Tailoring the interplay between electromagnetic fields and nanomaterials toward applications in life sciences : a review

Continuous advances in the field of bionanotechnology, particularly in the areas of synthesis and functionalization of colloidal inorganic nanoparticles with novel physicochemical properties, allow the development of innovative and/or enhanced approaches for medical solutions. Many of the present and future applications of bionanotechnology rely on the ability of nanoparticles to efficiently interact with electromagnetic (EM) fields and subsequently to produce a response via scattering or absorption of the interacting field. The crosssections of nanoparticles are typically orders of magnitude larger than organic molecules, which provide the means for manipulating EM fields and, thereby, enable applications in therapy (e.g., photothermal therapy, hyperthermia, drug release, etc.), sensing (e.g., surface plasmon resonance, surface-enhanced Raman, energy transfer, etc.), and imaging (e.g., magnetic resonance, optoacoustic, photothermal, etc.). Herein, an overview of the most relevant parameters and promising applications of EM-active nanoparticles for applications in life science are discussed with a view toward tailoring the interaction of nanoparticles with EM fields. © The Authors. Published by SPIE under a Creative Commons Attribution 3.0 Unported License. Distribution or reproduction of this work in whole or in part requires full attribution of the original publication, including its DOI. [DOI: 10.1117/1.JBO.19.10.101507]

Tailoring the interplay between electromagnetic fields and nanomaterials toward applications in life sciences: a review 1 Introduction Nanoparticles, in the following referred to as NPs, exhibit outstanding physicochemical properties in contrast to their bulk counterparts, i.e., non-nanostructured materials. Indeed, NPs can be considered as fundamentally new materials owing to distinct size-dependent properties, which some materials present in the size range of ca. 1 to 200 nm. In the nanoscale, properties such as size, shape, and crystallinity can dramatically affect the optical, magnetic, and/or catalytic properties of NPs. In general, the spatial confinement of electrons, phonons, and electric fields in and around the NPs determine most of these novel "nano" properties. 1 Control of the NP properties allows us to anticipate their response to electromagnetic (EM) fields of a particular frequency and intensity. For instance, the optical properties of noble metal 2 and semiconductor 3 NPs and the magnetic properties of ferrite NPs 4 can be tailored by changing both their size and shape.
The cross-sections of NPs are typically orders of magnitude larger than those of organic molecules, and thus, absorption and scattering processes are key for certain applications. 5 Metallic NPs are, for instance, much more efficient scatterers (>10 6fold) than any organic molecule. Clearly, this has important implications in the context of biomedical applications, which are based on the response of materials to EM fields. Moreover, current synthetic bottom-up approaches allow for tailoring the interaction of NPs with a specific EM field by simply adjusting the composition, size, and shape. 6,7 NPs can thus be engineered for a specific application. For example, NPs aimed at deep body imaging have to absorb and emit in the so-called biological window in the near-infrared (NIR), i.e., in the wavelength range of 800 to 1100 nm. Otherwise, both excitation and detection of the signal would be impaired due to scattering by the surrounding physiological components. For the case of noble metal NPs and quantum-dots (QDs), these optical features can be achieved by increasing the anisotropy of the NPs (e.g., nanorods, nanoprisms, nanostars, etc.) 6 and by controlling the composition and diameter of the QDs, 3 respectively. In the biological window, EM fields interact minimally with physiological components, such as blood, water, and fat. 8 Quoting the words of Kotov: "the only way is up," which makes reference to the tunability of the optical properties in the NIR of upconverting nanoparticles (UCNPs) for bioimaging. 9 Bioimaging using shortwave infrared (SWIR) light with wavelengths from 0.9 to 1.7 microns represents another recent example in this direction. The recent development of indium gallium arsenide sensors has made SWIR imaging technically possible. Likewise, NIR and SWIR bioimaging benefit NPs, which can interact efficiently with these wavelengths. [10][11][12] In the following, some important parameters and relevant examples with regard to EM-active NPs will be discussed. The definitions of NPs and nanomaterials are relatively broad, and thus, to simplify, this review will focus on (1) colloidal NPs based on inorganic materials and (2) some relevant bioapplications with regard to the interaction of NPs with EM fields, i.e., bioapplications based on EM-active NPs. Please forgive me for the important omissions, as there will be plenty due to the wide scope of this topic. This review is intended for nonspecialists in any specific bioapplication of NPs. I hope it will provide an ample overview about the opportunities that EMactive NPs can offer in life science applications.

Biochemistry and Biomolecular Processes
Occur at the Nano-and Microscale In addition to the ability of NPs to interact efficiently with EM fields, the size scale where this interaction occurs is of utmost importance and suitability for biological processes. Being able to remotely manipulate nanomaterials with EM fields opens up a variety of opportunities in life sciences because biochemistry is actually governed by nano-and microscale processes, such as biomolecular recognition, molecular gradients, signaling pathways, cellular uptake, etc. The ability to control ion channels and neurons through heating of NPs is one remarkable example, which illustrates how EM-active NPs can control biological processes. 21 Clearly, the size scale in this example is as important as the heating properties of the NPs, i.e., heating is required in the nanoscale only. The Gueroui group has reported other fascinating examples with regard to EM control over cellular fate by using functionalized magnetic NPs, [22][23][24] which were able to induce gradients of signaling proteins in the microscale by using magnetic field gradients. EM-active NPs can be combined with one or more components from a library of molecules of biological relevance, including proteins, peptides, carbohydrates, nucleic acids, peptides, antifouling polymers, etc. 25,26 The capability to perform more than one simple task is one of the most promising aspects of bionanotechnology, i.e., the so-called multifunctional NPs. The examples in the literature about multifunctional NPs are manifold, 15,[27][28][29][30][31] and the combinations of molecules and NPs are very diverse. Figure 1 schematically depicts several possible functionalities, which are categorized depending on the NPs' design. As previously acknowledged, this review focuses on EM-active NPs, and thus, several NP models and applications will not be discussed. This review will not cover NP models whose imaging, therapeutic, or sensing features are based on organic ligands/components, such as purely organic nanostructured materials, NPs as carriers of functional molecules, etc. As summarized in Table 1, three different categories of EM-active NPs will be considered, i.e., imaging, therapeutic, and biosensing agents. The range of functions provided to NPs by molecules can be as broad as the diversity of molecules. Therefore, and for the sake of simplicity, the molecules typically used to add biofunctionality(ies) to NPs could be categorized according to three main functions, i.e., targeting, antifouling, and treatment with drugs. Table 2 summarizes some of the most widely used molecules in combination with NPs.
In the literature, there are plenty of examples about multifunctional NPs designed to perform complex tasks simultaneously, using plasmonic, 76 upconversion, 77 semiconductor, 78 or magnetic 79 EM-active NPs as scaffolds upon which a diverse range of multifunctionality can be built. Furthermore, many NPs absorb light, which matches their plasmon band; the absorbed energy is transferred to the crystal lattice of the NPs, which upon relaxation get hot. Heating in the nanoscale occurs by thermal diffusion.
Magnetic heating Magnetic NPs 41 The magnetic moment of magnetic NPs couples to alternating magnetic fields of radiofrequency radiation; upon magnetic reversal, the absorbed energy is dissipated in the form of heat.

Photodynamic
UCNPs 42 NPs absorb more than one photon, which triggers the emission of one photon at higher energy by anti-Stoke emission.
Sensing Optical readout Noble metal NPs [43][44][45] The optical, magnetic, or electric properties of NPs are affected by the detection of the analyte. In general, the NPs are modified with biomolecules, which can recognize a specific analyte. The actual change in these properties is related to the amount of analyte.

Magnetic readout Magnetic NPs 46
Electric readout Noble metal, carbon nanotubes (CNTs) 47,48  NP models have been shown to work simultaneously as imaging and therapy agents, that is, multifunctional NPs typically referred to as theranostic probes. As we shall see in the following sections, NP models such as QDs, plasmonic, upconversion, and magnetic NPs can work as therapeutic and imaging agents. Although many of the most promising NP models have been shown to exhibit theranostic features, to simplify, this review will independently treat imaging and therapeutic NPs. Table 3 summarizes some examples of theranostic agents based on EM-active NPs.

Nanoimaging
In this section, basic principles and opportunities with regard to the use of NPs as imaging nanoantennas will be discussed. Three main phenomena will be covered, that is, photoluminescence (PL), magnetic resonance imaging (MRI) contrast, and optoacoustic imaging (OI) contrast. Others also exist, such as thermal imaging, 89,90 Raman mapping, 91,92 x-ray computed tomography (CT), 87 radionuclide-based imaging, 80 or nonlinear optical phenomena, such as two-photon luminiscence; 93 however, to date, they are less widely spread than the techniques covered herein. Please notice that this review focuses on imaging based on the physicochemical properties of the inorganic core of EM-active NPs and, therefore, imaging techniques based on NPs labeled with organic fluorophores or radio-labeled will not be discussed.

Photoluminescence
Equivalent to common organic fluorophores, upon absorption of light, various NP models can emit light typically of longer wavelengths. Indeed, the colloidal synthesis of QDs was probably one of the main triggers of the current nanohype. These NPs made of semiconductor materials exhibit extraordinary fluorescence when they are illuminated at wavelengths intrinsically related to their composition, structure (core or core@shell), size, and surface chemistry. 3 Actually, QDs present several enhanced optical features compared to organic fluorophores, such as size tunability of absorption and emission, high quantum yield, photostability, etc. For a detailed comparison between QDs and common dyes, the reader is referred to the work of Resch-Genger et al. 5 Since the original works of Brus, 94 Bawendy, 95 Alivisatos, 96 Weller et al., 97 and others, 98,99 tremendous advances have been done with controlling the optoelectronic properties of QDs, that is, how QDs respond to light excitation. These have been achieved mainly by the steady developments in colloidal chemistry, which have enabled one to finely control the size, shape, composition, coating, etc., of QDs. Currently, synthetic methods permit us to choose QDs with almost any emission from the UV to the SWIR. [100][101][102][103] Yet, although QDs are very bright and photostable, their range of application in vivo has been traditionally limited due to toxicity concerns. Traditional QDs for in vivo imaging contain toxic ions, such as Cd, Hg, Te, Pb, etc., which can be released upon corrosion. Thus, less toxic compositions, such as InP/ZnS, Ag 2 S, or CuInS 2 ∕ZnS QDs, have been investigated as alternatives to provide new opportunities in the medical field. 104 Coating methods have also been refined toward limiting the release of toxic ions, preserving the optical properties [high quantum yield (QY)], and enabling the colloidal stability of QDs in aqueous solution. 105,106 Carbon nanomaterials, such as nanotubes, graphene, nanodots, and nanodiamonds, can also be used for bioimaging owing to their optical response. 107,108 However, several aspects, such as cytotoxicity concerns, emission wavelength, or low extinction, which depend on the NP models, currently limit their use for bioimaging purposes. 109 As an alternative to QDs, UCNPs represent a relatively new and exciting type of imaging agent. The upconversion phenomena involve the combined absorption of more than one photon, which triggers the emission of one photon at higher energy by anti-Stoke emission. 9,110 UCNPs used for bioimaging are typically composed of a host matrix (e.g., Y 2 O 3 , Y 2 O 2 S, LaF 3 , NaYF 4 , and NaGdF 4 ), doping lanthanide ions (e.g., Er 3þ , Tm 3þ , and Ho 3þ , which are the actual absorbers), and Yb 3þ for enhancing the emission efficiency. Although the optical properties of UCNPs can be readily tailored by design, there is an important drawback with respect to QDs, that is, very low QY (0.005 to 0.3%). 111 This point represents a challenging hurdle as it has a difficult solution due to the low extinction of the lanthanides.
Last in this section, a very new class of fluorescence probes is discussed, that is, fluorescence metal nanoclusters (NCs). 112 NCs are made of few to hundreds of Ag and/or Au atoms (core size <2 nm) capped with molecules, which also importantly affect their optical features. 35 In contrast to bulk or NPs made of Au or Ag, the radiative decay of these NCs is very efficient. Indeed, the QY of NCs can be up to nine orders of magnitude larger than QY of the corresponding bulk material. NCs present good photostability in physiological media, high QY (although, in general, smaller than for QDs), broad tunability from the UV to the NIR, and large Stokes shifts. 113,114 The opportunities that these materials open in the context of bioapplications are manifold; however, much work is still needed with regard to the impact of these materials on physiological environments. Au ions are toxic as Cd or Ag. However, Au NPs are believed to be among the safest NPs since they do not decompose readily because Au is the noblest metal. Clearly, in the case of Au NCs, which have an extreme surface-to-volume ratio, their corrosion behavior and cytotoxicity should be reevaluated. Understanding the biological fate and impact on proteins, organelles, etc., of NCs requires further investigations.

MRI Contrast
MRI is widely used in the clinic and presents several advantages over other techniques, such as CT or positron emission tomography, which utilize ionizing radiation. MRI contrast is given by the distinct magnetic relaxation processes of the nuclear spins of hydrogen atoms in water and fat, the major hydrogen-containing components of the human body. Other elements can also be used, including 3 He, 13 C, 19 F, 17 O, 23 Na, 31 P, and 129 Xe. MRI is noninvasive, nondestructive, and allows for full-body three-dimensional reconstruction with high spatial resolution and excellent soft tissue contrast. 4 Nevertheless, MRI as an endogenous technique suffers from poor sensitivity. Therefore, contrast agents, such as gadolinium-complexes and iron oxide NPs, are widely used, enabling superior sensitivity. It should be noted that gadolinium-complexes might release toxic ions and suffer from low circulation time. 4 Iron oxide NPs are actually used to induce local field inhomogeneities (as nanoantennas) that affect the relaxation time of protons, leading to positive or negative contrast. Indeed, tailoring the size of these NPs from <4 nm to ca. 40 nm can be used to produce distinct effects on the relaxivity, called longitudinal or T 1 , and transversal or T 2 . Furthermore, doping with transition metal ions or clustering of NPs induces very high contrast (in this case via enhancement of T 2 ). The tunability, biocompatibility, and versatility make iron oxide NPs, in terms of biofunctionalization, a promising candidate for being widely used in the clinic. We refer to the original work of Lee and Hyeon for an extended review of this topic. 4

Optoacoustic Imaging
The newest and most exciting technique is discussed last in the imaging section, owing to the simplicity of its principles and the fact that it is based on already developed technologies, i.e., sonography and tomography. 115 OI is a hybrid technique that listens to light. The principle relies on detecting acoustic waves upon thermal relaxation of a photoabsorber excited with an intense pulse of NIR light, cf. Fig. 2. The two main advantages are the penetration depth, owing to NIR excitation, and the fact that resolution is not affected by scattering, as it is based on ultrasound. However, sensitivity can be poor due to limited endogenous contrast. Thus, NPs are ideal probes as OI contrast agents, with improved sensitivity, optical tunability by Fig. 2 (a) Schematics of an optoacoustic imaging (OI) system. The slight fiber bundle angle allows illumination of the sample exactly above the transducer array. The animal holder allows the sample to be moved and the recording of sequential slices in precise and predefined steps. (b) OI system used in the work of Bao et al., which reported the OI bioimaging of gastrointestinal cancer by using gold nanoprisms. 37 The clear illumination ring on the animal, which is wrapped in a thin transparent plastic foil and placed in a water tank, allows homogeneous light energy to be transferred around the focal point of the transducer array. Reproduced with permission from Ref. 37. chemical design, and biotargeting capabilities. To date, carbon nanotubes (CNTs) and anisotropic metal nanoparticles (Au and Ag) have been tested as OI contrast agents with excellent results in vivo. 36,37,[116][117][118] The main requirement of these probes is that they should be able to absorb in the NIR; clearly, materials with highest absorption will produce best contrast. 119 Current synthetic methods can be used to synthesize NPs with absorption bands in the range where OI operates. For instance, gold nanoprisms and nanorods, 37,120 whose plasmon band can be adjusted along the NIR by chemical design, have been used as OI contrast agents. Actually, expanding the excitation sources used by OI into the SWIR will improve the possibilities of this technique owing to deeper tissue penetration. 3,121 As summary of the imaging section, Table 4 shows important parameters for the different imaging modalities and selected EM-active NP models.

Nanotherapy
Most of the therapeutic uses of NPs, i.e., nanomedicine, are based on loading the NPs with a variety of functional molecules, thereby enabling multifunctional NPs. 68,130 Cancer treatment has been one major area where NPs have been extensively applied. 131 Ideally, multifunctional NPs should circulate in the bloodstream undetected by the immune system, reach the targeted region, and release or expose a drug or stimuli. Multifunctional NPs present not only several advantages compared to common therapeutic drugs, such as prolonged circulation time, targeting capabilities, superior stability, and enhanced pharmacokinetics, but also potential theranostic features. The diversity of therapeutic NPs can be as broad as the potential drug molecules that can be loaded on the NPs, including anticancer drugs, proteins, and nucleic acids. 132 Although the therapeutic opportunities of NPs are manifold, this review focuses on EM-active NPs alone, and thus, two main therapeutic processes will be discussed: nanoheating and photodynamic therapy (PDT). The therapeutic gain relies on the interaction of the inorganic core of NPs with EM fields. Although other NP-based therapeutic approaches exist, nanoheating and PDT are among the most widely investigated.
A variety of nanomaterials show great promise as nanoheaters, that is, NPs able to produce heat locally (at its surroundings) upon EM irradiation. In many applications based, for example, on plasmonics or in MRI, nanoheating is actually an unwanted phenomenon. 133 Several bioapplications based on nanoheating using NPs have been mainly developed in the last decade. Applications such as negative index of refraction, focusing and imaging with subwavelength resolution, invisibility cloaks, etc., require low-loss plasmonic materials, i.e., graphene, alkali metals, etc., as alternatives to noble metals, such as Au and Ag, which are indeed excellent for nanoheating. 133 The capability of being able to release heat upon remote EM exposure has opened new opportunities for a variety of goals in life sciences. Local heating with colloidal NPs has been used for killing tumoral cells, 134 drug-release applications, 135,136 ultralow detection of tumoral markers, 45 imaging in vivo 37 and in vitro, 137 or even sterilization. 138 In the frame of oncological hyperthermia, both magnetic and plasmonic NPs have been investigated as nanoheaters. Either can be remotely activated by radiation that do not or minimally interact with physiological tissues and fluids. Actually, the major challenge concerning colloidal chemistry within this framework resides in being able to produce NPs that absorb in EM regions where tissue absorption remains minimum, i.e., biological windows. Engineered nanomaterials with tailored heating performance, as well as suitable organic coatings, are continuously developed toward more efficient interactions with EM radiation and the performance of more complex tasks in biological environments. As previously discussed, these materials can be used simultaneously as contrast agents by using imaging techniques that rely on their magnetic (e.g., MRI) or plasmonic behavior (e.g., OI), thereby enabling theranostic NPs. Moreover, plasmonic nanoheating can be used in combination with other therapeutic and imaging approaches. Chen, Nie, and coworkers have recently reported on plasmonic nanoheating combined with PDT. 63 They proposed a theranostic nanoplatform based on plasmonic photosensitizeloaded vesicles, which, in addition, can be used as triple-imaging agents, i.e., NIR fluorescence, thermal and photoacoustic imaging, cf. Fig. 3. Previous works have demonstrated the feasibility of dual imaging-therapeutic NPs based on active NPs by using magnetic and resonant light excitation. 139,140 Two main decay processes (heat losses) can be accountable for nanoheating, i.e., magnetic relaxation and plasmonic relaxation. Both are based on the capability of magnetic and plasmonic NPs to couple to the magnetic component of radiofrequency (RF) radiation or the electric component of light, respectively. Thus, local heating can be produced upon dissipation of the absorbed energy.  139 and graphene nanomaterials. 144 The variety of compositions, sizes (from 10 nm to hundreds of nanometers), and shapes (coreshell, rod-like, cube, star-like, prismatic, triangular, etc.) is remarkable. Their common property is NIR activity. As previously discussed, ideal plasmonic nanoheaters should absorb as much light as possible and they should also exhibit high heating losses (high conductivity). Thus, Au represents an ideal nanoheater, in principle safer than Ag due to toxicity concerns. Ag NPs are more readily oxidized than Au, releasing toxic Ag ions. Therefore, most up-to-date plasmonic nanoheating studies have been carried out with Au anisotropic NPs, such as nanorods 134 or nanoprims 39 , and gold nanoshells. 145 The light-to-heat mechanism involves the resonant absorption of light by the conduction electrons of the NPs, which couple to the incoming radiation. Scattering between the hot electrons and the crystal lattice enables energy dissipation by phonon-phonon scattering. For details about the plasmonic photothermal effect, the reader is referred to an excellent review by Baffou and Quidant, which discusses many important parameters, including thermal diffusion, continuous or pulse illumination, coupling effects, and thermal confinement, among others. 19 Plasmonic nanoheating therapy has been explored using different approaches, ablation of tumoral cells being the most straightforward and widely reported to date. 146,147 The use of plasmonic nanoheating for cancer treatment has indeed reached clinical trials (see AuroLase® Therapy). The principle is simple, as it only requires surface modification of the nanoprobes to enable cellular uptake and prolonged circulation time, and a light source. Different biofunctionalization strategies using biomolecules have been reported to date, enabling specific targeting. Though simple, this approach is very powerful, due to the spatiotemporal control of the illumination of the tissue's areas to be destroyed.

Plasmonic Heating
Other more elegant approaches employ nanoheating to promote drug release, which can then be used for killing or treating the targeted cells. Halas and coworkers used the nanoheatingrelease approach by using nanorods and nanoshells. 136,[148][149][150] In one particularly interesting example, they functionalized the nanoheaters with complementary nucleic acid strands, which upon light excitation can undergo dehybridization of the complementary strand, i.e., the therapeutic drug siRNA, inducing gene silencing. 136 Other approaches have been employed in organic microparticles (layer-by-layer capsules, 151 liposomes, 152 polymer particles, 75 etc.) functionalized with plasmonic NPs, which upon illumination can undergo a phase transition, enabling the release of the caged drug inside the cells. Parak and coworkers have used plasmonic polyelectrolyte capsules to release different drugs, including polymers, 153 pH indicators, and proteins, 154 as well as nucleic acids, 67 inside the cytosol of cells at the level of single cells.

Magnetic Heating
Another type of nanoheaters involves the coupling of the alternating magnetic field of radio-frequency radiation to the magnetic moment of magnetic NPs. Magnetic dipoles result from the spinning of some of the NP electrons. These polarized electrons can align parallel or antiparallel with respect to the neighboring ones and respond very differently to an applied magnetic field. This, in turn, defines how materials are classified as paramagnets, ferromagnets, ferrimagnets, or antiferromagnets. Falling into one of these categories depends on the size of the material, and thus, the magnetic behavior of a particular material can be tuned by adjusting its size. 155 Indeed, superparamagnetism is intrinsically linked to the nanometer range. In contrast to ferromagnetic and ferrimagnetic (FM) NPs, in the absence of a magnetic field, superparamagnetic (SPM) NPs are not magnetized. This actually prevents magnetic coupling and, subsequently, unwanted agglomeration of NPs. Obviously, magnetic agglomeration should be prevented to preserve the colloidal stability and properties of the materials. This is especially important for biological applications where agglomeration can impede the performance of the nanomaterial. Furthermore, the magnetic dipole of SPM NPs and single-domain small FM NPs can couple to RF radiation using relative low field amplitudes, which enables the heating of their local environment upon energy dissipation. This is the basis of magnetic fluid hyperthermia, a technique investigated for decades in the field of cancer treatment. [156][157][158][159][160] The pillars of this therapeutical technique are (1) tumors are inherently more susceptible to increased temperatures than healthy tissue; (2) magnetic NPs can produce heat upon excitation with RF radiation; and (3) the intensity and energy required for exciting these NPs is in a physiological regime, which typically requires frequencies and field amplitudes <1 MHz and 250 G, respectively. Tissue surrounding tumors and nontargeted with NPs will not be damaged upon RF exposure.
Many of the research efforts concerning magnetic hyperthermia have been devoted to the development of NPs with the highest specific absorption rate (SAR), i.e., the capability of the magnetic fluids to absorb and heat as much as possible.
There are different models that aim to explain magnetic relaxation processes involved in magnetic heating. Ultimately, the source of nanoheating is the magnetic reversal of the magnetic moment of a single NP. 161 Magnetic reversal is actually influenced by several properties, such as magnetic anisotropy, size, shape, composition, and coupling effects, which ought to be synthetically tailored for specific RF excitations. Traditionally, iron oxide NPs (magnetite and/or maghemite) with diameters <15 nm have been used for magnetic fluid hyperthermia. The company MagForce AG (Berlin, Germany) utilizes ca. 15-nm iron oxide cores with an aminosilane coating in clinical trials. However, SAR of these NPs is extremely low and, therefore, large doses are required for achieving hyperthermic temperatures in tumors. Employing NPs with optimized SAR values can substantially reduce the required doses, and therefore, many recent works have focused on the development of more efficient magnetic nanoheaters, such as exchange-coupled magnetic NPs (e.g., CoFe 2 O 4 @MnFe 2 O 4 ), 7 iron oxide nanocubes, 162,163 iron NPs, 164 and iron carbide NPs. 165 Interestingly, magnetosomes produced by magnetotactic bacterium have been reported to produce unusually large SAR values compared to similar magnetic NPs produced by synthetic colloidal methods. 166,167 This indeed indicates that there is still space for improving synthetic methods to produce more efficient materials.
As was the case for plasmonic heating, most of the reports on magnetic nanoheating involve the destruction of tissue by heating. 168,169 Many efforts have been devoted to achieving active targeted therapy by functionalization with specific targeting ligands or by using specific cells as Trojan horses. 41,170,171 Figure 4 shows a classical example of magnetic hyperthermia in which clusters of iron oxide NPs-functionalized with folic acid and polyethylene glycol (PEG) to enhance tumor accumulation-act as nanoheaters and MRI contrast agents. 41 Nevertheless, the most widely used phenomenon toward targeting tumors makes profit of a passive response, i.e., enhanced permeability and retention effect, by which particles typically >100 nm are retained inside tumors due to their characteristic vascularity. 172 Yet, although to a lesser extent than plasmonic heating, other works have attempted to use magnetic heating as a route to promote drug release, 173 or even targeting of specific cellular receptors, which can be altered by magnetic nanoheating. 21,174 Figure 5 shows one interesting example of RFdriven smart release. 175 In this work, Aoyagi and coworkers reported a smart hyperthermia nanofiber, which combines heat generation and drug release to induce skin cancer apoptosis.

Photodynamic Therapy
PDT has been investigated to treat cancers for >100 years. 176 The therapeutic action of PDT relies on the excitation of photosensitizers by light in the presence of oxygen, which enables the production of singlet oxygen in the illuminated areas and, thus, the killing of treated cells. As for plasmonic nanoheating, its therapeutic power resides in the use of light as stimulating triggers for a great degree of spatiotemporal control. Several photosensitizers have been developed, 177 even activatable photosensitizers that require molecular activation, such as quenching, pH, solvent, or hydrophobicity. 178 Also, some hybrid materials that combine photosensitizers and NPs have been described. [179][180][181][182] In all the above examples, PDT is achieved by direct light excitation of photosensitizers, which may be or may not be tagged on passive NPs, cf. Fig. 3. 63,183 This review will focus on PDT by EM-active NPs in which reactive oxygen species (ROS) is directly produced by illumination of NPs or by activation of photosensitizers through illumination of NPs. TiO 2 NPs, a widely used material in consumer products, 184 have been shown to produce ROS upon UV illumination. 185,186 However, the use of UV light in cytotoxicity studies is rather challenging due to the intrinsic toxicity of UV light. To overcome this, the doping of TiO 2 NPs with different elements has been explored for allowing the TiO 2 NPs' photocatalytic properties into the visible. 187,188 To date, light activation of TiO 2 NPs represents a major safety concern for nanotoxicology rather than a therapeutic opportunity, 185 although some reports exist. [189][190][191] Equivalent to common photosensitizers, QDs have also been proposed as intrinsic PDT agents. [192][193][194] However, the QYs of the singlet oxygen of QDs are very low compared to the ones of common photosensitizers (ca. 1 to 5% versus 20 to 30%). Therefore, most of the reports regarding PDT and QDs describe the use of common photosensitizers in combination with QDs. [195][196][197] Upon light absorption, photosensitizer-derivatized QDs can produce singlet oxygen. In this case, QDs act as an intermediate of the activation, also known as Förster (fluorescence) resonant energy transfer (FRET) donor. By both oneor two-photon absorption, QDs can be excited with the appropriate light source, and they can subsequently activate photosensitizers, which absorb in the UV-visible. Please notice that the use of PDT has been traditionally limited to surface tumors because usual photosensitizers require low-penetration UV-visible light. Two-photon NIR excitation of photosensitizer-loaded QDs would be highly beneficial for PDT in vivo. 198 Both mechanisms, i.e., FRET-based or direct activation of QDs, result in the generation of reactive singlet oxygen species that can be used for PDT cancer therapy.
On the other hand, the combination of UCNPs and photosensitizers shows great feasibility for cancer treatment, as reported by several works. 42,77,[199][200][201][202] UCNPs have been widely used as FRET donors in PDT, which can be activated by NIR light. Photosensitizers are typically embedded in the silica coating of UCNPs, which can undergo energy transfer to the photosensitizers upon NIR excitation, thereby enabling the production of singlet oxygen. Indeed, the unique NIR features of UCNPs make them the ideal platform for PDT. UCNPs solve two of the most important limitations of photosensitizers, i.e., low solubility in aqueous solution and UV-visible activity. Several reports have shown the feasibility of using UCNPs as NIR transducers in PDT in vivo. 42,77,199 The interested reader is referred to the recent work of Arguinzoniz et al., which has covered in detail the most important aspects of PDT driven by NPs. 177 To finish this section, I would like to emphasize that there are many other NP models, EM-active or passive, which can be used for therapeutic purposes though they remain less explored to date. Other therapeutic approaches can also be achieved with the NP models discussed so far. In addition to PDT, for instance, the upconversion phenomenon can be used for NIR-driven release of photocaged compounds. 34,82 The most common role of NPs in therapy is as drug carriers, 203 which has not been discussed in this review because the therapeutic function of NPs does not rely on the interaction with fields in most of the cases. Therapeutic agents with low solubility (e.g., chemotherapeutic drugs 74 or photosensitizers 204 ) and/or susceptible to quick degradation (e.g., nucleic acids 68 ) can be loaded into NPs with high yield and in combination with other molecules.

Nanobiosensing
The capability of EM-active NPs to act as biosensors relies on changes of their physical properties upon analyte recognition, which can be detected by means of a quantifiable optical, thermal, electric, or magnetic signal. Herein, some sound examples of biosensing using EM-active NPs will be discussed. Biosensing using NPs for mass amplification of the signal upon recognition will not be discussed, i.e., microcantilevers or quartz crystal microbalance technology, as in this case, the role of NPs is passive, meaning not active in terms of interaction with fields.

Optical Readout
The most straightforward and widely used biosensor based on active NPs relies on a change of color (energy resonance), i.e., colorimetric sensors. These have been used to detect the presence of an analyte by simple visual inspection (yes or no sensor), or by spectroscopic means (e.g., plasmon resonance and surface-enhanced Raman). Au and Ag NPs display absorption band(s) in the visible range, which makes them very suitable probes for visual inspection. The plasmon band is mainly determined by the composition, size, and shape of the NPs. 2 However, changes in the dielectric environment also affect the resonance. This is the basis of localized surface plasmon resonance (LSPR) sensing based on LSPR shifts, where metallic NPs anchored to a substrate produce an LSPR shift upon analyte detection. The most suitable probes are those more sensitive to dielectric changes, such as gold nanorods. 205 This technique can be used to detect almost any analyte as long as the colloids are derivatized with catching biomolecules, such as nucleic acids, 206 antibodies, [207][208][209] carbohydrates, 210 etc. Figure 6 shows a schematic representation of the basic process of agglomeration, which is caused by the recognition of analytes, and its detection by LSPR. 211 The interested reader in refractometric nanoplasmonic biosensors is referred to two recent reviews from the group of Lechuga. 212,213 Yet though LSPR sensing is very versatile, colloidal agglomeration as a colorimetric sensor is extremely sensitive and straightforward, allowing for even naked eye detection. The group of Mirkin has reported pioneering works regarding colorimetric biosensors based on DNA-modified Au NPs. 43,[69][70][71][72][73]214 The original work describing this concept reports on NP agglomeration (color change) driven by detection of DNA sequences complementary to two DNA-modified Au NPs. 215 Since this pioneering work, the group of Mirkin has extensively explored the use of ligand-modified Au NPs for biosensing applications. Figure 7 shows one example, which illustrates the versatility and power of this sensing approach, where biobarcoded NPs were used for multiplexed detection of protein cancer markers. 43 This type of assays has developed into more complex assays, such as colorimetric logic gates. 216,217 The strength of colorimetric assays relies on the sensitivity of surface plasmons when there is coupling between plasmonic colloids. For instance, dimers of Au NPs (20 nm) with interparticle distances of <10 nm will have a significant impact on the plasmon resonances, both in the cross-section and wavelength. 6 Another type of optical biosensors is based on FRET. The process requires donor-acceptor pairs in close proximity (1 to 10 nm). Au NPs and QDs have been extensively used in FRET bioassays, the former as donors, which can quench fluorescence of an acceptor due to their high extinction coefficients. 218 In this way, several different approaches can be used to recover fluorescence upon analyte binding, for example, by using a competitive fluorescence molecule (quenched on the surface of the NP) whereby release occurs (fluorescence recovered) upon analyte detection. 219 Likewise, plasmonic NPs can be used to enhance the fluorescence of dye molecules placed at ca. 10 nm from the NPs' surface. 220 Actually, fluorescence quenching or enhancement depends on the distance between the NPs and the dye. 221 In a recent elegant work on DNA-directed nanoantennas, 117-fold fluorescence enhancement was observed for a dye molecule positioned in the 23-nm gap between 100-nm gold NPs. 222 In contrast to Au NPs, QDs are typically employed as energy donor molecules in FRET assays. The tunability of QD emission wavelength, large Stokes shifts, and wide absorption spectra enables them to be used as multiplexing agents. [223][224][225] As in the case of Au NPs, QDs can be functionalized with biomolecules, which allow for competitive assays or simply analyte recognition whereby the fluorescence is "switch on/off". 226 Next in this section, basic principles and some examples regarding surface-enhanced Raman scattering (SERS) biosensing are discussed. However, for details about the opportunities and challenges that this ultrasensitive technique can offer, the reader is referred to a recent work of Alvarez-Puebla and coworkers. [227][228][229][230] Briefly, SERS sensing is based on a strong enhancement of the Raman signals upon analyte detection by plasmonic NPs (typically Au and Ag NPs). In principle, this technique can detect single molecules. 231 The Raman signal depends strongly on the distance of the Raman reporter to the surface of the NPs, quickly extinguishing as the reporter moves away from the surface. Larger enhancements occur in the gap within agglomerates of NPs, allowing for ultrasensitive sensing. Obviously, between 1-and 2-nm gaps, the range of biomolecules that can fit is very limited. SERS-encoded NPs have been used for multiplex imaging in vivo, 44,232 which could be used for detection of multiple biomarkers associated with a specific disease. The company Oxonica Materials commercializes highly versatile signal-reliable SERS-encoded NPs. These consist of silica-coated agglomerates of Au NPs, which can be encoded with different SERS tags. As an alternative to agglomerates, many investigations have been focused on developing synthetic methods to produce anisotropic plasmonic NPs with sharp apexes (nanostars, nanorods, nanoprims, etc.), which can have hot-spots that concentrate large field enhancements. 233 By synthetic design, anisotropic plasmonic NPs can be used for energy concentration at the nanoscale. This is actually a fascinating property of plasmonics using EM-active NPs, with many applications besides SERS sensing. 228 Controlled assemblies of plasmonic NPs have been reported to improve SERS sensitivity, whether it is formed by few NPs 234 or by two-dimensional self-assemblies in large supports. 235 Last, photothermal biosensing is briefly introduced. Though the signal is, in this case, thermal, this is achieved due to photoheating using plasmonic NPs. The principle is simple, that is, plasmonic NPs are functionalized with catching biomolecules, which upon recognition immobilize the plasmonic complex in a support. Then, light excitation can produce a thermal signal, which can be detected by simple visual inspection on a thermosensitive support or by a thermal camera. 236 The sensitivity, which can be up to attomolar range in serum of patients, 45 and the simplicity of this method are astonishing. The principles and protocol of this novel approach are schematically represented in Fig. 8, for the case of detection of a common cancer marker, i.e., carcinoembryonic antigen. 45

Magnetic Readout
As in the case of colorimetric biosensors, magnetic NPs can be driven to agglomeration upon analyte recognition, which, in this case, will affect the magnetic relaxation of the surrounding proton spins (as in MRI). As previously mentioned, tailoring the magnetic properties of NPs and, thus, their interaction with RF radiation can be achieved by adjusting the size, shape, and composition of the NPs. This principle can be used to investigate biomolecular interactions, such as DNA-DNA, protein-protein,  Step-by-step processes for the formation of the immunocomplex using anti-carcinoembryonic antigen derivatized nanoprism (NPRs), enabling thermal sensing upon near-infrared illumination. Reproduced with permission from Ref. 45. protein-small molecule, and enzyme reactions. 46 Importantly, though these measurements require magnetic resonance equipment, which is an obvious disadvantage compared to colorimetric visual inspection, they can be carried out in dirty environments, meaning that sample pretreatment, such as purification, is not required.

Electric Readout
The electrical detection of target analytes by EM-active NPs, such as CNTs or Au NPs, is based on the measurement of their conductivity and impedance properties upon target recognition. Indeed, CNTs and Au NPs have been widely used as transducers in potentiometric analysis. [236][237][238] As in the different sensing methods discussed so far, the most important concept here is that NPs are functionalized with catching molecules, which, upon recognition, change the properties of the transducers. For instance, an aptamer-based CNT potentiometric sensor has been used to detect ultralow concentrations of bacteria. 48 Another type of electrical sensors is based on the photochemistry of QDs whereby charge carriers can be injected into redox reactions upon light excitation. Thus, current changes depend on whether a reaction occurs, in a quantitative manner, and upon light excitation, which allows one to investigate spatiotemporal-driven reactions. 47,239,240

Conclusions and Outlook
The applications of NPs in life science are growing dramatically. As the control over the synthesis of complex NPs evolves, new applications and opportunities can be explored. Two main concepts are to be highlighted: first, EM radiation can be absorbed by inorganic NPs, enabling many highly useful responses, like photoluminescence, nanoheating, magnetic coupling, etc. Second, hybrid NPs composed of inorganic EM-active cores and molecules of biological relevance are required for bioperformance enhancement.
Though the field of nanobiotechnology is now in the forefront of science, there are still fundamental issues that have to be deeply addressed, such as the impact of NPs on life, including biocompatibility, toxicity, ecotoxicity, etc.; in vivo targeting of specific diseases, markers, etc.; prevention of the unspecific interaction with proteins and accumulation in liver and spleen; and understanding of energy relaxation on NPs and related topics, like hot electrons, magnetic relaxation, heat diffusion in the nanoscale, etc., to mention just few. More work is needed on the development of multifunctionaltheranostic NPs, which can perform more than one simple task or serve for more than a proof of principle. As many proof of principles are already established in this area, more efforts should be put into achieving real medical solutions.