,189,190 ] or magnetic spectroscopy of nanoparticle Brownian motion (MSB) [ 148 ] can provide quantitative binding measurements in a homogeneous format. Magnetic relaxa-tion measurements can likewise enable detection and receptor profi ling of cancer cells, [ 118,131 ] and MNP-cellular interaction

can also be quantifi ed by fl ow cytometry, provided the nano-particles also bear a suitable fl uorescent marker. [ 183 ] Various microscopy techniques have proven invaluable for assessing MNP interactions with cancer cells. Surface associated and intracellular MNPs can be visualized at high resolution using electron microscopy. [ 48,128 ] Prussian blue staining and light microscopy enable lower resolution imaging, [ 48,183 ] and appro-priately functionalized MNPs are also amenable to fl uorescence microscopy. [ 123,183 ] As a whole, there exists an array of tech-niques and tools with which to characterize the biochemical, biophysical, and performance parameters of functionalized MNP constructs.

4. Immunomagnetic Screening Assay

4.1. Challenges of Rare Cell Separation

The most challenging obstacle in the separation of rare cancer cells are their extremely low concentration. A few CTCs may be presented in 1 mL of whole blood among billions of normal red and white blood cells, even during the early stage of cancer when the primary tumor is not detectable by current available methods. [ 9,191 ] Another tremendous challenge is the hetero-geneity of cancer cells, even the cells disseminated from the same primary tumor. [ 192 ] Therefore, screening technologies with high sensitivity, effi ciency, recovery, and purity are needed while it is also required to keep the cells alive and intact for downstream characterizations. Furthermore, for clinical appli-cations, screening of cancer cells must be performed in a high throughput format. The immunomagnetic approach is advanta-geous for this aspect as the magnetic fi eld can be applied over a broad spatial domain. Last but not least, as the immunomag-netic assay is based on the affi nity binding, selection of bio-markers for the surface conjugation of the magnetic materials largely determines the capability of the immunomagnetic sepa-ration assay. Therefore, the validation of new cancer biomarkers is important for the development of effi cient cancer screening methods. Recent advances of cancer biomarker development have been reviewed elsewhere. [ 193 ]

Numerous immunomagnetic methods have been developed for rare cell separation and many of them have been commer-cialized. CellSearch is the most well-known system for the detection of CTCs and is one of the few systems that have been approved by the U.S. Food and Drug Administration for clinical diagnostics of breast, colorectal and lung cancers. [ 194 ] Most of the commercial assays, including CellSearch, solely rely on one common carcinoma biomarker—epithelial cell adhesion mol-ecule (EpCAM). [ 195 ] However, a particularly important fi nding recently is that invasive tumor cells tend to lose their epithelical antigens by an epithelial-to-mesenchymal transition (EMT) pro-cess. [ 196 ] In addition, it is also known that nontumor epithelial cells can also be presented in blood. Therefore, the detection capability of EpCAM-dependent detection platforms in clinical screening is limited. [ 197 ] Consequently, the development of multiple ligands conjugated magnetic nanomaterials (which was also mentioned in Section 90 ) will allow capturing the phenotypically heterogonous cancer cells and will signifi cantly improve cell separation effi ciency. [ 198,199 ]

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There are several paramount considerations to assess the effi cacy of a cell separation system, including capture effi ciency (e.g., recovery rate), purity and viability. Capture effi ciency is the ratio of captured target cells to the total number of target cells in the input sample. A system with high capture effi ciency can be used to estimate the target cell number in patient's sample. Purity is the ratio of captured target cells to the total captured cells including both target cells and background cells. Purity is an important parameter for post-capture analyses of cells such as gene expression profi ling. Finally, the viability is clearly important when downstream application or analysis is required. Furthermore, in clinical applications there are addi-tional metrics that need to be considered. Throughput is a parameter indicating the speed at which the system can process a sample, which is usually recorded by either the volumetric fl ow rate or the number of cells that can be processed per unit time. Increased throughput can reduce the screening time and cost. Furthermore, the captured cells need to be collected and processed easily so as to reduce the sample preparation time and enable an integrated workfl ow.

MNPs association with CTCs could infl uence downstream events and processes, such as cell viability, proliferation, and metabolic state. It has been observed that nanoparticles attached to cells will eventually be taken into the cell cytoplasm through a process called endocytosis, over a span of a few hours to days. [ 200,201 ] The effects of nanoparticle accumulation within cells are critically dependent on the route of cellular entry, cell type, nanoparticle type, and nanoparticle coating as well as the length of time the cells have been exposed to the MNPs. Many research conducted on the toxicity of MNPs have drawn the generalized conclusion that the viability of the labeled cells is infl uenced by the particle concentration and exposure time. [ 202,203 ] Interested readers are referred to specifi c reviews and emerging studies on this topic. [ 204–207 ]

4.2. Multiscale Immunomagnetic Assay

MNPs have been widely employed in cancer diagnosis, imaging and therapy both in vitro and in vivo. For cell separation applica-tions, there is a debate on whether to use microbeads or nano-particles. The choice really depends on the specifi c applications for which these materials are used for. However, nanoparticles have the following advantages compared to microbeads. [ 208 ] First, nanoparticles have a large surface to volume ratio, which leads to higher binding capacity and effi ciency. Second, the nanoparticles have been shown to have faster transport to the target cells compared to microbeads and, thus, they reduce the capture time. Furthermore, the nanoscale dimensions allow multiple nanoparticles attached to one cells without cell aggre-gation, which has been observed with microbeads.

Magnetic separation can be applied in both macroscale and microscale systems, as the magnetic fi eld can be span a large range across these scales. In clinical settings, the analysis of a large blood volume is preferable, especially at the early stage of cancer. Therefore, a variety of separation methods have been introduced in macroscopic magnetic separation systems for large blood volumes. [ 32,209 ] Commercial magnetic activated cell sorting (MACS) systems have been developed, such as the

automated MACS systems developed by Miltenyi Biotec. [ 29 ] The principle of a MACs system is shown in Figure 8 a. Cells of interest are magnetically labelled with MACS microbeads (which are actually 50 nm superparamagnetic nanoparti-cles), and then attached to the inner wall of a column placed in a MACS separator. The column is fi lled with ferromagnetic microspheres and can be used to amplify the external magnetic fi eld. The fl ow-through fraction can be collected as the nega-tive fraction depleted from the labeled cells. Once the column is removed from the separator, the retained cells are eluted as the enriched and positively selected cell fraction. A similar auto-mated technology using a magnetic sweeper device, the Mag-Sweeper, was developed recently (Figure 8 b). [ 210 ] In this system, the diluted blood samples are prelabeled with magnetic beads. Magnetic rods covered with plastic sheaths are swept through the well of blood cells to magnetically accumulate target cells on the rod surface. The captured cells are collected through several wash steps. In a following work, these authors per-formed single cell gene expression profi ling of the isolated cells by MagSweeper using high throughput qRT-PCR arrays. [ 211 ] Another mode of magnetic cell separation was introduced by Zborowski and co-workers (Figure 8 c), which was referred to as a quadrupole magnetic fl ow sorter (QMS). [ 212 ] Four pole magnet pieces are arranged in N–S–N–S confi guration, and the magnetic force in this quadrupole fi eld acts as a “magnetic cen-trifuge” which pushing the magnetic particle labelled cells to the cylinder wall, while the nonmagnetic cell fraction are col-lected in the fl ow (Figure 8 d). Thus, the QMS system separates the cell population in a single and continuous process.

Because MNPs can be rendered with multiple functions in the synthesis and surface engineering process, the cancer cells isolated by the nanoparticles can be performed with other pro-cesses such as targeted imaging and photothermal therapy. Fan et al. synthesized plasmonic gold shell–magnetic core nanopar-ticles, and used the particles for selective isolation and photo-thermal destruction of SKBR-3 cells in a mixture of cancer cell lines. Furthermore, the capturing of rare cancer cells is usually performed in vitro using collected blood sample. A method to overcome this limitation is to target the cells directly in vivo. Galanzha et al. [ 213 ] developed a method to capture CTCs directly in blood stream using functionalized MNPs, and the captured CTCs were detected by rapid photoacoustic imaging ( Figure 9 a). To improve the detection sensitivity and specifi city, gold-coated carbon nanotubes were used as a contrast agent for photoacoustic imaging.

We have shown in an earlier review paper that the strength of magnetic fi eld could be increased by reducing the height of the capture region and increasing the cross-sectional area of the device. [ 32 ] Therefore, using miniaturized immunomag-netic devices may be important to increase the cell capture effi -ciency. With the development of microfabrication technology, various types of microfl uidic based screening devices have been explored for rare cancer cell detection, so as to provide control over the fl ow and transportation and reduce the sample volume and analyte footprint. [ 20,215,216 ] Microfl uidic devices also have the advantages of high throughput and integration of several processes (i.e., sample loading, separation, and identifi cation) into one chip. A microfl uidic device can be integrated with immunomagnetic assay using MNPs, in which the particles

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are conjugated with specifi c ligands to capture the cells, and an external magnetic fi eld is applied to either fi x the captured cells on the substrate or drive the cells to be collected at different outlets.

The methodologies of microfl uidic based cell separation have been developed and reviewed comprehensively in recent years. [ 217,218 ] The combination of magnetic particles-based immunoassay with microfl uidic device has been shown in pub-lications. [ 219 ] A typical example is the CTC-iChip developed by Toner and co-workers [ 220 ] (Figure 9 c,d) which integrated a size-based fi ltration assay and immunomagnetic assay for effi cient capturing of both EpCAM positive and EpCAM negative cancer cells. The iChip contains three stages, including debulking to separate nucleated cells, alignment of cells using inertial focusing, and defl ection of magnetically captured cells into a collection channel. The authors also demonstrated that the CTCs isolated using the iChip could be analyzed on the single-cell level. For nanosized particles, we have developed a system combining MNPs with microfl uidic devices (Figure 9 b). Rare cancer cells (≈5 cells per mL) with very low tumor cell to blood cell ratios (1:10 9 ,) were successfully separated by a magnetic

gradient created in the microfl uidic channel, with capture rates of 90% and 86% for COLO205 and SKBR3 cells, respectively. Kim et al. [ 221 ] also showed a similar system to separate CTCs based on both magnetophoresis and immunomagnetic nano-particles. CTCs from peripheral blood of patients with breast and lung cancers were isolated, and the results were compared with those of healthy donors.

4.3. Positive Enrichment versus Negative Depletion

Based on the nature of biomarkers on the magnetic particles, the current separation methods can be broadly categorized into positive selection (i.e., capturing CTCs and eluting blood cells) and negative depletion (i.e., capturing blood cells and eluting CTCs). Positive selection uses antibodies corresponding to sur-face antigens of the cancer cells to be enriched. Popular recog-nition molecules include EpCAM and cytokeratin families. [ 194 ] In order to increase the capture effi ciency, it is important to improve the interaction of cells and the antibody-coated sur-face. Microfl uidic devices have been developed to facilitate the

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Figure 8. Principles of major macroscopic cell separation systems using magnetic particle. a) MACS system. [ 29 ] Cells labeled with MNPs (black dots) are attracted to the tube wall by external magnetic force, while the unlabeled cells (white dots) are eluted. The attracted cells can be released by removing the magnetic fi eld. Reproduced with permission. [ 29 ] b) MagSweeper system. [ 211 ] Plastic sheaths are swept through the well of blood cells to magnetically accumulate target cells on the rod surface. Reproduced with permission under the tems of the Creative Commons Attribution license. [ 211 ] Copright 2012, Public Library of Science. c) QMS system. [ 209 ] Four pole magnet pieces are arranged in N–S–N–S confi guration, and the magnetic force in this quadrupole fi eld acts as a “magnetic centrifuge” which pushes the magnetic particle labelled cells to the cylinder wall, while the nonmagnetic cell fraction is collected in the fl ow. Reproduced with permission. [ 209 ] Copyright 2011, American Chemical Society. d) The magnetic fi eld distribution of the QMS system. Reproduced with permission. [ 209 ] Copyright 2011, American Chemical Society.

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effective contact between cells and the antibody-coated surface, by introducing grooves, nanostructured surface or micro/nano-particles into the microfl uidic channels.

Although positive enrichment has been shown to isolate cancer cells with high purity, it has several limitations. First, as mentioned in Section 130 , because of the heterogeneous nature of cancer cells, the target cells do not all express the same anti-gens even within the same origin cell lines. Therefore, insuf-fi cient expression or down-regulation of surface markers of target cells limits the positive selection of immunomagnetic assay. Some target cells with unknown surface biomarkers may be excluded during the positive screening, which affects the accuracy of enumeration and downstream analysis of captured cells. Second, it is a big challenge to remove MNPs from the captured target cells and keep the cells intact, while the infl u-ence of MNPs to the cells and downstream event is still an on-going topic as mentioned in Section 130 . In addition, because of the low number of the cancer cells, the positive enriched cells are very diffi cult to recover.

Because of the above reasons, negative depletion methods have been introduced and their popularity is growing. Various label-free isolation methods for negative depletion have been developed, [ 17 ] which is beyond the scope of this review. The immunomagnetic-based negative depletion approach usually use anti-CD45 antibody, as CD45 is a standard protein crite-rion expressed on the surface of leukocyte cells. An important

advantage of the negative depletion is that the target cancer cells can be left intact for further molecular analysis. [ 222 ] How-ever, the negative depletion approach may cause the loss of rare cell through the multiple steps required in the sea of normal blood cells. [ 223 ] A combination of both positive and negative modes may achieve high effi ciency, purity, and throughput, as demonstrated by Toner et al. [ 220 ] in their work on the CTC-iChip mentioned in Section 140 .

4.4. Characterization of Separated Cells

The separated rare cancer cells have to be characterized to pro-vide detailed information for a better understanding of the cell functions and molecular information, which are key steps for the development of personalized medicine, the understanding of metastasis processes and the discovery of new biomarkers, etc. Characterization includes identifi cation and enumeration of the cells and related molecular analysis. Most of the current CTC identifi cation approaches are immunocytochemical based assays and polymerase chain reaction (PCR)-based assays. For the identifi cation and enumeration, the captured cells are usu-ally stained with fl uorescence dyes and observed by microscopy. To date, cytokeratin have become the most widely accepted biomarker for the detection of epithelial tumor cells in mesen-chymal tissues such as blood, bone marrow, or lymph nodes.

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Figure 9. a) Schematic of in vivo magnetic enrichment with photoacoustic detection of CTCs by Galanzha et al. Reproduced with permission. [ 213 ] Copyright 2009, Nature Publishing Group. The laser beam is delivered either close to the external magnet or through a hole in the magnet using a fi ber-based delivery system. b) Microchip based immunomagnetic CTC screening device we have developed. Permanent magnets are placed beneath the microchannel to capture and retain the cells on the substrate. Reproduced with permission. [ 214 ] Copyright 2012, Springer. c) CTC-iChip for both posi-tive enrichment and negative depletion, including the steps of hydrodynamic cell sorting, inertial focusing. Reproduced with permission. [ 32 ] Copyright 2013, Royal Society of Chemistry. d) Picture of the integrated iChip system. Reproduced with permission. [ 32 ] Copyright 2013, Royal Society of Chemistry.

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The widely accepted identifi cation of CTCs was the presence of epithelial markers (EpCAM or cytokeration) and the absence of the leukocyte marker CD45. Furthermore, the use of an auto-mated device for microscopic detection and analysis of large number of immunostained slides will signifi cantly increase the screening speed and reproducibility.

Molecular profi ling of CTCs reveals the relationship with metastatic tumors, oncogenic pathway activation and thera-peutic sensitivity. [ 110 ] While tumors shed many cells into the bloodstream, only a few cancer cells would possibly give rise to metastases. Thus, sampling of collected cancer cells for downstream analyses prior to and during the treatment can guide appropriate therapy. With respect to PCR-based assays for DNA profi ling, reverse transcription PCR (RT-PCR) is usu-ally employed with RNA markers such as CK18, MUC1 and CEA. [ 224–226 ] In addition, a protein profi ling techniques with an adaption of enzyme-linked immunospot technology is employed for the detection of viable CTCs after negative deple-tion, called EPISPOT (EPithelial ImmunoSPOT), and the pro-cedure is shown in Figure 10 a. [ 227 ] In the EPISPOT assay, the bottom of the plate is coated with a specifi c antibody, followed by seeding and culture of the isolated cells. The cells are then removed by washing, and the presence of the released protein is revealed by the addition of a fl uorescence labeling antibody. The fl uorescent immunospot are counted with an automated reader, with one spot corresponding to the footprint left by one viable cells. Furthermore, the genetic instability of CTCs derived from the primary tumor can be assessed by fl uores-cence in situ hybridization (FISH), which is a well-established cytogenetic method using labeled DNA probes to interrogate CTCs for changes in individual genes, including gene copy numbers, arrangement and deletion (Figure 10 b). [ 228 ] FISH is now the current gold standard for monitoring of HER2 status,

which is commonly overexpressed in breast and prostate cancer CTCs. [ 229–231 ]

Conventional molecular analyses are performed in bulk scale on the order of 10 3 –10 6 cells. Such a demand suggests that multiple enrichment steps are needed. In addition, con-ventional methods provide only averaged information from the population which average out the cell heterogeneity that is actually important to the understanding of the captured cells. Therefore, the development of single cell analysis of isolated cancer cells will be very important. Furthermore, if the isolated cells are cultured to expand the cell numbers, the cancer cells tend to modify their characteristics for surviving as the sur-rounding environment is changed. Thus, the development of single cell analysis in parallel with the isolation will be of great importance. A recommended method is to capture the cancer cells at single-cell resolution and stain them on-chip for specifi c biomarkers or cytogenetic evaluation using FISH. Figure 10 b shows an example of FISH analysis of individual CTCs iso-lated from patients with late stage metastatic/recurrent breast cancer carried out by Mayer et al. [ 228 ] The results from HER2 gene amplifi cation by FISH were consistent with the presence of multiple CTC phenotypes, and strongly indicated that some CK-/CD45- cells are in fact CTCs that have presumably down-regulated CK expression below visible detection levels.

5. Conclusions and Outlook

In summary, we discuss the synthesis of MNPs and their surface engineering, as well as their applications in immuno-assay-based rare cancer cell screening. We illustrate how the morphology and the interaction between MNPs infl uence their magnetization, with the goal to guide the selection of MNPs

for cancer cell separations. In addition, with the recent surge of interest in stem cells, the magnet-based cell separation is now re-engi-neered to address this niche fi eld. Although MNP-based cell separation has been well developed, more efforts are still required to translate these techniques to clinical settings with desired specifi city and sensitivity. Here we aim to briefl y discuss the current chal-lenges and future directions of MNP-based cell separation systems.

Although there are several well-known features of cancer cells, such as surface antigen expression and size, many features of cancer cells have not yet been character-ized precisely, and their correlations with metastasis have not yet been identifi ed clini-cally. Thus, separating cancer cells by either one or several criteria may have signifi cant limitations. In addition, for downstream processes, the ability to release the captured cells from magnetic particles has remained one of the largest challenges in the cell iso-lation fi eld. For these reasons, the negative depletion based enrichment approach seems to be more feasible for the unbiased capture

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Figure 10. a) The EPISPOT assay. [ 227 ] Day 1, the bottom of the plates are coated with a specifi c antibody; Day 2–3, the cells are seeded in wells and cultured for 48 h. The released proteins from the cells are immunocaptured by the immobilized antibody. After the cells are removed, the presence of the proteins released from cells is revealed by the addition of a fl uorescence-labeling antibody; Day 4, fl uorescent immunospots are counted with an automated reader. One immunospot corresponds to the fi ngerprint by one viable cell releasing the marker protein. Reproduced with permission. [ 227 ] Copyright 2009, American Society of Clinical Pathology. b) FISH analysis of individual CTCs isolated from patients with late stage metastatic/recur-rent breast cancer. [ 228 ] Top panel shows HER2 amplifi cation compared to centromere 17 in a CK+ cell; bottom panel shows HER2 amplifi cation compared to centromere 17 in a CK-cell. Reproduced with permission. [ 228 ] Copyright 2011, Elsevier.

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of tumor cells, especially those subpopulations that lack cur-rent markers. However, the purities of these methods need to be improved for an accurate analysis. In fact, the idea of com-bining positive selection with negative depletion in one system is very attractive to achieve both high purity and more specifi c isolation of target cells. A promising method is the combina-tion of magnetic particles with microfl uidic devices for multiple isolations. The integrating of several separation processes into a miniaturized device will improve the purity and throughput, and it also allows the adoption outside of the clinic and labo-ratory settings in the developing world. Furthermore, as the screening of cells is only the fi rst step in complex biologic work-fl ows, the simplifi cation of this process by the development of automation, hands-free and user-friendly instrumentation will accelerate its application in clinical settings of diagnosis and therapeutic monitoring of cancers and numerous diseases.

Toward clinical applications, the mechanism and interplay of EMT and MET are only partially understood, and their rel-evance in cancer patients is unclear. Mesenchymal-like cancer cells are present in the blood stream of cancer patients, yet these cells are missed by most of the current assays which are based on epithelial markers. Some reports even suggested that current assays based on epithelial markers may miss the most aggressive CTC subpopulations. [ 232 ] Therefore, CTC isolation techniques need to be optimized to capture both mesenchymal and epithelial CTCs using multiple biomarkers. The specifi city and selectivity of MNP-based separation will also be improved with a better understanding of the antigen expression of target cells as well as the discovery of new biomarkers and corre-sponding targeting ligands.

Fueled by continuing advances in component technologies and their integration, MNP-based immunoassays are poised to enable breakthrough discoveries in our fundamental under-standing of rare cell populations and subsequent translation to a variety of clinical applications.

Acknowledgements The authors are grateful for the fi nancial support from the National Institute of Health (NIH) National Cancer Institute (NCI) Cancer Diagnosis Program under grants 1R01CA139070 and 1U54CA151662-01.

Received: September 30, 2015 Revised: November 1, 2015

Published online: February 8, 2016

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