Surface Plasmon Resonance Imaging: What Next? - The Journal of

Perspective pubs.acs.org/JPCL

Surface Plasmon Resonance Imaging: What Next? Giuseppe Spoto†,‡ and Maria Minunni*,§ †

Dipartimento di Scienze Chimiche, Università di Catania, Viale Andrea Doria 6, 95125 Catania, Italy Istituto Nazionale di Biostrutture e Biosistemi, Catania, Italy § Dipartimento di Chimica e CSGI, Università di Firenze, Via della Lastruccia, 3 50019 Sesto F.no (FI), Italy ‡

ABSTRACT: This Perspective discusses recent advances in the field of surface plasmon resonance imaging (SPRi) for the label-free, multiplex, and sensitive study of biomolecular systems. Large efforts have been made during the past decade with the aim of developing even more sensitive and specific SPRi-based platforms. Metal nanostructures have been used to enhance SPRi sensitivity and to build a specific SPR-active surface, while special effects such as long-range SPR have been investigated to develop more effective SPRi platforms. Here, we review some of the significant work performed with SPRi for the ultrasensitive detection of biomolecular systems and provide a perspective on the challenges that need to be overcome to enable the wide use of SPRi in emerging key areas such as health diagnostics and antidoping controls.

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which the real part of the two dielectric constants must be of opposite signs (e.g., metal/liquid or metal/gas), are confined at the metal/dielectric boundary and vanish at both sides of the metal surface. Plasmon excitation by a photon requires the conservation of both the energy and the momentum. A similar condition is obtained when wavevectors for photons (klight) and plasmons (kx) are equal in magnitude and direction. The dielectric constant of the medium at the metal interface directly influences the magnitude of the wavevector. This specific property has important consequences for the application of the SPR phenomenon in biosensing. SPs are nonradiative and cannot be excited by directly irradiating the metallic surface with light traveling through the medium at the interface because under similar conditions, wavevectors (kx and klight, respectively) are different. SP−photon coupling at a given energy requires a Δkx increase of the SP's wavevector, which can be obtained when light travels through a medium with a refractive index higher than that of the medium at the interface with the metal surface. SP−photon coupling can be also obtained by exploiting diffraction effects. It is for the abovediscussed reasons that couplers like prisms, gratings, fiber optics, or waveguides are needed for SPR-based sensors. Prism couplers are widely adopted for SPR biosensing by using a variety of different detection approaches, which include angle modulation,4 wavelength modulation,5 and intensity modulation. The latter, also known as SPRi, uses 2D detectors to measure differences in the intensity of the reflected light (expressed as the percent reflectivity % R) at a fixed angle and wavelength. The SPRi measurement approach differs from the spectral SPR approach because parallel interaction events are detected in real time from the spatially resolved functionalized

ver the last 2 decades, sensors based on optical excitation of surface plasmons proved to hold vast potential for the study of biomolecular interactions and became central tools in life science and pharmaceutical research.1 Surface plasmon resonance (SPR) biosensors take advantage of label-free and real time detection capabilities and operate with multiplexed formats when used in combination with the imaging capability offered by SPRi.

An overview on ultrasensitive and challenging SPRi uses, a perspective on emerging key areas, including health diagnostics, theranostics, membrane studies, and antidoping, as well as an update on SPRi coupled to nanotechnology. SPRi, also known as SPR microscopy,2 was first introduced by Rothenhäusler and Knoll in the 1988.3 Since then, SPRi has been proposed to investigate interactions with biomolecules arrayed onto chemically modified metal surfaces. SPRi enables biomolecular interactions to be monitored in real time and without the use of any label, as in classical SPR sensing. However, by using a CCD camera for signal detection, both sensorgrams (i.e., resonance signal vs time) and SPR images of the arrayed surface can be recorded, allowing simultaneous analysis of many interactions (up to hundreds). These facilities have important impact in high-throughput analysis. SPR biosensors detect optical signals generated as a consequence of the excitation of surface plasmons (SPs) at the interface of a metal and a dielectric substance, typically either a liquid or air. SPs, which are associated with coherent oscillation of electrons at the boundary between two media for © 2012 American Chemical Society

Received: July 28, 2012 Accepted: September 7, 2012 Published: September 7, 2012 2682

dx.doi.org/10.1021/jz301053n | J. Phys. Chem. Lett. 2012, 3, 2682−2691

The Journal of Physical Chemistry Letters

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SPRi metallic surface.6 Differences in the chemical composition or the thickness of the layer close to the metallic surface introduce changes in the local dielectric constant values, which generate the image contrast. Binding events are also detected by collecting difference images, obtained by the subtraction of a reference image from a postbinding image. A similar approach allows simultaneous and independent measurements to be made on different locations of the same sensor surface.

SPR-based sensing presents decisive advantages over other popular biosensing technologies, including the possibility to study kinetics of biomolecular interactions and to characterize weakly bonded molecular complexes at the surface. Figure 1. SPR image of immobilized BSA. Bright spots correspond to areas with the immobilized BSA. Vertical bands correspond to referencing areas. A BSA surface coverage sensitivity of 0.2 pg mm−2 was estimated. Reprinted with permission from Elsevier, ref 10, Copyright (2008).

SPR-based sensing presents decisive advantages over other popular biosensing technologies, including the possibility to study kinetics of biomolecular interactions and to characterize weakly bonded molecular complexes at the surface. SPRi adds further possibilities because parallel analysis of many interactions (up to hundreds) can be performed at the same time. This characteristic allows one to perform and compare different surface modifications at the same time, under the same experimental conditions, that is, in terms of chemistry applied on the same receptor or response of different receptors to the same ligand added in solution, thus providing homogeneous data comparison and speeding up the analysis process. As paradigmatic example, we can mention the drug screening from a library of compounds, both natural7 or of synthetic origin,8 with potential therapeutic interest. In this Perspective, we highlight a number of recent developments in SPRi technology, with particular attention to assay design strategies aimed at improving SPRi detection capabilities in terms of sensitivity, specificity, and application in real matrixes. The role played by metal nanoparticles (NPs) and their interactions in achieving enhanced sensitivity will also be discussed. Efforts paid in the development of computational supports and in the technology required for the fabrication of advanced SPRi platforms will also be discussed. Advances in SPRi Technology. Enhancing the sensitivity of optical detection platforms represents a challenge in SPRi. It is important when the development of advanced detection protocols useful for complex bioanalytical applications, such as the direct parallel detection of molecular biomarkers from human samples, is going to be investigated. The most widely available optical platforms for SPRi biosensing are based on prism couplers. Despite the technological simplicity of these platforms, excellent refractive index resolution (2 × 10−7 refractive index unit, RIU)9 has been obtained by combining the prism-based SPRi configuration with suitable polarization contrast and signal referencing.10 The latter, in particular, has been performed by detecting optical signals from two regions of the base of the prism used to block and to reflect portions of the incident light. The enhanced SPRi sensitivity achieved has been demonstrated by imaging a bovine serum albumin (BSA) 100 spot array (Figure 1).

An alternative approach for SPRi has been developed by detecting the 2π phase shift of p-polarized light waves interacting with a SP in the region of the SPR angle.11 An interference fringe image on the interface with a polarizer− depolarizer system was created, and changes in the fringe pattern caused by the adsorption of biomolecular system on the metal surface were detected. This SPRi approach has been proved to be able to detect ssDNAs with an increased sensitivity with respect to conventional prism-based SPRi. Further opportunities for enhanced SPRi detection are offered by optical multilayer structures supporting long-range surface plasmons (LRSPs).12 LRSPs are generated when a specific layered structure, in which a metal film is sandwiched between two dielectrics having similar dielectric constants, is fabricated. A metal film thickness comparable to the distance where the SP evanescent field intensity falls to 1/e (the latter is called penetration depth, Lp) is required to obtain the coupling of SPs at each interfaces. Two SP modes, referred to as LRSPs and short-range surface plasmons (SRSPs), are thus generated. The dispersion relation of LRSPs and SRSPs is the following: SRSPs:

⎛k d ⎞ εdkz m + εmkzd coth⎜ z m m ⎟ = 0 ⎝ 2i ⎠

LRSPs:

⎛k d ⎞ εdkz m + εmkzd tanh⎜ z m m ⎟ = 0 ⎝ 2i ⎠

where kzm and kzd denote the z-components of wavevectors of SPs in metals or dielectrics, respectively, εd and εm denote the dielectric constants for the dielectric or metal, and dm denotes the thickness of the metal layer. LRSP attenuation is lower than that of conventional SPs, thus resulting in longer propagation lengths.13 Moreover, LRSPs produce higher surface electric field strengths and narrower angular resonance curves. The specific properties of LRSPs have stimulated a large number of studies aimed at investigating possibilities in 2683



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different fields including biosensing, surface-enhanced Raman spectroscopy, fluorescence, nonlinear interactions, and molecular scattering.13 Special attention has been given to the development of photovoltaic devices, amplifiers, and lasers based on SPs.14,15 In particular, LRSPs have attracted special attention for the development of innovative amplifiers and lasers as a consequence of the reduced material gain needed.16 Multilayered structures supporting LRSPRs in combination with a conventional SPR apparatus have been used to demonstrate an ultrahigh refractive index resolution (2.5 × 10−8 RIU),17 while SPRi based on LRSPs has been obtained by using an optically transparent fluoropolymer with a refractive index very close to that of water.18 In the latter case, the optical apparatus has been shown to be slightly more sensitive than conventional SPRi for DNA hybridization detection. Grating couplers offer a number of attractive features in comparison to prism-based couplers, including the possibility to fabricate low-cost SPR sensors. A gold grating coupler combined with gold nanoparticles (AuNPs) has produced a 10 fM sensitive SPRi detection of ssDNA.19 The sensitivity, in this case, was comparable to that obtained with standard NPenhanced SPRi. SPRi Coupled to Nanotechnology. Open challenges in SPRi technology are improvement of detection limits and application to direct analysis (i.e., in real matrices) on the one hand and system miniaturization and low-cost production on the other hand. Both aspects benefit of possibilities offered by nanotechnology.20 SPRi detects changes within the metal boundary in the submicrometer range possessing the maximum sensitivity on the metal surface and being characterized by an exponential decay of sensitivity with increasing distance from the surface. The decay of sensitivity is a consequence of the SP penetration depth (Ld) that is expressed as Ld =

solving Maxwell’s equations using a quasi-static approximation and is given by ⎡ ε − εout ⎤ 3 Eout(x , y , z) = E0 z ̂ − ⎢ in ⎥a E 0 ⎣ (εin + 2εout) ⎦ ⎤ ⎡ ẑ 3z ⎢⎣ 3 − 5 (x x̂ + y ŷ + z z)̂ ⎥⎦ r r

where εin is the dielectric constant of the NP, εout is the dielectric constant of the medium where the NP is embedded, and E0 is the applied field magnitude. εin, which is dependent on λ, plays an important role in determining the resonant condition for the NP, but also the size of the NP (a) and the NP-surrounding medium (εout) influence the magnitude of the field outside of the particle.22 The extinction E(λ) spectrum (the sum of absorption and scattering cross sections) of spheroid NPs also depends on the NP (εr and εi are real and imaginary components, respectively) and surrounding medium (εout) dielectric constants E (λ ) =

3/2 ⎡ ⎤ 24π 2Na3εout εi(λ) ⎢ ⎥ 2 2 λ ln(10) ⎣ (εr(λ) + χεout) + εi(λ) ⎦

The form factor χ is 2 for spherical NPs and takes higher values for particle forms with high aspect ratios.22 The above-reported equations show that light-LSP resonant conditions and electromagnetic field enhancement are strongly dependent not only on the shape, size, and composition of metallic NPs but also on the dielectric constant of the proximate medium. The most widely used SPRi experimental setups are based on prism couplers. In this case, the combination of extended and localized SPs phenomenon obtained by employing metallic NPs or nanorods in the design of sandwich-like assays has been shown to effectively enhance the SPRi sensitivity.23,24 The signal enhancement is caused by both the effective mass of metallic NPs in the evanescent sensing layer as well as the interaction between extended and localized SPs established when NPs are close to the flat SPRi metallic surface. Metallic NP clustering on the SPRi metallic surface is expected to further contribute to signal enhancement. In fact, clusters of spherical metal−dielectric colloids have been shown to exhibit strong electric, magnetic, and Fano-like resonances25 that arise from the electromagnetic coupling between closely spaced particles.26 The extended and localized SP resonance is dependent on the distance between the metal film and the NP. AuNPs in close proximity to a gold film produce a vertically polarized light scattering and a red-shifted plasmonic resonance. These effects are responsive to nanometer-scale changes in the distance between the film and the NPs and are observed under total internal reflectance configurations.27 NP dielectric constants and extinction coefficients are also influenced by the distance between the NP and the film. When AuNPs are deposited almost directly onto gold films, the NP dielectric function shows anomalous dispersion that disappears at longer distances. NP dielectric constants and extinction coefficients, which are a function of excitation wavelength, are important for quantitative applications but not easily determined.28 The first investigated approaches using metal nanostructures were applied to the SPRi transduction for DNA-based sensing and involved the use of metallic nanostructures properly modified for molecular probe attachment.29 For example, for

⎛ c ⎞ |ε ′ | + ε 1 =⎜ ⎟ m 2 d ⎝ω⎠ |k z i | εi

where i stands for metal (m) or dielectric (d), respectively. Sensing performances can thus be improved by introducing signal enhancers at the proximity of the metal surface. Metallic nanostructures have been widely used to enhance SPRi sensitivity by exploiting specific effects generated when metallic nanostructures are close to the SPRi metallic surface. These structures allow strong optical coupling of electromagnetic radiations to localized SPs (LSPs). The latter differs from the extended (or propagating) SPs that are described as a longitudinal electromagnetic wave that exists on the interface between metals and dielectrics because they are excited in metallic structures with dimensions less than half of the wavelength of the exciting electromagnetic wave.21 When a NP has dimensions much smaller than the wavelength of excitation, a resonance condition (localized surface plasmon resonance, LSPRs) can occur at which the elastic scattering cross section of the particle increases dramatically and the local electromagnetic fields are strongly enhanced. Under similar conditions, the incident radiation frequency is resonant with the collective oscillation of the conduction electrons confined in the volume of the NP. The magnitude of the electric field outside (Eout) of a spherical NP of radius a that is irradiated by z-polarized light of wavelength λ (with a much smaller than λ) can be obtained by 2684



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Figure 2. Time-dependent SPRI curves obtained after the adsorption of conjugated AuNPs on normal (a), heterozygous (b), and homozygous (c) DNAs previously adsorbed to the surface-immobilized PNA-N (red curve) and PNA-M (blue curve) probes. Solutions of 5 pg μL−1 genomic DNAs were used for the experiments. A representative SPR difference image (d) demonstrating the DNA parallel detection is also shown. Reprinted with permission from the American Chemical Society, ref 35, Copyright (2011).

AuNPs clustering on the SPRi surface driven by the change of surface charge conditions caused by the surface adsorption of large genomic DNA fragments (Figure 2). The combined use of NP-enhanced SPRi and surface enzymatic amplification procedures has been shown to offer further possibilities for nucleic acid detection.3637 A variety of different SPR-active metal nanostructures have also been used to enhance SPRi-based immunosensing or nucleic acid detection.38−40 LSPR excited by metallic surface nanogratings have been investigated with the aim to enhance sensitivity in DNA detection.41 Similar approaches involve complex nanofabrication procedures that significantly limit the current widespread applicability of similar SPR-based devices.42 Semiconductor nanocrystals (quantum dots, QDs) have also been used to enhance SPRi sensitivity in biomolecular detection.43 The enhancement phenomenon in this case involves not only the mass-loading effect but also the coupling between SPs and excited states of nanocrystals.44 Computational Approaches for SPRi. Progress in NP-enhanced SPRi detection require adequate modeling approaches, which can describe the electromagnetic field distribution over the space and expected SPRi responses. Several methods are available today in plasmonic simulation, which are applicable to

DNA target sequence detection, nanorods modified with poly T tails, 25 bases in length, were used in sandwich-like assays to produce large increases in SPRi signals. In this specific case, the probe immobilization chemistry was based on amineterminated ssDNA to bind silica-coated gold nanorods.30 Similarly, the reflectivity change observed for the adsorption of a monolayer of gold nanorods was comparable to the value previously reported for the adsorption of a monolayer of AuNPs, which was approximately 15 times larger than the response observed for the hybridization adsorption of a monolayer of DNA.31 The attomolar DNA detection achieved by using NPenhanced SPRi has been proposed as an effective way to perform DNA sequence detection directly in genomic samples, bypassing the polymerase chain reaction (PCR)-based amplification procedure.32 The detection of target sequences in real samples of genomic DNA from plants and human blood, in sandwiched AuNPbased assays, has been demonstrated. In this case, peptide nucleic acid (PNA) probes in combination with functionalized AuNPs33 and continuous flow control were used for ultrasensitive SPRi DNA detection.34,35 Here, a further contribution to the enhanced sensitivity is proposed to be provided by 2685



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Figure 3. (a) ROI selection: (A) Image of the array of receptors on a biochip surface by the CCD camera. At this stage, no ROIs are defined, and thus, no signal can be recorded. (B) Green circles define the initial grid of ROIs. Each ROI is coincident with one single spot. (C) The grid of the second sampling method applied, “C”, consists of three ROIs for each spot, arbitrarily selected by the operator. (D) Grid obtained using the developed method “D”. (b) SPRi results obtained for each spot of the array by comparing the three signal sampling methods (B−D) displayed in Figure 4a. SDs are the average calculated on the total number of the selected ROIs for each considered method and for 3 ppm hIgG injections in replicates (n = 3). Reprinted with permission from Elsevier, ref 55, Copyright (2010).

31.52°. DSM does not require any integration procedures because the scattered field, everywhere outside of the NP, is constructed as a finite linear combination of fields resulting from multipoles distributed inside of the scatterer. The Green’s tensor of a layered interface was used to account for the EM interaction of the NP with the multilayered interface. The computational simulation described a sharp increase of the scattered intensity for P-polarized light excitation, which occurs in the evanescent wave region behind the critical angle. This effect was named as the extreme scattering effect (ESE).50 The enhancement effect involves both the transmitted and backscattered directions and is strongly affected by the film material. Even more advanced computational capabilities are highly desirable in this area. The above-discussed NP/layered simulated systems are still too simple in comparison to the NP-enhanced SPRi experimental systems adopted to achieve ultrasensitive DNA detections because they lack in implementing the surface-immobilized probe−target interacting system. Computational methods for probe selection in DNA-based sensing experiments are of great importance in improving detection performances.51,52 In this direction, an SPRi experimentally validated computational-assisted approach for probe design, that is, in silico selection, to be used in DNA sensing has been also described. It is also expected that more sophisticated in silico models, designed on purpose, may better mimic the biochip environment, thus providing a useful tool for the design of even more

particles of arbitrary shapes. These include the boundary element method (BEM), the discrete dipole approximation (DDA), the finite difference time domain method (FDTD), and the finite element method (FEM).45,46 Most of these methods are straightforward to implement but are burdened by the additional computational load caused by the need to model the NP surrounding space.47 Nevertheless, they have been implemented in order to simulate 1D or 2D periodic repetition of NPs or a NP on or near a semi-infinite substrate. In the latter case, the semi-infinite substrate role has been accounted for by incorporating the Green’s tensor of the substrate structure.48 In particular, BEM and DDA have been shown to be able to solve the problem by discretizing only the NP, once the Green’s tensor is adjusted to account for the substrate. BEM expresses the electromagnetic field scattered by a NP in terms of boundary charges and currents, which leads to a system of surface-integral equations that is discretized using a set of N representative points distributed at the boundaries. DDA, instead, describes the NP as an array of polarizable dipolar elements organized on a lattice. Only recently, the interaction of a P- or S-polarized plane wave (λ = 670 nm) with a AuNP (52 nm in diameter) deposited above a multilayered structure, composed of a Au film (thicknesses of 53 and 46 nm) deposited on a LASF46A prism, has been simulated by using the discrete source method (DSM).49 The simulated system considered the upper half-space filled with air. The critical angle at which evanescent waves were generated was calculated to be 2686



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efficient SPRi assays. Molecular dynamics simulations of surface-immobilized simple DNA complexes provide similar possibilities even though the time scale of simulated events and the length of the DNA complexes are still far from those involved in standard SPRi assays.53 A further interesting aspect is SPRi signal sampling and management. In SPRi, bioreceptors are tethered on a chip gold surface in array format, as circular or square spots or even as microchannels,54 and any portion of the biochip could be suitable for SPR signal sampling because the only limit is represented by the SPR image lateral resolution. As a consequence, any area having a diameter equal to or greater than the image resolution may be associated with a SPRi signal, leaving the operator completely free to select number, location, and type of sensing areas undergoing signal sampling. Although this selection is a key step for the subsequent analysis, there is a lack of common criteria for doing it, despite the high number of papers that have recently appeared in the literature on SPRi. Valuable information provided by imaging data analysis would be more efficiently exploited if coupled with well-defined criteria for signal sampling and affinity binding data management. Moreover, standardization of both procedures would eventually build up a common platform for homogeneous comparison of SPRi results obtained in different contexts. A method for a rational and reproducible strategy in selecting sensing areas on the biochip (regions of interest, ROIs) for SPRi signal sampling based on a two-step approach, first performing a fine mapping of the array followed by identification and selection of the best ROIs to be used for measurements, was reported, referred to as a direct data acquisition method.55 Improved analytical performance is obtained by using this rational ROI fine mapping approach. According to the postprocessing method, SPR images are instead acquired and computer-stored for subsequent ROI selection, while relevant interaction processes are established on the SPRi sensor surface. The direct data acquisition method is a less computationally demanding approach that avoids SPR image storage, thus requiring an optimized ROI selection to be performed before the interaction process is established. In Figure 3, as proof of principle for ROI selection in direct data acquisition, a paradigmatic immunosensing example is reported. The peculiarity of this approach was that final ROI selection was made after a real and fast preanalysis with the specific target analyte (human IgG). The comparison with conventional ROI selections, in terms of improvement of analytical parameters, demonstrates the validity of this approach. Finally, in the postprocessing method, automated analysis of binding data, associated to each spot, can be applied to differential images with further signal analysis. In this case, different statistical approaches for data analysis have been validated, including the principal component analysis, the multidimensional scaling, and the hierarchical clustering.56 Interestingly, the mentioned approaches have been shown to give rise to the similar spot classification with reliable identification of replicates independently on their position, shape, or size. In addition to spot selection, a global model taking into account transport and diffusion mechanisms into the aqueous phase and accessibility and hybridization mechanisms into the chemically modified surface has been developed.56 The model has been used to define critical assembly parameters and to derive relations between microscopic and observable events in

Figure 4. Schematic representation of the on-chip synthesis of protein from a DNA microarray. mRNA was transcribed with an RNA polymerase by encoding the dsDNA sequence that was covalently attached to the gold surface of the generator element. Translated Histagged protein diffused to the adjacent detector elements and was captured by the Cu(II)-NTA surface. The whole process was imaged with SPRi. Reprinted with permission from the American Chemical Society, ref 61, Copyright (2012).

experimental SPRi conditions. Fluidic and matrix heterogeneities within cell can affect kinetic behaviors and interfere with automated identification of molecular interactions, thus limiting the multiplexing power of imaging. Particularly, transport and diffusion mechanisms within the aqueous or polymers’ layer, eventually coating the chip gold surface for receptor attachment, might significantly differ between the microchannel and large fluidic cell setups. In the affinity binding reaction, it is of paramount importance, in fact, to evaluate the presence of mass transport limitations events for correct calculation of kinetic parameters of the interaction (association rate constant ka and dissociation rate constant kd). Apparent constants can be obtained with SPRi measurement, using different suitable fittings, and one has to be confident that the interaction is not affected by analyte diffusion limitation from the solution to the interface where binding occurs. For this reason, modeling is also welcome. The global model (GM) takes into account cell properties as well as the diffusion mechanisms, density of grafting, and the hybridization kinetics. The model was validated using experimental parameters and solved using finite element simulation approaches.56

Possibilities offered by carbonbased surfaces, onto which biomolecules carrying alkene moieties can be covalently immobilized, have been investigated with the principal aim to overcome the above-mentioned limitations. SPRi Chip Surfaces and in Situ Receptor Production. Conditions for SP resonance are obtained in the infrared and visible wavelength regions for air/metal and water/metal interfaces. Different metals including aluminum, silver, and gold satisfy required conditions for SPR. However, differences in relevant parameters for such metals such as the SP propagation length are related to the imaginary part value of the metal dielectric constant. The relatively low imaginary part of gold dielectric constant is one of the reason why gold is the 2687



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mentary sequence. The final RNA aptamers were subsequently used in SPRi measurements for the bioaffinity detection of protein biomarkers, thus creating a multiplexed aptamer microarray. The interest in all of these approaches is represented by the use “molecular laboratories” on chips, with the advantage of overcoming problems related to sample manipulation and addition of reagents, often leading to contamination and loss of sample material. Furthermore, DNA microarrays are invaluable tools for the detection and identification of nucleic acids in biosensing applications.

metal of choice for SPRi. Gold also offers other advantages, including surface chemical purity and its compatibility with simple, effective, and reliable surface chemistry. However, gold surfaces introduce important limitations in array fabrication caused by the easy photodecomposition and oxidation of the gold−sulfur bond. Improvements in array fabrication for SPRi detection have been obtained with the use of gold SPRi substrates coated with a few nanometer thick silicate layer.57 However, the silane chemistry, involved when immobilization procedures on glassified surfaces are required, introduces other limitations. In fact, harsh environments and increased temperatures promote siloxane bond hydrolysis. Possibilities offered by carbon-based surfaces, onto which biomolecules carrying alkene moieties can be covalently immobilized, have been investigated with the principal aim to overcome the above-mentioned limitations. Thin layers of amorphous carbon have been deposited at room temperature on SPR metallic layers, and in situ fabrication of oligonucleotide arrays utilizing photochemically protected oligonucleotide building blocks has been performed.58,59 The method has also been used to synthesize 500−700 oligonucleotide arrays on carbon-on-gold substrates in situ in order to identify secondary structures of RNA molecules with SPRi.60 Interestingly, a similar in situ fabrication method is not compatible with bare gold substrates due to the extended exposure to UV radiation and oxidizing chemical conditions. The in situ probe array fabrication for SPRi detection idea has recently evolved into the combined receptor in situ synthesis and its capture on side surfaces. This approach represents a way to use SPRi technology for parallel detection, eventually overcoming limitations related to the storage of surfaces modified with biomolecules. The approach has been demonstrated for protein microarrays enzymatic fabrication from double-stranded DNA (dsDNA) microarrays.61 The protein fabrication step was combined with a parallel SPRi antibody detection in an on-chip microfluidic format. More specifically, a dsDNA microarray that encodes either a His-tagged green fluorescent protein (GFP) or a His-tagged luciferase protein was utilized to create multiple copies of mRNA in a surface RNA polymerase reaction. The mRNA transcripts were then translated into proteins by cell-free protein synthesis. The His-tagged proteins diffuse to adjacent Cu(II)-NTA microarray elements where they are specifically adsorbed and from which SPRi detection was performed. Similarly, in situ RNA surface transcription was achieved by RNA polymerase. The RNA created on this “generator” element was then detected by specific adsorption onto an adjacent “detector” element of ssDNA complementary to one end of the ssRNA transcript. SPRi was then used to detect the subsequent hybridization of cDNA-coated AuNPs with the surface-bound RNA. This RNA transcription-based method is useful to detect ssDNA down to a concentration of 1 fM in a volume of 25 μL.62 Finally another example is given by microarrays of RNA aptamers fabricated in one step by multiplexed enzymatic synthesis on gold thin films then employed in the detection of protein biomarker measurement by SPRi.63 In this case, ssRNA oligonucleotides were transcribed on-chip from dsDNA templates attached to microarray elements by the surface transcription reaction of T7 RNA polymerase. As they were synthesized, the ssRNA oligonucleotides were hybridized onto adjacent ssDNA microarray elements carrying the comple-

This convenient on-chip protein microarray fabrication method can be implemented for multiplexed SPRi biosensing by coupling different reactions on the same chip, of interest for measurements in both clinical and research applications. This convenient on-chip protein microarray fabrication method can be implemented for multiplexed SPRi biosensing by coupling different reactions on the same chip, of interest for measurements in both clinical and research applications. Emerging Application Fields. Recent work has exploited the possibility of achieving sensitive nucleic acid detection coupled with sensor reuse, allowing one to speed-up the measurement process and to reduce the biochip preparation time.64 As a model study, the target analyte was identified in a DNA sequence mapping of the human ABCB1 gene (protein coding, ATP-binding cassette), directly detected in human genomic DNA extracted from human lymphocytes with an experimental detection limit of 140 aM. This particular gene has been identified to be of importance to address the personalized medicine issue, and SPRi was applied to study the polymorphism of ABCB1 by identifying a particular SNP, the socalled rs1045642, a key target in pharmacogenomics.65 The detection of SNPs in target genes is an attractive goal for diagnostics and theranostics. For example, SNPs could confer increased susceptibility to a particular disease or may elicit different responses to drugs. This approach has a general validity and can be transferred to other genotypes of rs1045642 SNP, as well as other SNPs. Moreover, the SPRi multiarray asset may lead to simultaneous analysis of several SNPs on the same genomic sample, providing once more a useful tool for screening purposes when parallel analysis is a necessity, coupled with easy to use, label-free, and fast responses. Behind nucleic acid detection, SPRi may play a certain role in membrane-related studies. In biological membranes, various membrane proteins and lipids are laterally organized so as to establish highly efficient reaction systems. Assembling a mechanism, including the kinetics of the event, can be understood by label-free and real time approaches. Supported lipid membranes (SLMs) have received considerable interest as sensing interfaces in the past years,66 and their applications in sensor arrays by label-free sensing such quartz crystal microbalances (QCM)67 and conventional SPR68 have also been reported. More recently, findings reporting SLM studies by SPRi represent an important advancement for SPRi 2688



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application to binding studies in lipid systems.69 Phosphoinositides and their phosphorylated derivatives (PIPs) were studied by SPRi for their binding properties to proteins with a lipid microarray. A limit of detection of proteins on lipid membranes was determined to be on the order of nM. These results open interesting possibilities for simple construction of lipid membrane arrays for the label-free study of membrane proteins. Finally, looking at future and demanding applications of SPRi biosensors, we may propose the antidoping control field as an emerging area where recent advances may open new interest. New frontiers in antidoping controls are the detection of prohibited methods and drugs to enhance athletic performance. Among prohibited methods, one interesting field is defined by the detection of gene doping, meaning the misuse of gene therapy. Fast analytical innovative approaches such as biosensors are welcome in this area,70,71 and SPRi may eventually play a role. In particular, SPRi was applied to detect transgene sequences in human embryo kidney transformed cell cultures, used as model systems.72 Monitoring the presence of different sequences identified as transgenosis markers simultaneously, as allowed by SPRi, coupled to bioinformatics may be of certain help for gene doping screening purposes. Moreover, increased use of peptide hormones has been reported. In particular, very recently, the World Antidoping Agency (WADA) has focused attention on the challenging detection of peptide hormones and related substances in doping controls.73

analysis of different peptides, can be strategic to fulfill the analytical requirement in a field where controls are increasing in the number of samples and fast response.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: maria.minunni@unifi.it. Notes

The authors declare no competing financial interest. Biographies Giuseppe Spoto, Ph.D., is a Professor of Analytical Chemistry at the Chemistry Department, University of Catania, and a research associate at Istituto Nazionale di Biostrutture e Biosistemi, Italy. His research work over the last 20 years has focused on the study of surface and interface processes, with specific emphasis on bioanalytical detection processes. His main research interests are currently with surface plasmon resonance imaging, mass spectrometry, and microfluidics for the study of biomolecular interactions. Maria Minunni, Ph.D., is an Associate Professor of Analytical Chemistry at the Faculty of Science, University of Florence, Italy. Since 1990, she has been involved in biosensor research using different transduction principles. Her research activity covered mainly the development of optical, that is, surface plasmon resonance and piezoelectric affinity-based sensing (ABBs), immuno-, nucleic acid-, and aptamer-based sensors with different applications, from environmental, food, and pharmaceutical analysis to clinical diagnostic and, more recently, antidoping analysis.

The detection in body fluids of small peptides such as hormones and their relative releasing factors for enhancing athletic performance represents a challenge for analytical chemists, and SPRi may represent a new analytical tool for parallel analysis of different prohibited substances.

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ACKNOWLEDGMENTS MIUR (PRIN 20093N774P and 2009MB4AYL003) and the World Antidoping Agency (Research Grant −2010, “Detection of Hepcidin As A New Biomarker of Erythropoiesis Stimulators Abuse: A Pilot Study”) are acknowledged for partial financial support.

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REFERENCES

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The detection in body fluids of small peptides such as hormones and their relative releasing factors for enhancing athletic performance represents a challenge for analytical chemists, and SPRi may represent a new analytical tool for parallel analysis of different prohibited substances. In particular, to detect illegal use of peptide in antidoping, analytical approaches should be able to detect the presence of exogenous peptides versus the endogenous one. Many peptides may be of recombinant origin; thus, the sensor should possibly recognize differently the native molecule from the recombinant one. For this purposes, the availability of suitable receptors such as antibodies, synthetic peptides, aptamers, or molecular imprinted polymers may be of key importance. In our experience, SPR has been applied to insulin74 and to hepcidin detection,75 and the latter is indicated as a new biomarker of erythropoiesis stimulators abuse. Further extension of this work may be to translate the approaches to parallel detection of different prohibited peptide targets, as indicated in the prohibited list by the WADA. To give an idea of the phenomenon, just think to recent EPO-positive athletes, even at the last London 2012 Olympic games. In this sense, SPRi, combining label-free, real time monitoring and parallel 2689



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