Plasmonic nanoparticles in 2D for biological applications: Toward active multipurpose platforms

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REVIEW

Plasmonic nanoparticles in 2D for biological applications: Toward active multipurpose platforms Juan J. Giner-Casares a, Luis M. Liz-Marzán a,b,∗ a b

CIC biomaGUNE, Paseo de Miramón 182, 20009 Donostia-San Sebastián, Spain Ikerbasque, Basque Foundation for Science, 48011 Bilbao, Spain

Received 5 March 2014; received in revised form 27 May 2014; accepted 29 May 2014

KEYWORDS Plamonics; Self-assembly; SERS; Fluorescence; Cell attachment; Cell imaging

Summary Biomedicine and clinical practice are entering a new era in which nanotechnology plays a core role. Plasmonic nanoparticles are most interesting in this regard, given the possibility of fine-tuning light-matter interactions at the nanoscale. The assembly of plasmonic nanoparticles onto planar ensembles with short interparticle distances leads to novel and unique optoelectronic features due to long-range plasmon coupling over large areas. These extended plasmonic structures incorporated onto substrates are proposed as advanced platforms to address biological and medical challenges. In this review we analyze the state-of-the art of plasmonic substrates with respect to their feasibility toward applications in biomedicine. We first consider plasmonic sensing of biomolecules and living cells, which is subsequently complemented by a further step toward the manipulation of living cells. We propose that plasmonic substrates will undoubtedly enrich biomedical practice for generalized use in the near future. © 2014 Elsevier Ltd. All rights reserved.

Introduction Biomedicine undoubtedly benefits from nanotechnology. Indeed, the interplay between these two disciplines is gradually increasing, thereby offering new possibilities and opening new challenges [1,2]. In particular, plasmonics

∗ Corresponding author at: CIC biomaGUNE, Paseo de Miramón 182, 20009 Donostia-San Sebastián, Spain. Tel.: +34 943005300. E-mail address: [email protected] (L.M. Liz-Marzán).

offers a unique opportunity for manipulating the interaction between light and matter at the nanoscale. An impressive array of methods for the preparation of plasmonic nanoparticles has been reported in the literature, and intense research is still being performed in this direction [3]. Two principal applications that are expected from plasmonic structures in biomedicine are sensing (diagnostics) and manipulation of biological systems. Biological objects range from small biomolecules in dilute solutions to large living cells and tissues within crowded environments. In addition, the large complexity of the biological samples is an additional challenge: the composition of biological

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samples is not only complex, but also typically evolves with time. Therefore, a simple and well-defined architecture of the plasmonic nanoparticles on a 2D substrate significantly reduces the experimental variables in understanding and controlling the interactions of the biological sample with the plasmonic substrate. Moreover, the organization of a superstructure made of plasmonic nanoparticles is more closely controlled in a 2D arrangement when compared to bulk solution, where local variations of concentration with time at different places of the solution are unavoidable. The use of plasmonic substrates provides an advanced toolbox in probing the biological objects by experimental methods, mostly related to spectroscopy and imaging. Beyond sensing, the directed actuation on living cells is a most interesting option that we also discuss herein. The concept of designing active substrates that allow the manipulation of living matter in addition to biosensing is most promising if we are to push nanoplasmonics for actual use in biomedicine. We aim herein to demonstrate the growing relevance of 2D plasmonic substrates in biomedicine, offering not only improved features, but also novel concepts.

Assembling plasmonic nanoparticles onto planar substrates Brief comment on ‘‘top-down’’ and ‘‘bottom-up’’ approaches Up to now, nanofabrication has relied on two standard approaches: ‘‘bottom-up’’ and ‘‘top-down’’. In spite of being usually presented as competing procedures, these two approaches are in fact compatible and combinations of both approaches have been used in a number of cases. Such a central discussion of the prevalent general approach in nanotechnology is beyond the scope of this review; we however present a few ideas. The ‘‘bottom-up’’ approach may be defined as the realization of a superstructure from the self-assembly of suitable building blocks. Bottom-up fabrication thus leads to finely defined nanostructures, which in turn results in a narrow distribution of the physical properties of the assembled nanostructures. Bottom-up procedures are experimentally simple, yet achieving precise positioning of the nanoparticles. In the case of interest herein, spatial and temporal manipulation of nanoparticles into the desired structure is conceptually and technically challenging, but significant achievements have been made [4—7]. On the other hand, the ‘‘top-down’’ approach can be defined as the partition of a larger piece of matter into the smaller nanostructure. Top-down processes appear easier to be implemented in large-scale production, nanolithography probably being the most popular technique [8]. We have included in this review some study cases based on either ‘‘bottom-up’’, ‘‘top-down’’, or a combination of both approaches, concerning the fabrication of plasmonic substrates. We conclude that each approach poses different advantages and shortcomings, and the choice should ultimately depend on the required application.

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Order and interparticle distance: nanoplasmonics in 2D Plasmonics is now consolidated as a prominent area in nanotechnology and materials science, since exceptional results have been achieved toward important applications such as energy conversion [9], sensing [10], and biology [11]. In metal nanoparticles, light can induce localized surface plasmon resonances (LSPRs), i.e. collective oscillations of conduction electrons. Such collective oscillations are stimulated by an electromagnetic radiation of a given frequency that is resonant with the frequency of electron oscillation [12]. LSPR frequencies can be conveniently tuned by varying the size, shape, and chemical nature of the nanoparticles. Additionally, LSPRs can be significantly affected by the physicochemical environment of the nanoparticle, which is of special relevance in the applications that we discuss herein, i.e. in biological environments. Given the dependence of the LSPR signal of a plasmonic nanoparticle with the surrounding dielectric constant, surface plasmon resonance sensing has been implemented in biological detection. In this approach, the adsorption of the analyte on top of a plasmonic surface leads to a change in the local refractive index, thereby affecting the LSPR frequency [13,14]. Plasmonic substrates have been purposefully designed for this sensing technique, with large success [15—17]. Note that LSPR sensing is beyond the scope of this review, so we discuss the fabrication and use of plasmonic substrates focusing on the enhancement effect and unique optoelectronic environment provided by the intense plasmonic field in planar (2D) substrates. An ensemble of plasmonic nanoparticles in a 2D substrate displays modified optoelectronic features with respect to those of individual nanoparticles in bulk solution [18]. These unique optoelectronic features are most relevant when considering the usage of plasmonic substrates for biological applications. The advantages of using 2D instead of 3D architectures are many-fold. First, self-assembly of plasmonic nanoparticles can be realized in the bulk, leading to 3D structures [19,20]. On the other hand, 2D assemblies of plasmonic nanoparticles allow for a more effective coupling of the individual plasmon oscillations. An interparticle separation in the nanometer range is required for strong plasmon coupling, which greatly enhances the electromagnetic field at the gaps between nanoparticles, leading to so-called ‘‘hot spots’’. The distance required for plasmon coupling depends on the size and shape of the nanoparticles, being longer when increasing the particle size. As a rule of thumb, NPs should be separated by a distance typically shorter than their radius [21,22]. Monodisperse and well-ordered plasmonic nanoparticles are desirable to maximize plasmon coupling. Second, extended assemblies of plasmonic nanoparticles on 2D substrates may achieve a well-defined nanostructure over the microscopic or even the macroscopic scales. Therefore, the integration of the nanoscale design of nanoparticle ensembles within a macroscopic device is feasible. Eventually, the usage of the plasmonic devices in biomedical practice will require a user-friendly configuration, which can indeed be achieved on the basis of 2D plasmonic devices.

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Figure 1 Assembly of plasmonic nanoparticles on 2D substrates provides a unique platform for studying and applying plasmonic fields to biomedical problems. Short interparticle distances lead to significant modification of their optoelectronic properties through plasmon coupling, which can be finely defined in the nanoscale over large areas in the macroscopic range. 2D plasmonic substrates interacting with biological bodies show a reduced number of interactions, thus the biophysical mechanisms are easier to understand.

Third, the interaction of plasmonic nanoparticles with biomolecules or cells is difficult to characterize in fine detail in bulk solution, given the large degree of freedom of both nanoparticles and analyte. On the other hand, a 2D architecture usually offers both a simpler and more finely defined platform. This simplicity is the main advantage for effective characterization at the interface between the plasmonic substrate and biomolecules or cells. Additionally, the position and structure of nanoparticles can be much better controlled within a 2D arrangement, as compared to bulk solution, where local inhomogeneities with time will be present. Well-defined plasmon coupling over large areas can be achieved on solid substrates, whereas Brownian motion in bulk solution impedes such stable nanoparticle arrangements. 2D plasmonic substrates constitute simpler physical models for studying the mechanisms of interaction between biological and plasmonic entities, as compared to their 3D counterparts. We thus conclude that, 2D plasmonic devices provide unique features related to plasmon enhancement that render them particularly useful for studying interactions between biological bodies and plasmonic interfaces, ultimately leading to useful devices in biomedical practice. Fig. 1 summarizes these ideas.

Deposition and growth of plasmonic nanoparticles on substrates A plethora of fabrication methods for 2D plasmonic substrates are available in the literature, including different procedures based on both bottom-up and top-down approaches. An exhaustive description of such methods deserves a separate focus, and is beyond the scope of the present review. We restrict ourselves to discussing a selection of relevant procedures for the fabrication of plasmonic Please cite this article in press as: J.J. http://dx.doi.org/10.1016/j.nantod.2014.05.004

Figure 2 Top-down and bottom-up approaches have been successfully used to fabricate 2D plasmonic substrates. (a) Nanosphere lithography based on metal evaporation through a monolayer of colloidal particles [24]. Copyright from American Chemical Society, 2001. (b) Nanopillars constructed by etching a nanostructured substrate [31]. Copyright 2013 American Chemical Society. (c) Substrates fabricated by deposition of metal onto an previously patterned substrates [33]. Copyright 2013 Wiley-VCH. (d) Standing superlattices of Au nanorods fabricated by drop casting [5]. Copyright 2009 Wiley-VCH. (e) Block copolymer micelle nanolithography, i.e. deposition of micelles containing an Au salt and subsequent removal and Au reduction by plasma treatment [34]. Copyright 2013 Wiley-VCH. (f) Chemical growth of patterned Au NPs substrates [35]. Copyright 2010 Elsevier.

substrates to our personal view. Special attention is given to the further applicability of these substrates in biologically related aspects. Van Duyne’s team has undoubtedly contributed to qualitative advances in the field of plasmonic substrates. Although considering several fields of research, a special focus was devoted to LSPR sensing [13,23]. One of the most illustrative and successful procedures is the so-called nanosphere lithography method (NSL, see Fig. 2A). The NSL is based on the deposition of one or two layers of colloidal spheres onto a bare substrate, which are subsequently used as a lithographic mask [24—27]. Subsequently, the desired metal is deposited by physical techniques such as electron beam deposition or thermal vacuum evaporation. The resulting substrate exhibits periodic patterning of nanoparticles, which might include different sizes and shapes, as well as different LSPR features. NSL has been applied with great success to prepare a wide variety of nanoparticles, to be applied to different problems in chemical and biochemical sensing [23,28]. The NSL method fulfills the requirements in plasmonic efficiency and large-area coverage for mass-scale production of plasmonic substrates, and can be regarded as a smart combination of top-down and bottom-up approaches. On a side note, we highlight a study from this group using Al as the plasmonic metal for patterning of nanotriangles [29]. Aluminum is inexpensive and in principle also biocompatible, thus the possibility of using these plasmonic substrates within coating implants or other in vivo devices

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could be envisioned. Aluminum however suffers from poor chemical stability, and this issue has to be dealt with in case of launching a research line for application of Al-based plasmonic substrates for biomedicine [30], considering in particular the harsh physicochemical conditions that in vivo devices are subjected to, i.e. during fabrication, sterilization and within a living body. On a different line of research, the Boisen group designed a plasmonic substrate based on Au nanopillars for biomedical sensing. The nanopillars were fabricated by first etching a silicon wafer and subsequently depositing Au by electron beam deposition (Fig. 2B). Such plasmonic nanopillar substrates exhibited good performance as spectroscopybased substrates against a hormone relevant in hemorrhagic shock. This hormone, vasopressin, typically occurs in concentrations up to 10 pM in plasma under healthy conditions, whereas this concentration increases up to 350 pM in case of hemorrhage. Therefore, quantitative detection of this hormone would be of great interest in first-aid clinical practice. Vasopressin-specific aptamers were also incorporated on the surface of the Au nanopillars, thus adding selectivity in the attachment of vasopressin to the Au nanopillars and its subsequent detection. The most remarkable feature of these nanopillars is the possibility of controlling their relative tilting, which helps trapping the analyte and therefore creating hot spots that maximize the spectroscopical signal. The creation of hot spots was induced in this case during solvent evaporation, resulting in leaning of the nanopillars against each other [31,32]. This study nicely illustrates the dynamic approach for fabrication of 2D plasmonic substrates, in which the nanostructure is responsive to a given stimulus provided by the biological system. In this case the formation of the hot spots is triggered by the presence of the analyte, but within the same approach different stimuli can be used, e.g. pH, temperature or ionic strength, all of them closely related with cell media and physiological environment. A combination of the top-down and bottom-up approaches has also been presented by Kneipp, by subjecting a patterned Au substrate obtained by electron beam lithography to Au evaporation and thermal annealing (Fig. 2C). Additional deposition and annealing cycles lead to growth of the Au nanoparticles, thereby decreasing the interparticle distance and eventually reaching a situation in which the Au nanoparticles merge into Au islands. In this case, the main idea was to combine both lithographic and deposition approaches to achieve a large spectroscopic enhancement through an intense plasmonic field, thereby demonstrating the feasibility of this mixed approach, which can also be considered promising for large-scale production of plasmonic substrates [33]. The bottom-up approach toward the controlled selfassembly of nanoparticles on a substrate originates from colloid and interface science. The number of available experimental procedures for fabricating plasmonic substrates is large, and still under constant increase. An example of a robust self-assembly method for the fabrication of plasmonic substrates is the simple drop casting procedure, for which a drop of the nanoparticle solution is placed on a substrate, followed by evaporation of the solvent under controlled conditions of humidity and temperature. The nanoparticles achieve a certain order during Please cite this article in press as: J.J. http://dx.doi.org/10.1016/j.nantod.2014.05.004

the drying process, eventually self-assembling into superlattices. Although these superlattices might have thicknesses in the order of microns, substrates covered with these plasmonic assemblies can be used as 2D systems from the point of view of biological applications, given the confinement of the plasmonic nanoparticles to a solid interface, as well as their well-defined arrangement. The assembly can be induced by appropriate capping agents, as has been demonstrated for aqueous solutions of gemini surfactant-coated Au nanorods (Fig. 2D). In this case, Au nanorod growth was assisted by the gemini surfactant, and subsequent assembly was based on the interaction between surfactant layers in neighboring nanorods. Given the perpendicular arrangement of the nanorods with respect to the substrate, preferential coupling of the transverse plasmon band was obtained [5]. The universality of a fabrication procedure is highly desirable when considering the biomedical application of the plasmonic substrates; one of the foremost advantages of the drop casting method is that it can be used for basically any type of nanoparticles. Additionally, when the drop casting method is applied under controlled conditions of temperature and humidity, it may be implemented for large-scale production. Probably the major drawback of this procedure is the comparatively small size of the obtained supercrystals, typically up to tens of micrometers. A reliable method for producing 2D plasmonic substrates over large areas is the block copolymer micelle nanolithography method (BCML), developed by Spatz and co-workers (Fig. 2D) [36]. BCML is actually a bottom-up approach, even though it includes the term ‘‘lithography’’. BCML is based on dip coating block-copolymer micelles containing Au3+ , which yields a hexagonal arrangement of the micelles onto the substrate. Subsequent application of an intense plasma treatment leads to removal of the micelles while the Au3+ ions get reduced into Au nanoparticles, which keep the hexagonal arrangement of the micelles and are partly embedded in the substrate [37]. Although it requires a post-deposition treatment, this BCML method successfully leads to a pattern of Au nanoparticles that can be subsequently modified, e.g. by including bioactive chemical groups [38], Ag coating [35], or Au deposition [39]. Note that biochemical functionalization as a post-deposition step widens the applicability of these plasmonic substrates to a great extent. This method has been extended to the formation of large homogeneous nanopatterned areas by spin-coating of polystyrene-coated nanoparticles [34] or by Langmuir—Blodgett deposition of microgel-coated spheres and rods [40]. The question of which of these deposition methods will be chosen for large-scale production remains open. These two bottom-up procedures are based on soft interactions, leading to the self-assembly of plasmonic nanoparticles into a structure with a certain order. An alternative concept is the functionalization of the substrate surface with chemical moieties that can bind the plasmonic nanoparticles. For example, glass surfaces modified with silanes such as (3-aminopropyl)trimethoxysilane (APTMS) readily adsorb Au nanoparticles, the coverage depending on the exposure time to the nanoparticle solution [41]. Significant plasmon coupling can be achieved with this procedure, but a full monolayer is still difficult to obtain. An alternative route to overcome this problem has been realized by

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chemically growing Au sharp tips from patterned Au nanoparticles [39]. Interparticle distance can be modulated on plasmonic substrates by a number of strategies, e.g. layer-by-layer polyelectrolyte assembly, as reported by Fery and Möhwald. In this case, Au NPs were functionalized by poly(acrylic acid) and deposition on the substrate was mediated by layers of poly(ethylene oxide). Biocompatible polymers can also be used, anyway keeping the electrostatic interactions responsible for the assembly. A red-shifted LSPR due to plasmon coupling was observed, whereas a thermal annealing step could reverse the plasmon band to that of isolated nanoparticles [42]. Interestingly, Au nanoparticles can be prepared at selected interparticle distances on plasmonic substrates, as reported by Mulvaney et al. by coating the Au nanoparticles with silica shells. This procedure is also interesting given the excellent biocompatibility of silica. The interparticle distance on the substrate then depends on silica shell thickness, which is pre-defined during the synthesis of the nanoparticles, therefore reducing the variability of the self-assembly process [43,44].

Assembly of plasmonic nanoparticles at air/liquid and liquid/liquid interfaces Probably the most relevant drawback of the ‘‘bottom-up’’ approach for the fabrication of plasmonic substrates is the difficulty to fabricate uniform self-assembled structures over large areas. The classical Langmuir trough [45,46] has inspired many modern routes for assembling inorganic nanoparticles at air/liquid interfaces [47], since the air/liquid interface is a most interesting platform for assembling nanoparticles over large areas with high periodicity. Murray has greatly contributed to this idea, showing the successful assembly of different inorganic nanoparticles into highly ordered supercrystals, thereby demonstrating the broad applicability of this method to nanoparticles with different compositions and shapes [4,48—52]. The binary self-assembly of two different nanoparticles at the air/liquid interface has also been demonstrated, not only using nanospheres of different sizes [4], but also combining different sizes and shapes [53]. This possibility is interesting for biological applications, because different nanoparticles can simultaneously fulfill different tasks, e.g. cell recognition and spectroscopic enhancement. Therefore, we consider the interfacial self-assembly of plasmonic nanoparticles as a classical approach that will be considered for the design of plasmonic substrates aimed at biological applications, mainly on the basis of the following features; (a) simplicity, (b) large areas, (c) precise arrangement of the nanoparticles, and (d) possibility of including different nanoparticles with different biofunctionalities. The liquid/liquid interface has also been exploited for self-assembly of plasmonic nanoparticles and subsequent transfer into solid substrates, with different purposes [54,55]. The use of the liquid/liquid interface requires an additional experimental parameter as compared to the air/liquid interface, namely the upper liquid phase. Given the successful self-assembly of nanoparticles at the air/liquid interface, this new liquid phase might appear unnecessary in the first instance. However, the liquid/liquid interface provides a unique environment for in situ studies Please cite this article in press as: J.J. http://dx.doi.org/10.1016/j.nantod.2014.05.004

[56,57], since it benefits from being a soft interface, in which reactants and analytes can be easily incorporated from any of the two liquid phases. Self-renovation of the interfacial structures with direct incorporation of active plasmonic material is also possible. Given the ubiquitous presence of aqueous phases in biological systems, 2D substrates at the liquid/liquid interface are highly interesting in terms of modeling interactions at the cell membrane, where both the inner and the outer media are liquid. Indeed, the construction of plasmonic substrates at the liquid/liquid interface has been realized for in situ measurements, either electrochemical [57] or spectroscopic [58—60]. Therefore, the exploitation of self-assembled structures based on plasmonic nanoparticles at liquid/liquid interfaces for biologically oriented studies is envisioned in the near future. Indeed, biological processes take place in aqueous solutions. These self-assembled structures will however display a different physicochemical behavior from those deposited on solid substrates. Note that plasmonic superstructures assembled at the liquid/liquid interface benefit from well-defined long-range order, while retaining some degree of translational freedom. Novel perspectives of plasmonic studies for biomedicine are expected due to such unique features: in our opinion, using 2D plasmonic substrates in a fluid phase will open new ways of studying biological objects, mainly living cells. In liquid phase closely mimicking their natural environment, living cells can freely move and interact. Therefore, liquid/liquid assembled plasmonic substrates can provide a more realistic and biologically relevant view of the actual state of living cells, compared to the insights obtained using solid substrates.

Sensing of biomolecules and living cells Surface enhanced Raman scattering Surface enhanced Raman scattering (SERS) mediated by plasmonic nanostructured substrates is rapidly being established as a reliable detection and analysis spectroscopic technique in the biological and medical fields [10]. Therefore, suitable substrates for SERS analysis of biological samples are most desirable. Although the ultrasensitive limits of detection provided by SERS might be achieved through other analytical techniques such as chromatography coupled with mass spectrometry, such techniques require expensive instrumentation and chemicals, relatively long analysis time, and highly trained personnel, whereas SERS requires much less time and more limited resources. SERS analysis of biomedical samples acquires maximum interest when the plasmonic substrates not only provide the enhancement of the Raman signal, but also additional functionalities, such as cell manipulation. Concerning detection of target biomolecules, probably the first and most relevant difficulty is the complexity of biological fluids, including extremely different chemical moieties, ranging from small inorganic ions to micrometer-size aggregates of proteins and genetic material. Therefore, separation of the target material to include the analyte into the hot spots for effective SERS detection is required. SERS analysis of complex biological samples has been reported using Au nanorod

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supercrystals as plasmonic nanostructures [61,62]. This substrate was applied to the detection of scrambled prions, i.e. harmful proteins, in complex biological media such as serum or blood. In this case, the key for successful prion detection was the chemical affinity of prions for the Au surfaces, together with the extremely efficient plasmonic enhancement of the supercrystals through antenna effects (Fig. 4). The separation of the prion from the chemically complex plasma was feasible due to the 2D confinement of the plasmonic nanoparticles on a planar substrate. In case of a bulk solution experiment, the characteristic Raman signals from the prion would be screened with the Raman signals coming from such a complex mixture as plasma. Short detection times were achieved, in the range of minutes, which are remarkably shorter than those obtained with standard clinical methods, usually requiring hundreds of hours. This study definitely constitutes a good example of the application of plasmonic substrates to a biomedical problem, of relevance in clinical practice. In a different approach, composite substrates have been realized, in which mesoporous silica and titania thin films were spin-coated on top of a layer of plasmonic nanoparticles [63]. The mesoporous layer excludes large molecules from reaching the plasmonic nanoparticles and thus from interfering in the SERS signal for the detection of small molecules in biological fluids. Using such a physical filter to separate the vast number of biomolecules present in biological fluids is most attractive for the practical application of these substrates in clinical practice, where different matrix compositions can be found. We therefore propose the use of such a physical filter separating the plasmonic surface from the studied biological mixture as a relevant option in studying biological complex fluids. In a further step toward the application of SERS to biological problems, the detection and classification of living cells has been attempted [64,65]. Plasmonic substrates can trap a certain type of cells by means of their surface antibodies [66]. Though interesting, this biochemical modification introduces an additional functionalization step involving antibodies, which usually requires expensive biochemical reagents. In an approach involving broad categories of cells, i.e. bacteria and eukaryotic cells, the plasmonic nanostructure can be covered with vancomycin, a glycopeptide that selectively binds to bacteria through interactions with the bacterial membrane. Therefore, the plasmonic substrates covered with vancomycin would preferentially capture bacteria over eukaryotic cells within a mixture [67]. Although the latter approach is promising for clinical applications in the detection of infections, a recognition unit is still required when targeting eukaryotic cells. Thus, the applicability of this method would probably be restricted to bacterial cells. Label-free SERS analysis of tumor cells has been reported by Reinhard, who showed that malignant cells can be distinguished from their non-malignant counterpart by comparing the SERS intensity ratio of characteristic Raman bands from the cell membrane, see Fig. 3 [68]. Such a relatively simple analysis from a complex sample is largely appealing. In this sense, chemometrics treatment of complex SERS signals from mixed cell colonies might allow the classification of the different types of cells present in the sample. Optimally, a certain degree of quantification would be highly desirable as Please cite this article in press as: J.J. http://dx.doi.org/10.1016/j.nantod.2014.05.004

well. We suggest that the latter approach, i.e. methods not requiring specific biorecognition groups and rather simple spectroscopic analysis, are key to successful implementation of plasmonic substrates and SERS analysis in standard clinical practice.

Plasmon enhanced fluorescence Plasmon enhanced fluorescence (PEF) has also attracted considerable attention in biological applications. On the contrary to SERS, where a minimum distance between the Raman active molecule and the plasmonic surface is pursued, in the case of PEF a short distance between the plasmonic nanostructure and the fluorophore results in quenching of the fluorescence. Thus, a certain distance is required, usually at least 5 nm, to avoid fluorescence quenching, whereas a longer distance between the fluorophore and the plasmonic nanostructure results in no interaction. Therefore, a delicate balance of the plasmonic field—fluorophore separation is required to achieve the maximum enhancement [69—71]. PEF has also been used for cell imaging, as discussed below. PEF and fluorescence quenching on 2D plasmonic substrates have been used for detection of DNA requiring no label by Miller. A fluorophore is linked to a planar plasmonic substrate via DNA hairpin probe. The hairpin structure retains the fluorophore in close distance to the planar plasmonic substrate, thus quenching the fluorescence. However, in the presence of the complementary DNA, the fluorophore is located a few nm away from the plasmonic surface, thereby leading to a significant increase in the fluorescence intensity [72]. This strategy is interesting, since the distance between the fluorophore and the plasmonic nanoparticle dynamically changes in the presence of the analyte. Note that this label-free approach can be readily incorporated in biosensors requiring the attachment of the plasmonic nanoparticles onto a planar substrate. An interesting concept based on distance-dependent quenching was developed by Salaita and co-workers [73]. Therein, Au NPs were aimed to induce the translation of chemical stimuli at the micrometer scale into a spectroscopic signal at the nanometer scale, i.e. pulling a protein from a living cell translates into a fluorescence signal. Interestingly, the ability of the Au NPs to quench the fluorescence was exploited to obtain turn-on detection with a certain Au NP—fluorophore distance. Through this concept, the researchers achieved actual imaging of the surface tension exerted by the living cell on the biofunctionalized substrate. This approach requires planar plasmonic substrates, since the mechanical information on the membrane of living cells is rather inaccessible by other experimental techniques. Although biosensing concerning living cells is usually focused on detecting certain types of cells or biomolecules, this information on the biomechanics of the cell membrane is expected to be of high interest in biophysics.

Electrochemical sensing Biosensing based on an electrochemical response benefits from the use of inexpensive, fairly simple, and fast response instrumentation. Plasmonic features are affected by

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Figure 3 Left: Dark field optical microscopy image of a prostate cancer cell on top of a Ag-based plasmonic substrate. SERS spectra of breast cells, healthy and cancerous are shown in blue and red, respectively. Right: Comparing the intensity ratio of the SERS peaks at 722 and 655 cm−1 allows the recognition of cancer cells on plasmonic substrates [68]. Copyright 2010, American Chemical Society.

surface charge density, this experimental parameter being at the core of the electrochemical response. An additional advantage of electrochemical modification is the specific functionalization of selected areas down to the nanometer scale. Spatz reported template-fabricated Au nanowires for plasmon sensing. The LSPR signal was monitored while applying a certain voltage, thus inducing selective protein adsorption (Fig. 4) [75]. Although the detection method is label-free and offers good results, a further step concerning the control of non-specific adsorption of biomolecules onto the electrodes needs to be taken for application in biological fluids. As commented above, the possibility of including a separating filter as part of the electrode might provide a solution to this issue. The combined use of electrochemistry and plasmonics is promising for biological applications and still almost unexplored; we therefore encourage studies in this direction.

coupling is prevented for better observation of the plasmon shift. Given the broad public acceptance of colorimetric sensing, a significant research effort is expected within this field when pursuing commercial applications of plasmonic biosensing for mass usage.

Cell manipulation: tissue engineering The first approach of nanotechnology toward biology was concerned with rather simple biomimetic systems and nanoparticles containing small biomolecules, such as lipid bilayers and small proteins. However, for the future use of nanotechnology in biology and medicine, a further step toward the basic unit of life, living cells, remains to be taken. We discuss in this section how 2D plasmonics can be effectively used to study and manipulate living cells.

Adhesion and viability of living cells on plasmonic substrates Colorimetric sensors Colorimetric sensors in which the presence of a certain analyte originates color changes that are readily observed with the naked eye, are undoubtedly of unmatched success in mass-scale usage. The enzyme-linked immunosorbent assay (ELISA) is probably the most extended colorimetric technique in biosciences. Käll et al. extended the ELISA concept to plasmonic substrates by showing the ultrasensitivity of an ELISA test based on LSPR shifts as the readout signal. Lithography fabricated Au nanorods on a large substrate were used for the ELISA test as read by dark-field images. The plasmonic nanoparticles were functionalized with the enzyme horse radish peroxidase (HRP) via biotin—streptavidin bonding. HRP catalyzes the precipitation of 3,30-diaminobenzidine (DAB). Thus, when DAB was added to the medium, the precipitate on top of the nanoparticles induced an LSPR shift, which was imaged by dark-field microscopy [76]. In this case, a large area of plasmonic structures is fabricated onto a planar substrate, but plasmon Please cite this article in press as: J.J. http://dx.doi.org/10.1016/j.nantod.2014.05.004

The foremost question when designing a plasmonic substrate for use with living cells is: how are the cells going to interact with the substrate? In a first approximation, the most desirable features of such substrates are a good adhesion and viability of the cells on the substrate. Indeed, the interactions of living cells with surfaces constitute a rich field of research [77]. We restrict the discussion to the characteristic features of plasmonic substrates when including living cells. In this sense, the group of Spatz has probably been the most active one [38]. Au NPs were produced in situ and attached on substrates as anchoring points for biomolecules and subsequently for cells. Moreover, the substrates could be functionalized with poly(ethylene glycol) (PEG) for focusing the attachment of the cells at the functional groups located at the Au NPs. The PEG-covered surface avoids cell adhesion. It should be noted that the interparticle spacing in these substrates is too large to obtain plasmon coupling, therefore hindering the efficient use of plasmonic fields to enhance the spectroscopic signal. In these studies, the Au NPs were

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Figure 4 Three approaches for plasmon enhancement of standard experimental techniques are highlighted concerning the combination with sensing techniques for applications in biology and medicine. SERS is used to detect harmful prions [61]. Copyright 2011 National Academy of Sciences of the United States of America. Plasmon enhanced fluorescence allows for the detection and imaging of oligonucleotides. From left to right, the concentration of oligonucleotides is increased, thus larger fluorescence intensity is obtained [74]. Copyright 2006 American Chemical Society. A Au-based nanowires planar substrate is used as electrode to monitor the adsorption of proteins [75]. Copyright 2013 Royal Society of Chemistry.

therefore mostly used as precisely located anchoring points rather than plasmonic units. In any case, the architecture of these substrates renders them appealing candidates for post-production chemical modifications, leading to modified nanoparticles with plasmon coupling and enhanced spectroscopic signal. In recent studies, the patterning of substrates with bioreceptors allowed the precise activation of attached T cells, which are highly relevant agents in the immune system. Indeed, T-cells are used in the so-called adoptive cell

therapy (ACT), in which ex vivo T-cells are grown and activated. The T-cells are used for improving the immune response in treatments of cancer and viral infections, by transfusion of these T-cells to the patients. The idea is then to build a platform for growing and manipulating T-cells. In this case, attachment and growth of T-cells were provided by the Au nanoparticle monolayer on the substrate, which allows a well-defined distribution of the specific antibody to CD3, a component of the T-cell membrane. Manipulation and tuning of the activation of the T-cells were performed

Figure 5 Plasmonic substrates based on Au nanoparticles at different interparticle distance allow placing specific proteins for activation of T cells. Left top: schematics of the T cells attached to the plasmonic substrate. Left bottom: T cells seeded on the 2D plasmonic substrate. Right: The Au nanoparticle density is finely controlled, and quantitatively related with the index of activation of the T cells [79]. Copyright 2013, American Chemical Society.

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Patterning of living cells at the micrometer scale

Figure 6 Manipulation of living cells on plasmonic substrates allows for different functionalities. Nanoparticle uptake from living cells moving on planar plasmonic substrates is most relevant for a successful application of nanoplasmonics with living cells [80]. Controlled detachment of living cells from the substrates by remote laser irradiation for cell collection [81]. Using purposefully placed plasmonic nanoparticles, delivery activated by a plasmon assisted reaction at selected spots is feasible [82]. Copyright 2013, 2012, and 2013, respectively, American Chemical Society.

by placing a given surface density of class II proteins on the plasmonic substrate, see Fig. 5 [78,79]. The use of T-cells demonstrated that plasmonic substrates can be used not only with model cells, but also with cells that are currently relevant in medicine. We note that a usually overlooked phenomenon when designing plasmonic substrates for cell adhesion is the uptake of plasmonic nanoparticles by living cells, which most probably will affect the performance of the substrate as well as the living cycle of the cell. Murphy qualitatively studied the dependence of nanoparticle uptake on cell type, as well as on the size and shape of the nanoparticles (Fig. 6). A strong dependence was found on the nanoparticle uptake from the planar substrate with the surface charge of the nanoparticles. This strong influence is in fact expected, given that the uptake proceeds through interaction between the nanoparticle and the cell membrane in a first step. This study compared the uptake of Au nanospheres and nanorods, with the result that shape was not the most relevant factor concerning nanoparticle uptake by the cells [80]. Insights into the uptake of nanoparticles by living cells undoubtedly provide valuable information on the detailed interactions between nanoparticles and cell surface. Whereas uptake studies from plasmonic nanoparticles in bulk solution suffer from the uncertainty derived from local effects of local aggregation and variation on the concentration of nanoparticles within the solution and at the cell surface, having the plasmonic nanoparticles on a 2D substrate allows a large degree of control on the surface concentration of nanoparticles. Therefore, the uptake of plasmonic nanoparticles by living cells can be quantitatively monitored while accurately knowing the number of plasmonic nanoparticles interacting with the cell membrane per surface area and time unit. If plasmonic substrates are to be used with living Please cite this article in press as: J.J. http://dx.doi.org/10.1016/j.nantod.2014.05.004

Patterned substrates containing plasmonic nanoparticles can lead to a suitable design toward sorting of the attached cells. Among the vast array of procedures to prepare patterned substrates for cell attachment, the achieved nanostructure should fulfill the following requirements: (a) reproducible and large area patterning on the micrometer scale, so cell ordering is feasible; (b) presence of plasmonic nanoparticles exclusively on a given region of the substrate, ideally with ordered nanoparticles and at short interparticle distances on the range of 2—10 nm, for most effective plasmon coupling; (c) stability of the nanostructure under different physicochemical conditions, as biological studies are usually performed including harsh and varying pH and temperature conditions, incoming radiation, and high ionic strength; (d) biocompatibility of the building blocks. A useful concept based on patterning Au NPs and PEG by a combination of microstamping and chemical modification has been developed by Lensen [83]. In a possible application of these patterns to cell culture, the PEG regions can be used as non-adhesive areas for cell substrates (as previously applied by Spatz group), while Au NPs regions might be used for attachment and plasmonic manipulation of living cells. On a top-down approach using electron beam lithography, Delcea was able to effectively pattern surfaces with microarrays containing quantum dots, while attaching and capturing living cells onto the different regions of the substrate [8]. Although this study did not include plasmonic nanoparticles, the same procedure can be readily extended to patterning of these particles. Moreover, a large number of experimental procedures are also available to produce plasmonic nanoparticles by electron beam lithography. The patterning of living cells onto 2D plasmonic substrates would enable the delivery of certain biomolecules into specific cells or even at selected regions of the cell membrane. Such targeted delivery requires a hot spot from a plasmonic nanostructure, thus well-defined plasmonic nanostructures over large substrates, as described in this review, are required. Nanoantennas have been used in this sense, where the hot spots were used to trigger a biochemical reaction between biotin—BSA and streptavidin, specifically performing the biochemical reaction at the hot spots (Fig. 6) [82]. This possibility is extremely interesting to perform biochemical reactions at living cells. Patterning of cells on plasmonic substrates opens the way to manipulate living cells in a directed way, leading to farming and displacement of designed cells for different purposes, such as tissue regeneration.

Directed detachment of living cells Once adequate interactions between living cells and plasmonic substrates are achieved and the desired cell manipulation is completed, detachment of the living cells for further use is typically desired. Ideally, the detachment should be: fast, spatially defined, reversible, and not harmful to the cells. Skirtach and Möhwald reported the design

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Figure 7 Using plasmon-enhanced fluorescence, the distance between fluorophore and 2D plasmonic substrate can be quantified, as the plasmonic substrate reduces the fluorescence lifetime. The fluorophore is a modified green fluorescent protein, eGFP, forming a complex with the receptor CXCR4. The effective interaction between the cell membrane and the substrate through the recognition between the CXCR4 unit and the ligand CXCL12 present on the surface of the plasmonic substrate reduces the cell—substrate distance. The fluorescence lifetime of the CXCR4-eGFP is then shortened. After endocytosis of the ligand CXCL12 by the living cell, the distance between CXCR4-eGFP and the plasmonic substrate is increased, the fluorescence lifetime is subsequently also increased. Therefore, by mapping fluorescence lifetime, the distance between fluorophore and plasmonic substrate can be mapped in vivo [84]. Copyright 2013 from American Chemical Society.

of a substrate covered with Au nanoparticles for remote detachment of living cells (Fig. 6) [81]. By using laser irradiation in the visible range, controlled temperature variations were induced with micrometer scale resolution. This local heating is based on the resonance of the laser radiation with the plasmon modes of Au NPs aggregates, leading to the formation of reactive oxygen species (ROS). Cell detachment actually involves the production of ROS followed by cell signaling, thereby leading to conformational changes in the cells and their subsequent detachment. The complete process of cell detachment spans up to 24 h. Remarkably, the cells could migrate toward the emptied regions, which is a relevant feature when considering the reusability of the plasmonic substrates. Note however, that complete recovery of an empty surface of hundreds of micrometers with living cells took ca. 6 days, which might hinder the practical applications of these substrates.

Cell imaging Cell imaging is required for studying the spatial distribution of biomolecules within living cells under different biological environments, e.g. in different organelles or at the cell membrane. We highlight that kinetics and structural transformations at the cell membrane are most relevant for understanding and hopefully controlling ongoing processes at living cells, which can be considered a short-term research challenge for plasmonic substrates. On the other hand, intracellular imaging aided by plasmonic nanoparticles is currently being exploited by using nanoparticles Please cite this article in press as: J.J. http://dx.doi.org/10.1016/j.nantod.2014.05.004

that have been uptaken by the cell. The concepts behind 3D nanoparticle-based cell imaging can be readily translated into 2D plasmonic substrates. The 2D approach might actually be advantageous when studying composition and processes at the cell membrane. Intracellular imaging might require the release of plasmonic nanoparticles from the substrate into the cell medium, therefore restricting the reusability of the substrates. In any case, cell imaging can become an additional feature within a multifunctional plasmonic substrate. On the other hand, fluorescence, which might be amplified by plasmonic fields as described above, has also been applied in intracellular imaging. However, fluorophores in the intracellular space suffer from photobleaching, biochemical degradation, and fast transport across the cell, leading to fast variations in local concentration. Using fluorescence lifetime as a reporting signal in 2D plasmonic substrates has been realized by Richards, using a nanostructured Ag substrate that amplified the fluorescence signal of a model enhanced green fluorescent protein, eGFP [84]. See Fig. 7. The protein was covalently linked to a receptor at the cell membrane, the chemokine receptor CXCR4. Such fluorescent biolabeling facilitated the detection and imaging of this receptor. A detailed theoretical and experimental assessment of the variation of the fluorophore lifetime with the distance to the 2D plasmonic structures was provided, thus highlighting the relevance of the physical insights into the actual phenomenon responsible for the imaging. The plasmonic field enhances the intensity of the fluorescence while decreasing the emission lifetime. The fluorophore eGFP was anchored to the membrane

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receptor CXCR4, forming the complex CXCR4-eGFP. The effective interaction between CXCR4 and the ligand CXCL12 present on the surface of the plasmonic substrate leads to a short distance between the cell membrane and the substrate, thereby decreasing the fluorescence lifetime of the CXCR4-eGFP. After endocytosis of the ligand by the living cell, the complex is displaced within the cell. The distance between the complex and the plasmonic substrate increases, and the fluorescence lifetime is then increased. This technique is highly promising in studying biological processes at the membrane of living cells using plasmonic substrates. A qualitative step toward the application of plasmon enhanced fluorescent nanoparticles in cell characterization was taken by Nienhaus group. They used the temperature dependence of the fluorescence to map the temperature within the cell. This approach can be easily implemented in 2D substrates to monitor in situ temperature variations at the cell membrane, as a response to different chemical or physical stimuli [85]. For example, the release of adenosine 5 -triphosphate (ATP) from red blood cells has been shown to strongly depend on temperature [86]. This and other effects of subtle temperature variations on the biological activity of living cells can be conveniently studied by 2D plasmonic substrates using fluorescence signals. Whether produced by the cell or attached to the probing nanoparticle, the presence of the fluorophore is required in the above methods. Therefore, the applicability of fluorescence based methods may present advantages but might also be hindered in some cases, such as biological systems that can be significantly perturbed by the presence of the fluorophore, or quench the fluorescence within the crowded intracellular medium.

Conclusions Two-dimensional plasmonic substrates have proven useful both toward biosensing and manipulation of living cells. Their rather simple architecture is advantageous to control and understand the interface between biological objects and the surface of the plasmonic substrate. Several fabrication methods are available in the literature, using either top-down, bottom-up, or a combination of these approaches. Combined approaches are foreseen as most promising in large scale applications. From the bottomup procedures, drop casting and assembly at air/liquid interfaces have been highlighted. Both approaches can be applied to a broad range of nanoparticles, but the latter method presents a number of additional advantages, such as low sample consumption and long-range order that can be scaled up to large substrates. Plasmonic structures assembled at liquid/liquid interfaces are promising, but still require much detailed study. The challenge related to the large complexity of biological samples when concerning their study at plasmonic substrates is the focus of much research effort; the novel concept of using a physical filter imposing a distance between the biological matrix and the plasmonic surface is most interesting in this respect. The analysis of biological samples aided by plasmonics can be based on different experimental techniques, from which we have discussed mainly spectroscopy methods such as SERS and PEF, while electrochemical and colorimetric Please cite this article in press as: J.J. http://dx.doi.org/10.1016/j.nantod.2014.05.004

methods have been proposed as rich fields of study. Living cells have been studied and manipulated to a certain extent using plasmonic substrates. The label-free analysis of tumor cells using characteristic SERS signals allows their detection, even in the presence of healthy cells. Cell imaging is also feasible using fluorescence lifetime rather than intensity as a measuring parameter. Additionally, living cells have been grown following certain patterns, and in some cases can be detached under controlled conditions. Although the research on living cells at plasmonic substrates can be considered at its early stages, T cells have already been used, therefore evidencing the possibility to incorporate other cells that are relevant in current biomedical problems. In summary, plasmonic substrates provide enhanced features for the study and manipulation of biological entities, paving the way for the future application of plasmonics in biomedicine.

Challenges and outlook To predict the evolution of how plasmonic substrates will be fabricated and used in biomedicine is risky. One of the main challenges is the design of a synthesis and deposition method that can be implemented for large-scale production. As discussed above, a large number of experimental methods have been described, and it remains to be seen which one will win the competition for industrial use. In this regard, daily in-house usage will probably be dominated by colorimetric methods. More specialized studies will greatly benefit from methods that do not require specific biorecognition groups, most likely based on chemometric data analysis. Concerning the plasmonic substrates themselves, multifunctionality is expected to be pursued in greater effort. Therefore, different tasks might be accomplished in parallel, e.g. cell recognition, enhancement of spectroscopic signals, cell patterning, and so on. An additional and relevant but often overlooked factor is the reusability of the substrate, especially when considering the commercialization of the substrates. A more extended and fruitful combination of living cells with plasmonic substrates is expected. Cell imaging is one of the first targets in sight. The farming and manipulation of living cells, probably including cell recovery is still at an early stage, and development in this direction is to be expected. Ideally, the plasmonic field could be used to influence cell growth. Detachment and recovery of cells in a short time is also challenging, but if achieved, it could be used as an advanced platform for the production of cells for tissue regeneration. The manipulation and study of cells with prominent relevance in biomedicine has been achieved within a few examples in the literature, but it is still to be fully developed. In spite of the uncertainty and difficulties inherent to this field of study, we undoubtedly consider that plasmonic substrates will enrich the knowledge and technical possibilities of plasmonics in relation to biomedicine.

Acknowledgements The authors acknowledge financial support from the European Research Council (ERC Advanced Grant #267867 Plasmaquo). J.J. G.-C. acknowledges the Ministry of Economy

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and Competitiveness for a Juan de la Cierva fellowship (#JCI-2012-12517).

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[83] J. Chen, M. Arafeh, A. Guiet, D. Felkel, A. Loebus, S.M. Kelleher, A. Fischer, M.C. Lensen, J. Mater. Chem. C 1 (2013) 7709—7715. [84] N.I. Cade, G.O. Fruhwirth, T. Ng, D. Richards, J. Phys. Chem. Lett. 4 (2013) 3402—3406. [85] L. Shang, F. Stockmar, N. Azadfar, G.U. Nienhaus, Angew. Chem. Int. Ed. 52 (2013) 11154—11157. [86] K.K. Kalsi, J. González-Alonso, Exp. Physiol. 97 (2012) 419—432. Juan J. Giner-Casares obtained a Ph.D. in Chemistry in the group of Prof. Luis Camacho at the University of Córdoba (Spain) in 2009, where he studied Langmuir monolayers using experimental and computational methods. Then he joined the Max Planck Institute for Colloids and Interfaces as an Alexander von Humboldt fellow, under the supervision of Prof. Helmuth Möhwald, from 2009 to 2012, extending the Langmuir technique to new inorganic/organic polyoxometalate surfactants. Currently, he works as a ‘‘Juan de la Cierva’’ Postdoctoral Fellow at the BioNanoPlasmonics Lab of Prof. Luis Liz-Marzán at CIC biomaGUNE, where he is investigating the interfacial self-assembly of plasmonic nanoparticles with a biological focus. Luis M. Liz-Marzán has a Ph.D. from the University of Santiago de Compostela (1992) and was a postdoc at Utrecht University and visiting professor at various universities and research centers. After holding a chair in Physical Chemistry at the University of Vigo (1995—2012), he is currently an Ikerbasque Research Professor and Scientific Director of CIC biomaGUNE in San Sebastian. His current interests include nanoparticle synthesis and assembly, nanoplasmonics, and nanoparticlebased sensing and diagnostic tools.

Giner-Casares,

L.M.

Liz-Marzán,

Nano

Today

(2014),