Biomedicine with surface enhanced Raman scattering (SERS)

Biomedicine with surface enhanced Raman scattering (SERS)

5

K.W. Kho1, U.S. Dinish2, M. Olivo2,3 1 Imperial College London, London, UK; 2Singapore Bioimaging Consortium, A*STAR, Singapore; 3National University of Ireland, Galway, Ireland

5.1

Introduction

Medical diagnostics based on Raman spectroscopy, where diseases are classified according to their unique Raman signatures, has been well explored since the invention of a continuous laser. Unfortunately, owing to its low sensitivity, Raman-based diagnostics has not matured to a level for it to be utilized on a routine basis. Nonetheless, there are recent renewed interests in Raman spectroscopy, following the discovery of significant Raman enhancements on a corrugated metallic surface. Touted for their chemical specificity and superior sensitivity, surface enhanced Raman spectroscopy, or surface enhanced Raman scattering (SERS), is now being embraced as a new paradigm in optical-based medical diagnostics. This chapter is by no means intended to provide an exhaustive coverage of SERS, but instead will concentrate more on the recent developments in SERS and its future directions in biomedicine. First, a brief history of SERS is discussed in Section 5.2, followed by an explanation of the mechanisms behind the effect in Section 5.3. Section 5.4 includes various nanofabrication techniques commonly used for producing SERS-active nanostructures. Emphases on various biosensing schemes based on SERS are then given in Section 5.5, including the functionalization of SERS-active surfaces for the purpose of improving antigen-detection specificity. Recent examples of preclinical and clinical applications of SERS are discussed in Section 5.6. Lastly, in Section 5.7, the prospect of SERS will be looked into and commented on, particularly, in the context of translating the technology from a lab bench-based tool to a bedside biomedical device.

5.2

Surface enhanced Raman scattering

Several other optical processes can occur in a molecule under laser illumination, in addition to the relatively more pronounced elastic scattering effect known as the Rayleigh scattering. An inelastic scattering can arise when a transition between different vibrational states occurs within the probed molecule, which results in the emission of new photons with frequencies different or shifted from that of the excitation light. This optical effect, known as the Raman scattering (RS), was first observed experimentally by Raman and Krishnan (1928). Their studies lay the foundation Biophotonics for Medical Applications. http://dx.doi.org/10.1016/B978-0-85709-662-3.00005-1 Copyright © 2015 Elsevier Ltd. All rights reserved.

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for Raman spectroscopy, whereby the associated Raman shifts are directly correlated to the vibrational states of a molecular structure. While this offers a convenient and noninvasive means for molecular fingerprinting, the inevitable weak efficiency of RS has severely plagued its use in biomedicine. It is estimated that only about one-millionth of the incident photons impinging on any given molecule are “Raman-scattered,” hence, impeding trace biomarker analysis with RS. Fortunately, such a shortcoming can be overcome by Raman enhancements occurring in a molecule adsorbed on a plasmonic nanostructured metallic surface in a phenomenon known as SERS. SERS was discovered in 1974 by Fleischman et al. when they observed strong Raman enhancements on an electrochemically roughened silver electrode precoated with pyridine samples (Fleischmann et al., 1974). However, an explanation for the mechanism behind such an observation was not available until a more detailed study was carried out in 1977 by two separate groups, Jeanmaire and Van Duyne and Albrecht and Creighton, who attributed the observed enhancement to intensified electromagnetic fields and various chemical effects on the metallic surfaces (Jeanmaire and Van Duyne, 1977; Albrecht and Creighton, 1977). SERS then begun to gain interest with the same effect being reported for various other molecules adsorbed on metals such as Ag, Au, Cu, and Al.

5.3 5.3.1

Theory of surface enhanced Raman scattering Electromagnetic SERS (EM-SERS) enhancement

Unlike a dielectric medium, free surface charges (also referred to as surface plasmon) on a corrugated metal can be set to oscillate collectively by light with an appropriate frequency, bringing about a redistribution and nanofocusing of the photon-energy density at certain locations across the surface. Maximum electric fields can be reached at some of the focused spots when the incident light frequency matches the natural oscillating frequency of surface plasmon. This effect, known as surface plasmon resonance, is what contributes to the extraordinary Raman enhancements in molecules situated near these hot spots, owing to the strong optical excitations there (Dieringer et al., 2006; Moskovits, 1985). Coupled with the advancements in nanotechnology, EM-SERS has now become the most explored route to optimizing overall SERS enhancement through nanoscale surface engineering. While metallic nanotips and nanogaps (see Figure 5.1) have been the most sought-after geometry to maximize SERS enhancements, it was recently shown that other designs could be equally effective, or perhaps even more sophisticated in terms of optical functionality. This shall be included in the next section. All in all, regardless of the geometry of the nanostructures employed, the enhancement factor or EF (i.e., enhanced vs. unenhanced Raman intensity) obtainable with EM-SERS is typically around 104–1010 (Moskovits, 1985).

5.3.2

Chemical SERS (CM-SERS) enhancement

A secondary enhancement mechanism is the chemical enhancement (CM-SERS) primarily resulted from charge transfer between the adsorbate molecule and the metal surface. Although the exact nature of CM-SERS is still debatable, it is normally

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Figure 5.1 Numerical simulations showing intense electric field distributions (indicated in red) within nanogap (left panel) and nanotips (right panel).

believed to arise from the formation of Raman-active intermediates between the molecule and the metal surface which are able to couple resonantly with the excitation light to give rise to enhanced RS. Attainable SERS-EF in CM-SERS is typically in the range of two orders of magnitude (Liang and Kiefer, 1996). Unlike EM-SERS, the strength of chemical enhancement is generally affected only by the surface potential (Adrian, 1982).

5.4

SERS substrates

Among the plethora of SERS substrates developed, it is generally possible to categorize these substrates into two different types—namely, mobile substrates (MSs) and immobile substrates (ISs). In the former type, the substrates are typically in the form of submicron particulates bearing SERS-responsive metallic surfaces and are usually synthesized either by (mechanical and optical) ablations or by chemical means (Agarwal et al., 2011; Jeong et al., 2009). Although an MS is advantageous in that its small size permits in vivo semi-invasive/noninvasive spectroscopic applications, engineering the surface of such a substrate for optimized EM-SERS enhancement has been challenging due to its tendency to agglomerate. Nonetheless, the recent developments in particle encapsulation by a protective biomolecular layer have been able to alleviate such an issue without compromising overall SERS enhancement. Thus, it may not be difficult to predict ISs’ widespread uses in biomedicine, or perhaps their potentiality in rivaling biocompatible fluorescent counterparts. Immobilizing SERS-active surfaces on a solid platform, such as a metal-coated nanostructured surface, is an alternative approach to eliminating the issue of aggregations observed in MSs. While MSs achieve signal reproducibility via the averaging effects provided by their Brownian motions within the laser focus spot (MunizMiranda et al., 2011), ISs do so through closely packed arrays of localized plasmon fields on a periodically patterned surface structure, which can be obtained with various high-precision nanofabrication techniques, such as nanosphere lithography (Haynes and Van Duyne, 2001), deep-UV photolithography (Dinish et al., 2011), dip-pen lithography (Zhang and Mirkin, 2004), and e-beam writing (Clark et al., 2009). Owing

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to their structural flexibility, ISs have become the preferred platform in SERS spectroscopy (Deng et al., 2009). In the following sections, a brief discussion on various MSs, and different IS platforms is provided, emphasizing in particular their unique geometrical designs and optical properties in the viewpoint of biomedical applications.

5.4.1

Mobile substrates: SERS-active metallic nanoparticles

Spherical Au/Ag nanocolloids synthesized by salt reduction are the most extensively explored substrates in the earliest studies of SERS, owing to their geometrical simplicity, theoretically predictable optical characteristics, and good signal reproducibility. Unfortunately, such substrates generally suffer from low or moderate SERS-EFs (Li et al., 2009) and are prone to aggregation caused by nullification of their surface charges by oppositely charged bioanalytes in the sample. However, it is possible to stabilize an SERS nanoparticle against agglomerations by the use of a suitable protective encapsulating layer. Improving SERS-activeness, or rather the Raman EF, which is imperative for trace bioanalysis, on the other hand, would require a relatively more complex particle geometry, which can be challenging, but nonetheless attainable in some cases. This shall be briefly discussed below via some recent examples of novel SERS-active nanoparticles.

5.4.1.1

Multibranched Au nanoparticles

Metallic nanotips have been shown to elicit a “lightning rod” effect capable of creating strong and localized electromagnetic fields (Aizpurua et al., 2003). As such, multibranched, or star-shaped, Au nanoparticles synthesized by chemical reduction of HAuCl4 with hydrazine at room temperature in the presence of polyvinylpyrrolidone (PVP) should exhibit large Raman enhancements (see Figure 5.2) (Jeong et al., 2009; Calandra et al., 2006; Wu et al., 2006; Xie et al., 2007). Indeed, the SERS efficacy of such multibranched nanoparticles was found to be highly dependent upon the aspect ratio of their branches instead of the particle size. Experiments carried out with 4-nitrobenzenethiol have shown an SERS-EF of about 5.7
105 in multibranched Au-nanoparticle hydrosol, exceeding that of a nanoparticle thin film by one order of magnitude (Jeong et al., 2009).

5.4.1.2

SERS-active Au nanocrescent moon structures

The “lightning rod” effect can equally be achieved at the opened mouth lining the thin circular edge of an asymmetric nanosphere (see Figure 5.3) (Yu et al., 2005). The socalled colloidal Au nanocrescent moon structures fabricated by Yu et al. can be viewed as a rotational spread of sharp gold tip that extends the SERS “hot-spot site” over a circular line as elaborated by the field distribution plot shown in Figure 5.3 (bottom panel). By closing the two opposing “tips,” further enhancement can be attained, giving an overall SERS-EF of about 1010 as estimated using Rhodamine 6G as the sample molecule.

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Figure 5.2 (a) Low- and (b) high-magnification TEM images of the multibranched gold nanoparticles. (c) Histogram showing the distribution of nanoparticles sizes observed in the sample shown in panel (a). Adapted from Jeong et al. (2009)—reproduced by permission from Journal of Colloid and Interface Science.

5.4.1.3 SERS-active Ag dimers Extremely large Raman enhancements can also be observed at plasmonic nanogaps (see Figure 5.1), which are known to sustain intense localized electromagnetic fields. Implementing gap enhancements by bridging two isolated metallic nanoparticles with an organic linker has been attempted previously, but the approach suffers from a drawback that the linker molecule could prevent analytes from entering the hot spots within the gap (Li et al., 2009). Li et al. recently circumvented this difficulty by generating single-crystal truncated octahedron-shape Ag nanospheres that dimerize via the (1 1 1) facet by van der Waal attractive forces (Li et al., 2009). The resultant dimer structure consists of two Ag nanospheres each 30 nm in diameter and separated by 1.8 nm “unfilled” gap in the solution phase (see Figure 5.4). SERS measurements of 4-methyl-benzenethiol reveal the SERS-EF of such a colloidal substrate to be about 2.5
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Surface enhanced Raman scattering

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Figure 5.3 Gold nanocrescent moons with sharp edges. (Top panel) Conceptual schematics and TEM images of a nanocrescent moon SERS substrate. (Bottom panel) Local electric field amplitude distribution of a nanocrescent moon at one of its scattering peak wavelength. Adapted from Yu et al. (2005)—reproduced by permission from Nano Letters.

5.4.2 5.4.2.1

Immobile substrates: planar SERS-active solid platform Au bowtie nanoantenna

As mentioned earlier, an SERS-active nanostructured surface immobilized on a solid platform could not only provide stability against aggregations caused by ionic molecules in the samples, as occurred in bioanalysis based on MSs, but also offer a more flexible route to achieving optimized EM-SERS by permitting access to a wide variety

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Figure 5.4 (a and c) TEM images of silver nanosphere dimers at two different magnifications. (b) TEM image of the silver nanosphere dimers after their surface had been coated with silica. The dimers are highlighted by black ellipses. The inset in (b) shows a magnified TEM of the sample. The inset in (c) give a schematic illustration of the silver nanospheres in the dimer. (d) High-resolution TEM image of the gap in the dimer. Adapted from Li et al. (2009)—reproduced by permission from Nano Letters.

of surface morphologies. An interesting example is that demonstrated by Hatab et al., where periodic arrays of Au bowtie with 8-mm gaps (i.e., nanogap enhancement) were fabricated via a combination of e-beam lithography, metal deposition, lift-off, and reactive ion beam etching (see Figure 5.5). The nanobowtie substrate shows a maximum SERS-EF of about 1011 with a spot-to-spot variation of only about 10% based on SERS measurements of p-mercaptoaniline as a sample molecule (Hatab et al., 2010, 2011).

5.4.2.2 Nanodonut A periodic array of Au nanowells (Au-NWs) (as shown in Figure 5.6) fabricated with electron-beam lithography has been shown, through numerical simulations, to exhibit field structures favorable for Raman enhancements. The two essential elements in

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Figure 5.5 A typical SEM image of the gold bowtie arrays. The inset is an enlarged titled view of a gold bowtie array sitting on the top of silica posts. Adapted from Hatab et al. (2011)— reproduced by permission from Analyst.

Figure 5.6 (Top panel) SEM images of nanowells. (Bottom panel) Scalar cut of the electromagnetic field enhancement in a plane defined by the NW symmetry axis and the polarization direction. Adapted from Li et al. (2008)— reproduced by permission from Analytical Chemistry.

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such an NW are the Au nanodisc at the base of the well, and the Au nanodonut on top of it. Particularly, it was shown that, by altering the geometrical parameters, and hence the coupling strengths between the nanodonut and the nanodisc, one can tune the plasmon resonance spectra of the wells over the Vis–near-infrared (NIR) range (Li et al., 2008). This is crucial as SERS from certain biomolecules (e.g., cytochrome) can be more favorably observable only at a certain excitation frequency. Simulation studies (bottom panel of Figure 5.6) into the origin of SERS enhancements reveal active regions distributed over a large area on the well, including the lateral side of the Au nanodonut as well as around the edge of the Au-nanodiscs. An SERS-EF factor of about 1010 is estimated for Au-NWs.

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5.4.2.3 Nanoring The plasmonic split-ring resonator (as shown in Figure 5.7) fabricated by Clark et al. is another example of nanogap-based SERS structures, but unlike conventional nanogap systems (such as a metallic dimer particles), an important feature of the split-ring resonator is its multiple sharp resonance peaks, which correspond respectively to different SERS-active regions on the structure (see Figure 5.7) (Clark et al., 2009). By tuning the excitation frequencies as well as the incident polarization, Clark et al. were able to selectively activate these regions, and showed, as a proof-ofprinciple, multiplexed detection of molecules labeled with different Raman-active dyes. They do so by measuring SERRS, excited at two different wavelengths, from Cy5- and Cy7-labeled oligonucleotides that are hybridized to a complementary strand immobilized on the resonator. It is estimated that labeled oligonucleotide sequences are detectable, in phosphate saline buffer, down to 500–1000 molecules per resonator with equal fidelity.

5.4.3

Immobile substrates: SERS-active optical fibers

Earlier attempts to achieve in vivo real-time monitoring of biomolecules in an animal model by SERS required subcutaneous implantation of a thin 2D SERS substrate platform (Yuen et al., 2010). Certainly, a more practical adaptation of the technique must be devised prior to applications in a clinical setting. Transferring an SERS substrate onto the tip of an optical fiber offers a potential avenue in this aspect due to the small size, and this has been an interesting topic pursued by the SERS community. Owing to the great demands for in vivo sensing, several SERS studies have been directed toward the construction of SERS-active optical fibers, which offer the advantage of molecular fingerprinting ability of RS, the enormous Raman enhancement of SERS, and the flexibility of optical fibers. Typically, an SERS fiber would be configured in an “optrode” mode, in which a single optical fiber delivers both the exciting laser radiation and the returned SERS signal from molecules adsorbed onto the active substrate at the tip of the fiber. Here, an overview of some of the earlier design of SERS fibers will be provided, including highlights into one of the most recent progresses made in SERS fiber research using photonic crystal fibers (PCFs).

5.4.3.1 Conventional SERS optical fibers Earliest demonstrations of SERS fiber sensors generally involved forming metallic nanostructures on the fiber faucets, either by direct evaporation of metallic islandfilms or by vacuum deposition of metal films onto the fiber tips that are roughened by alumina, diamond nanoparticles, or sandblasting (Volkan et al., 2000; Viets and Hill, 1998; Stokes and Vo-Dinh, 2000). In another work, SERS-active fiber was realized by chemically immobilizing Ag nanoparticles onto the fiber tip with 3-aminopropyltrimethoxysilane. This configuration was able to record SERS spectra from a 1-nM solution of crystal violet (CV) in distilled water (Lucotti and Zerbi, 2007). However, since these techniques rely essentially on random assembly of

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Figure 5.7 SEM of a split-ring array showing a high level of array uniformity (top of panel a). Individual structure (bottom of panel a). (a) Graphical representation of the nanostructure’s field activities when the electric field of the incident light is polarized differently. (b) SEM images of a split-ring array. Adapted from Clark et al. (2009)—reproduced by permission from Journal of the American Chemical Society.

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nanoparticles, they often failed to generate strong SERS signals due to nonuniform coverage of particles. To circumvent this issue, a double substrate “sandwiching” structure was utilized (Shi et al., 2008), in which a layer of Ag nanoparticles were predeposited onto the fiber tip, followed by adsorption of the analyte molecules and a subsequent overlaying of Ag nanoparticles to form an Ag-Analyte-Ag sandwich. Due to the large plasmon fields experienced by the analyte at the junction between the two Ag nanoparticles substrates in the sandwich (i.e., nanogap enhancements), strong SERS signals can be obtained. Recently, Brolo and coworkers made a different attempt to improve the SERS efficacy of an Ag nanoparticles film immobilized on a single-mode fiber tip. Specifically, instead of depositing film comprising only a single layer of Ag nanoparticles, they found that SERS enhancement increased with increasing number of Ag nanoparticles layers deposited, reaching a maximum SERS enhancement when six layers of particles were used (Andrade et al., 2010). This type of design was successfully demonstrated for the real-time remote sensing of solution with a good sensitivity. SERS-active optical fibers can also be realized by coating a chemically etched fiber tip with a layer of Ag film, which then forms clearly defined triangular islands on a scale of 80 nm (White and Stoddart, 2005). These nanostructures show favorable SERS properties with a high EF of 106 when tested with thiophenol molecule. So far, the fabrication procedures discussed rely solely on the chemical properties of the fiber facets and thus lack the controllability over the resultant surface nanomorphology. For a more well-defined structural geometry, methods such as e-beam lithography have been used (Smythe et al., 2009). For example, a periodic array of nanoantennas can be defined by e-beam lithography on a Si wafer before being transferred onto the tip of a fiber and immobilized via van der Waals forces as shown in Figure 5.8. Such a lithographic fabrication and patterning procedure of nanoantennas provides a platform for producing two-dimensional arrays with well-defined geometry that allows (1) plasmonic tuning of the optical response of the probe and (2) the density of hot spots that determine the SERS enhancement to be controlled. The performance of the optimized geometry is studied with standard Raman-active molecule, benzenethiol and shown to possess an EF of 105. Fiber bundle has also been explored as a platform for in vivo measurements. For instance, Guieu et al. have recently achieved a fiber-based SERS sensor by etching the facet of a fiber bundle to create a collection of identical structures, such as microwells or nanotips (Guieu et al., 2008). The treated tip was subsequently coated with Au to facilitate Raman enhancements. An interesting feature of such a design is the possibility to generate high spatial resolution SERS imaging through the bundle of fibers, as Raman signals collected from each individual fiber can now be correlated to a specific position on the sample surface.

5.4.3.2 SERS platform based on photonic crystal fiber (PCF) Conventional optical fibers bearing an SERS-active tip as discussed above typically suffer from signal loss and low collection efficiency, making them unsuitable for remote sensing and sensing of chemicals at trace concentrations. In order to be of

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Figure 5.8 Schematic diagram of the optical setup used for the measurement with SERS fiber probe. A SEM image shows the array of gold optical antennas on the facet of a fiber. Adapted from Smythe et al. (2009)—reproduced by permission from Nano Letters.

use in biosensing applications, an improvement in the SERS-EF by at least three orders of magnitude would be required. To address this limitation, recently, hollow-core PCF (HCPCF) has been used to make the SERS probes. Unlike conventional fibers, propagating optical waves in an HCPCF are confined transversely in the central (hollow) core by surrounding it with many relatively narrower micro air channels running along the length of the fiber. It was suggested that by incorporating metallic nanoparticles into the core channel, one can enlarge the effective SERSactive area significantly due to the long-interaction length (as opposed to the active area on an SERS fiber tip) (Yan et al., 2006; Cox et al., 2007; Han et al., 2008), thereby leading to the possibility of working with extremely small sample volumes at low analyte concentrations. In the case of a sample in liquid form, the cladding air channels must be selectively collapsed to prevent the liquid from entering and causing a reduction of the refractive index gradient that affects the light confinement in the fiber core (Han et al., 2008; Zhang et al., 2007; Irizar et al., 2008).

5.5 5.5.1

Bioimaging and sensing with SERS SERS nanotags

Typically, molecular imaging of a small animal is carried out with techniques such as positron emission tomography, single photon emission computed tomography, magnetic resonance imaging, computed tomography, bioluminescence, fluorescence, and

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ultrasound. However, none of these techniques satisfies all the important requirements of high sensitivity, high spatial and temporal resolution, multiplexing capacity, and high-throughput. Among these techniques, optical modalities can provide high spatial resolution but at the expense of poor penetration depth. Fluorescence technique (FT) is a very promising tool, but it is limited by the availability of molecular fluorescent dyes in the NIR window with minimal spectral overlap. Moreover, fluorophores are inherently associated with photobleaching, which adds further challenge in developing as molecular imaging probes. Due to the broader spectral profile, fluorophores are often not suitable for multiplex imaging/sensing applications. In addition, background autofluorescence from superficial tissue layers also affects the sensitivity and image quality. MSs offer an alternative means to resolve this issue. By immobilizing a highly active Raman molecule (Raman reporter) on to metal colloids (Qian et al., 2008; Keren et al., 2008), MSs with “bright” Raman signatures can be constructed. Such a nanoparticle–Raman reporter assembly is sometime referred as SERS nanotag, or SERS dots in analogy with quantum dots, and is schematically shown in Figure 5.9. To facilitate biocompatibility, as well as to avoid bioanalyte-induced agglomerations, SERS nanotags must be encapsulated by a thin layer of silica coating (Keren et al., 2008; Kuestner et al., 2009), polyethylene glycol (PEG) (Qian et al., 2008; Maiti et al., 2010) or bovine serum albumin (BSA) (Samanta et al., 2011). This encapsulation helps to provide the physical robustness, stable signals, immunity to the biochemical environment, and means for bioconjugation. When forming a silica encapsulation, the thickness of the layer forms a crucial part. A thicker outer shell not only increases the size of the nanotag but also reduces the Raman signal generated from the reporter molecules. The sensitivity of an SERS nanotag will primarily depend on the signal intensity generated by the Raman reporter molecule. Previously, highly active Raman molecules such as CV, malachite green isothiocyanate (MGITC), 3,3-diethylthiatricarbocyanine iodide (DTTC), nile blue, 2-napthalenethiol, and various fluorescent dyes such as Cy3, Cy5, and Rhodamine, were used as reporter molecules (Qian et al., 2008; Maltzahn et al., 2009; Lee et al., 2009; Huang et al., 2009; Mahajan et al., 2008; Han et al., 2009). However, only a handful of such sensitive Raman reporters are presented in the literature. With the growing need for Raman reporter molecules that are easily identifiable in a multiplex analysis platform and capable of generating higher SERS intensity, a library of molecules has recently been developed through the technique of

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Figure 5.9 Schematic of the construction of SERS nanotags with various targeting ligands.

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combinatorial synthesis of triphenyl methine (TM) dyes that are then screened for their suitability in developing highly sensitive SERS nanotags. Cho et al. recently did just that and identified reporter molecules named as B2, B7, C3, and C7 that possess much higher SERS intensity than the reported best TM Raman reporter molecule, CV (Cho et al., 2010). It was found that these reporter molecules can bind to a colloidal surface in a noncovalent manner and, hence, the resulted intensity tends to fluctuate. To address this limitation these molecules are later linked with lipoic acid (LA) to form reporter molecules, B2LA, B7LA, C3LA, and C7LA that can be chemisorbed on citrate stabilized Au nanoparticles. These reporter molecules have shown excellent stability and produced stable SERS signals over a period of 1 month. Later, these reporter molecules were used to construct nanotags for detection of cancer biomarkers in vitro and in vivo (Maiti et al., 2010). Reporter molecules in TM library are ideally suited for visible light excitation and are most favorable for in vitro applications. Subsequently, a series of NIR-active Raman reporter (for resonant Raman) libraries were developed, and it was found that some of the hit compounds show a much higher SERS signal than other commercial reporter molecules (Samanta et al., 2011). SERS nanotags constructed with these reporter molecules were used for targeted in vivo cancer detection in a mouse model.

5.5.2

Sensing with SERS optical fiber

Functionalization of SERS HCPCF for the purpose of biomedical applications has been explored only relatively recently. In a more straightforward scheme, the liquid sample is simply injected into the central core to allow binding of the analyte of interest onto the SERS-active inner wall. Detection is then accomplished by analyzing the returned signals for Raman signatures corresponding to the analyte. As demonstrated by Yang et al., such a scheme is capable of highly sensitive SERS detection of Shewanella bacteria with a detection limit of about 106 cells/mL (Yang et al., 2011). In another scheme, sensitive SERS cancer protein detection was successfully demonstrated by immobilizing the epidermal growth factor receptors (EGFRs) in a lysate solution from human epithelial carcinoma cells into the HCPCF. Highly sensitive detection of this cancer biomarker was achieved using anti-EGFR antibody-conjugated SERS nanotag. This sensing probe was able to detect low amounts of proteins at 100 pg in a sample volume of 10 nL (Dinish et al., 2012). This detection is highly significant because the conventional enzyme-linked immunosorbent assay (ELISA) offers detection of cancer proteins at concentrations in the range of ng–mg/mL. The superior sensitivity of SERS HCPCF can be attributed to its long-interaction length.

5.6

Biomedical applications of SERS

As mentioned earlier, Raman spectroscopy has been utilized to monitor chemical changes in body fluids and tissue samples during disease progress (Feng et al., 2010; Bergholt et al., 2011). However, due to the extremely weak efficiency of RS, the technique has yet to achieve widespread uses in clinical settings. On the other

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hand, in the past few years, SERS has emerged as a suitable tool for trace chemical analysis (Dieringer et al., 2006) and also shown to exhibit single-molecule detection sensitivity (Guo, 2007; Mallory and Hajdu, 1990). Coupled with its insusceptibility to photobleaching, SERS is now anticipated to rival FTs in terms of reproducibility, sensitivity, photostability, ease of use, and total analysis time (Han et al., 2009). Unlike FTs or other optical techniques, SERS also possess the advantage of being immune to photoquenching by unexpected species present in the sample and hence offers the possibility to distinguish targeted molecules from interfering ones based on their Raman signatures (Kudelski, 2008). For instance, glucose-induced diffraction effects in boronic acids could be compounded by interfering species in blood (Yonzon et al., 2004), whereas a direct detection of glucose by its SERS spectra is less vulnerable to such problems. Additional benefits of SERS include suppressed fluorescence backgrounds by the SERS-active substrates (Kneipp et al., 1999) and the potential to operate at NIR, where undesired photochemical reactions as well as fluorescence—as could occur in a conventional FT with UV-to-visible excitation light source—can be avoided (Li et al., 2008; Kneipp et al., 1999). This is essential because strong a background signal has been a long challenging issue and can significantly reduce the limit of detection (Han et al., 2009). In this respect, SERS could possibly surpass FTs in analyzing complex biosamples. Another attractive feature of SERS is its ability to detect multiple analytes in parallel per assay due to the narrow (1 nm, full width at half maximum) bandwidth of Raman peaks, which minimize spectral overlap (Ni et al., 1999). Simultaneous detection of as many as 10 SERS spectra has been reported by Gambhir and coworkers (Zavaleta et al., 2009) in great contrast to fluorescence spectroscopy where broad spectra prohibit multiplexing (Graham and Goodacre, 2008). Coupled with the advance in pattern-recognition algorithm, automated identification of spectral components in a complex SERS spectrum may become a reality (Kastanos et al., 2010), allowing for fast analyte identification and eliminating the pitfalls of user bias. In terms of commercial prospect, the omission of reagents in SERS means real-time monitoring becomes possible as opposed to traditional methods, which have the disadvantages of being time-consuming, consuming large amounts of materials, and having poor yield like in ELISA (Westermeier and Vaven, 2002). Additionally, with SERS, only one single laser spot is needed to probe all analytes within a sample (Qu et al., 1999; Kho et al., 2012), thereby permitting the use of a much simplified or miniaturized optical system that requires less sample volumes. Due to this fact, together with its high sensitivity and multiplexing capability, SERS may emerge as a convenient point-of-care optical-diagnostic tool in the future.

5.6.1

Preclinical study

5.6.1.1 SERS detection based on mobile substrates Detection of circulating tumor cells Sha et al. used commercial SERS nanotag (Nanoplex biotags, a trademark of Oxonica, Inc.) for the direct detection of circulating tumor cells (CTCs) in whole blood. They used magnetic beads for capturing CTC and nanotags for rapid and sensitive detection

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directly in human whole blood (Sha et al., 2008). Magnetic beads conjugated to an epithelial cell-specific antibody (epithelial cell adhesion molecule, anti-EpCAM), and the SERS tags conjugated to an anti-HER 2 antibody that bind to a tumor cell. Since the breast cancer cell is of epithelial origin, the magnetic bead-EpCAM antibody complex will bind to this tumor cell but not to regular circulating blood cells, while HER 2 receptor is a cell membrane receptor, the anti-HER 2-SERS tag will specifically recognize these tumor cells. By adding this combination of magnetic beadEpCAM and SERS tag-HER 2 conjugates to a patient’s blood sample, circulating breast cancer cells (CTCs) were detected rapidly with good sensitivity in the presence of whole blood.

Detection of bronchioalveolar stem cells Woo et al. developed sensitive dual mode (SERS and fluorescence) antibodyconjugated spectroscopic dots (F-SERS dots) for the detection of three cellular proteins, including CD34, Sca-1, and SP-C (Woo et al., 2009). These proteins are simultaneously expressed in bronchioalveolar stem cells (BASCs) in the murine lung. F-SERS dots for this study were constructed using silver nanoparticle-embedded silica nanospheres with Raman reporters and fluorescent dyes. They used mercaptotoluene, benzenethiol, and naphthalenethiol as Raman reporters, while dye molecules such as fluorescein isothiocyanate and Alexafluoro 647 were used as fluorescent molecules in F-SERS dots. They could estimate the relative expression ratios of each protein in BASCs since external standards were used to evaluate SERS intensity in tissue. This was the first quantitative comparisons of multiple protein expression in tissue using SERS nanotags.

SERS imaging As was discussed earlier, the most promising advantage of using SERS nanotag over fluorescent nanoparticles (such as quantum dots) lies in the capability of multiplex detection. Multiplex detection of biologically relevant species is highly useful and can be easily achievable with SERS nanotags having various reporter molecules possessing distinct spectral profile and conjugated with different target moieties. In one of the earlier studies, the F-SERS dots developed by Woo et al. were successfully used for in vitro SERS multiplexing applications. They demonstrated highly selective and multifunctional characteristics for multiplex targeting, tracking, and imaging in cell lines. As described earlier, expression levels of three proteins (CD34, Sca-1, and SP-C) simultaneously expressed in BASCs in the murine lung, were quantitatively compared in the tissue using SERS-active nanoprobes (Woo et al., 2009). In a later study, Matschulat et al. demonstrated full exploitation of the multiplexing capability of SERS nanotags by applying cluster methods and principal components approaches for discrimination beyond the visual inspection of individual spectra. SERS spectra from five different nanotags were shown to be separable by hierarchical clustering and by principal components analysis (PCA) (Matschulat et al., 2010). In a duplex detection study in live cells, they demonstrated the imaging positions of different types of SERS probes along with the spectral information from cellular constituents by combining various spectral processing techniques.

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In one of the pioneering studies of the demonstration of SERS nanotags for in vivo imaging, Gambhir et al. used silica-coated nanoparticles (Keren et al., 2008). As a proof-of-principle, they demonstrated in vivo multiplex imaging using two different SERS nanotags by SERS mapping in mouse liver. In a follow-up study, the same research group showed the superb multiplexing capability of SERS nanotags by detecting 10 different tags, which were injected subcutaneously into a living mouse (Zavaleta et al., 2009). They also demonstrated the passively targeted detection in the liver using five most intense and spectrally unique SERS nanotags-injected intravenously. All five SERS nanotags were successfully identified and spectrally separated as shown in Figure 5.10 (Zavaleta et al., 2009). They could observe linear correlation of the Raman signal with concentrations of SERS nanotags for both subcutaneous and intravenous injection.

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(b) Figure 5.10 Demonstration of deep-tissue multiplexed imaging of five unique SERS nanotags simultaneously. (a) Graph depicting five unique Raman spectra, each associated with its own SERS batch: S420 (red), S421 (green), S440 (blue), S466 (yellow), and S470 (orange). (b) SERS image of liver overlaid on digital photo of mouse, showing accumulation of all five SERS nanots accumulating in the liver after 24 h post i.v. injection. Panels below depict separate channels associated with each of the injected SERS nanotags. Adapted from Zavaleta et al. (2009)—reproduced by permission from Proceedings of the National Academy of Sciences of the United States of America.

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In a recent study, Maiti et al. demonstrated the first proof-of-concept targeted multiplex detection of cancer in a living mouse. In this study, SERS nanotags were prepared with three in-house NIR reporter molecules—Cy7LA, Cy7.5LA, and CyNAMLA 381—followed by BSA encapsulation and antibody conjugation (Maiti et al., 2012). In this study, in vivo multiplex detection was carried out in a tumor xenograft having overexpression of EGFR receptor by injecting an equal amount of three bioconjugated nanotags through the tail vein of a living mouse. As shown in Figure 5.11, two nanotags conjugated with anti-EGFR antibodies were detected simultaneously in the tumor while all the three nanotags (two with anti-EGFR and one with anti-HER 2, which is a negative control) were accumulated in the liver via passive localization. These nanotags showed excellent stability over a period of 1 month on shelf with negligible SERS intensity fluctuation, which is a significant achievement especially when long-term monitoring of an SERS signal is required for both in vitro and in vivo applications. Furthermore, a phramacokinetic study of these nanotags in tumor and liver over a period of 8 days was carried out. This study revealed that stable SERS intensity from targeted nanotags in the tumor suggests its better localization and specificity. Also, in the liver, SERS intensity of these nanotags decreased steeply after 2 days, which indicates the possible excretion via fecal pathway. This research pertaining to kinetics of SERS nanotags will help in designing biocompatible nanoprobes for future sensitive in vivo imaging applications.

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Figure 5.11 In vivo multiplex detection in xenograft tumor: (a) SERS spectra from tumor site (peaks obtained at 503 and 586 cm1 from two EGFR-positive nanotags Cy7LA and Cy7.5LA); (b) SERS spectra from liver site (peaks obtained at 503, 523, and 586 cm1 from two EGFR-nanotags Cy7LA, Cy7.5LA and anti-HER 2 nanotag CyNAMLA-381); and (c) SERS spectra from dorsal region. SERS spectra were measured at 785 nm excitation, 30 mW power and integration time 20 s. Adapted from Maiti et al. (2012)—reproduced by permission from Nano Today.

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Imaging and sensing of EGFR and HER 2 cancer biomarkers

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Over expression of the EGFR and human EGFR 2 (HER 2) were found to be associated with number of solid tumors such as head and neck cancer, breast cancer, lung cancer, bladder cancer, and colon cancer. Moreover, EGFR expression level is associated with aggressive tumors and resistance to treatment with cytotoxic agents. Hence, these proteins are often used as biomarkers for various cancers. For instance, Qian et al. used SERS nanotags for noninvasive tumor detection that overexpresses EGFR in a mouse model (Qian et al., 2008). Their PEGylated SERS nanotags with NIR Raman reporters were found to be brighter than quantum dots. They conjugated SERS nanotags with single chain variable fragments (scFvs) that can target EGFR on a human cancer cell and in xenograft tumor models as shown in Figure 5.12 (Qian et al., 2008). In another work, EGF-peptide conjugated SERS nanotags were successfully demonstrated for the measurement of CTCs in the presence of white blood cells in SCC of the head and neck (Wang et al., 2011a). Recent work on affibody-functionalized fluorescent-SERS nanotags were used as effective multimodal contrast agents for molecular imaging of EGFR biomarkers. In their study, the signal from EGFR-positive tumors were found to be

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(c) Figure 5.12 In vivo cancer targeting using scFv antibody-conjugated SERS nanotags that recognize the tumor biomarker EGFR. (a and b) SERS spectra obtained from the tumor and the liver locations by using targeted (a) and nontargeted (b) nanoparticles. (c) Photographs showing laser beam focusing on the tumor site or on the anatomical location of liver. In vivo SERS spectra were obtained with 2-s signal integration and at 785 nm excitation (20 mW). Adapted from Qian et al. (2008)—reproduced by permission from Nature Biotechnology.

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much higher than that from the EGFR-negative tumors, which was later validated by competitive inhibition and in vitro flow cytometry analysis (Jokerst et al., 2011). There has been interesting work on the detection of HER 2 biomarkers using SERS nanotags. Lee et al. used antibody-conjugated hollow gold nanospheres (HGNs) with CV as a Raman reporter and applied for the imaging of HER 2 in cell culture (Lee et al., 2009). Their SERS mapping study showed that in comparison to silver nanoparticles, these HGNs exhibited significantly better and homogeneous scattering. This nanoprobe was also used as a multimodal agent for both dark-field imaging and SERS detection. An SERS nanotag was also constructed with gold nanorods (Au NRs) for the detection of cancer biomarkers. Park et al. demonstrated that antibody-conjugated gold nanorods with 4-mercaptopyridine reporter molecule for the imaging of HER 2 biomarker in cancer cells (Park et al., 2009). They conjugated anti-rabbit IgG onto the surface of the gold nanorods and treated with HER 2, overexpressing cells to demonstrate the sensitive detection. Maiti et al. also demonstrated a sensitive detection of HER 2 biomarker both in vitro and in vivo. In the first case, Raman reports were chemisorbed onto Au colloids and SERS screening was performed in cancer cell lines using anti-HER 2-antibody-conjugated nanotags. Very high SERS signals were obtained from HER 2-positive cells but not from the control cells (Maiti et al., 2010). In addition, in vivo SERS measurement was successfully carried out in a mouse model to detect subcutaneously injected SERS nanotag labeled cancer cells as shown in Figure 5.13. In the in vivo study, SERS nanotags were constructed with specially Skin

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Figure 5.13 Spectral comparison of functionalized SERS nanotag used in in vitro and in vivo studies. Pure tag: SERS spectra obtained from HER 2 antibody-conjugated nanotag in PBS suspension; subcutaneous injection: SERS spectra from SKBR-3 cell suspension with recognized antibody attached nanotag measured through the skin; cell (SKBR-3): spectrum of pure cell suspension only and skin: spectrum from the region far away from the location of subcutaneous injection. Measurement using 633 nm laser excitation at 6.2 mW. Adapted from Maiti et al. (2010)—reproduced by permission from Biosensors and Bioelectronics.

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synthesized sensitive NIR Raman reporters (absorption around 800 nm) and HER 2 detection was carried out using two HER 2-recognition motifs: a full anti-HER 2 monoclonal antibody (170 kDa) and a scFv anti-HER 2 (26 kDa) antibody. Initially, in vitro specificity was shown using HER 2-positive cancer cells. The target specificity of these nanotags in SKBR-3 cells (HER 2 positive) was confirmed by competition assays between antibody-conjugated nanotags and free HER 2-recognition motifs: a 10- to 15-fold decrease of the SERS signals in the presence of the competing anti-HER 2 antibodies was observed (Samanta et al., 2011). It was also found that signal intensities obtained with scFv-conjugated nanotags were at least 1.5 times stronger than those with the full-HER 2 antibody. To validate the detection by scFv-conjugated SERS nanotags in vivo, nanotags were injected into the tail vein of mice bearing xenografts generated from SKBR-3 cells. After 5 h of injection, the SERS spectra at the tumor site through the skin were acquired under a 785-nm laser excitation. The signal from the tumor site perfectly resembled the SERS spectra of the pure nanotag, whereas no SERS signal was detected from other anatomical locations such as upper dorsal. On the other hand, no significant SERS signal can be detected from the nanotags-injected mouse with xenograft prepared using HER 2-negative cancer cells (MDA-MB231).

Imaging of folate receptor Folic acid is a vitamin, which is essential for the proliferation and maintenance of all cells. Folate often acts as a limiting nutrient in human serum. Up-regulation of the folate receptor (FR) on cancer cells may enable malignant cells to compete more aggressively for this vitamin (Xia and Low, 2010). Many cancers, such as ovary, lung, kidney, breast, brain, and colon express high levels of FR and are used as biomarkers in many cases (Kelemen, 2006). Recently, Wang et al. constructed a dual-modal imaging probe for SERS (with reporter 2-nitrobenzoic acid) and fluorescence (with reporter Rhodamine B isothiocyanate) using mesoporous silica-coated gold nanorods for targeting FR in cancer cells (Wang et al., 2011b). Fluorescence and SERS signals were measured independently by using different excitation wavelengths, which enabled the investigation of the folic acid-conjugated probe in HeLa cells that overexpress FR. Cellular uptake of the probe by endocytosis is demonstrated by fluorescence imaging and SERS mapping.

Imaging of prostate-specific antigen (PSA) The prostate gland of the cells produces the prostate-specific antigen (PSA), and blood test for its measurement is considered as the gold standard for early detection of prostate cancer. Rising levels of PSA over time are associated with both localized and metastatic prostate cancer (Gonzalgo and Carter, 2007; Crawford and Abrahamsson, 2008). In this direction, Schlucker et al. used SERS microscopy for selective localization and detection of PSA in tissue specimens using a nanoconstructs with PSA antibody attached to a Raman reporter, 5,50 -dithiobis (succinimidyl-2nitrobenzoate), which is covalently attached to Au nanoparticles (Schlucker et al., 2006). This method combines the specificity of antigen–antibody interactions with the high sensitivity of SERS as a novel methodology for immunohistochemistry. In 2009, Jehn et al. used hydrophilic SERS labels for the construction of SERS nanotags

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and controlled conjugation of anti-PSA antibodies to the nanotag-enabled immunoSERS microscopy for imaging of PSA in prostate cancer tissues (Jehn et al., 2009).

Imaging of pH Measurement of intracellular pH with subcellular resolution is challenging, and it is critical to the understanding of various physiological processes. Kniepp et al. demonstrated spatially resolved probing and imaging of pH in live cells by biocompatible SERS nanotags with 4-mercaptobenzoic acid reporter anchored onto gold nanoaggregates. In this study, the team measured the relative intensity of pairs of spectrally narrow Raman lines in the same spectrum that allowed for quantitative measurement of pH without any correction regarding cellular background absorption/emission signals (Kneipp et al., 2007). This pH-sensitive SERS nanotag was used to measure and to image the pH values in subcellular structures in the range from 6.8 to 5.4. Surface enhanced hyper-Raman scattering (SEHRS) with two-photon excitation was shown to exhibit a spectral signature suitable for measurement and differentiation of pH values between 8 and 2. Such a wide response range obtained by the SEHRS-pH sensor may well be used for probing a variety of subcellular compartments, including those of extreme pH.

Apoptosis imaging Programmed cell death, or apoptosis, plays a critical role in the maintenance of cells by providing controlled cell deletion to balance cell proliferation. In a molecular level study, one can find that apoptosis represents a series of complicated pathways, which are directed by many proteins such as those in the family of bcl-2 (Rong and Distelhorst, 2008; Adams and Cory, 2007). Some of these proteins (e.g., bcl-2 and bcl-XL) are anti-apoptotic, while some others (e.g., Bad, Bax, or Bid) are pro-apoptotic. Nam Yu et al. developed multifunctional (fluorescent and SERS active) nanotags (F-SERS dots) with silver nanoparticle-embedded silica spheres with fluorescent organic dye and specific Raman labels for multiplex targeting, tracking, and imaging of cellular/molecular events in the living organism. 4-Aminothiophenol and 4-mercaptotoluene were used as Raman reporter molecules while fluoresceinisothiocyanate and Alexafluoro647 were used as fluorescent tagging chemicals (Yu et al., 2007). They detected apoptosis by specific target antibodies (BAX and BAD) conjugated F-SERS dots and could monitor the apoptosis effectively and simultaneously through fluorescence imaging and Raman signals, both in cells and in tissues with high selectivity.

Nuclear targeting A novel nuclear targeting nanoprobe based on peptide functionalized gold nanoparticles and its SERS detection in living cells was demonstrated by Xie et al. (2009). They probed SERS signals from the living cell nucleus by using high-selectivity functionalized nanotags. For a demonstration of this concept, they used 20-nm gold nanoparticles conjugated with SV-40 large T nuclear localization signal peptide successfully entered into the cell nucleus of HeLa cells. As a real-time detection technique, SERS-based imaging provided structural information for both nucleic acids

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and proteins without notable disturbance caused by the functionalization. This class of nanoprobe is expected to be nontoxic and biocompatible and, hence, could potentially be used for drug delivery and cancer therapy applications. Recently, Gregas et al. reported the development and application of a cofunctionalized nanoprobe and biodelivery platform combining a nuclear targeting peptide for improved cellular uptake and intracellular targeting application. Their SERS nanotag was constructed with 50-nm Ag nanoparticles tagged with paramercaptobenzoic acid as a reporter molecule functionalized with a cysteine-modified cell-penetrating peptide derived from the HIV-1 TAT peptide (Gregas et al., 2010). SERS mapping experiment proved that it was possible to track the spatial and temporal progress of the nanoparticle uptake in cancer cells at various time points. These nanotags showed significantly enhanced cellular uptake over the control nanoparticles without the targeting moiety. Such a nanoprobe for intracellular delivery has potential clinical applications in early detection and selective treatment of disease in affected cells. It could also potentially be used in basic research to understand the inner workings of living cells and how they respond to chemical and biological stimuli.

5.6.2

SERS detection based on immobile substrates

5.6.2.1 Detections without the use of Raman-active dyes While SERS-active metallic nanoparticles can be easily prepared, ISs are still the preferred platform for SERS studies owing to their stability against potential salt-induced aggregations (Muniz-Miranda et al., 2011; Kho et al., 2008). Quantitative detection of bioanalytes can simply be achieved by measuring their intrinsic SERS signals (i.e., without the use of Raman labelers) from samples deposited directly onto such a platform. Recent works, in this aspect, include those described by Yuen et al. (2010), Ackermann et al. (2007), Driskell et al. (2009), Wilson et al. (2010), Stokes et al. (2008), and Liu et al. (2009). For instance, Liu et al. were able to study antibiotic-induced chemical changes in the bacterial cell wall of Gram-positive bacteria Staphylococcus aureus by SERS measurements (see Figure 5.14), thus permitting a rapid identification of antibiotic resistant bacteria (Liu et al., 2009). Another interesting example is the transcutaneous SERS glucose sensing in a rat model by Yuen et al. (2010). In this particular work, nanostructured Ag-film coated with a layer of glucose-attracting film was prepared on a thin titanium substrate and, subsequently, implanted subcutaneously on the side-belly of an anesthetized rat. SERS spectra of glucose were then collected in vivo from the untreated side of the titanium substrate via an optical fiber bundle. Despite some variations in the data set, the glucose sensor was fairly linear over a physiological glucose concentration range of 100–500 mg/dL.

5.6.2.2 Detections with the use of Raman-active reporters Instead of detecting SERS signals generated directly from the analyte molecules, one can also carry out sensing based on sandwiched immunoassay with SERS-active reporters. In fact, such has been and currently is still the most common SERS detection

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scheme, due to the absence of photo-instability as would normally be observed in fluorescent dyes (Ni et al., 1999). Among such studies is a particularly interesting one by Chen et al., who used isotope-modified single-wall carbon nanowires as Raman labelers on an SERS-based micro-array (Chen et al., 2008). To render the nanowires analyte-selective, they were conjugated with an antibody that targets antigen molecules spotted onto the array (see Figure 5.15). By treating the micro-array with a thin layer of nanostructured Au film were detectable with a sensitivity of up to 10 fM. While the sensitivity of such a technique may supersede that of a conventional ELISA,

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Figure 5.15 Multicolor SWNT Raman labels for multiplexed protein detection. Adapted from Chen et al. (2008)—reproduced by permission Nature Biotechnology.

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the method still requires multiple washing steps, hence long analysis time. Alternatively, for rapid detection, one can functionalize an SERS-active substrate with a Raman-dye labeled aptamer that would displace from the SERS-active surface upon binding to the targeted analyte. As demonstrated by Cho et al., such an approach can be used to detect proteins such as thrombin in one single step, thereby considerably shortening the analysis time (Cho et al., 2008).

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Clinical study

5.6.3.1 Cancer diagnosis with SERS-active nanoparticles As cancer is now the top killer disease in the world, a simple and rapid diagnosis tool is highly desirable in order to detect cancer at its earliest stage as well as to reduce the costs for cancer patient management (Steward and Wild, 2014). Due to the many drawbacks associated with traditional diagnostics methods, many are beginning to turn to SERS as an alternative approach in the endeavor of cancer detection. For instance, in a clinical study carried out by Aydin et al. (2009) and Feng et al. (2009, 2010, 2011), SERS Ag-NPs were used to study cancerous samples ranging from blood plasma to homogenized brain tissues. In the case of SERS analysis of blood plasma samples, Feng et al. obtained samples from 32 gastric cancer patients and 33 healthy volunteers and compared SERS spectral data of these samples under four different excitation polarizations (nonpolarized, linearly polarized, left-handed circularly polarized, and right-handed circularly polarized) via a combination of PCA and linear discriminant analysis (LDA) (see Figure 5.16). A sensitivity and specificity ranging between 71–100% and 72–97%, respectively, were attained with lefthanded circularly polarized excitation yielding the highest accuracy (Feng et al., 2011). Feng et al. attributed the sensitivity to the polarization of light to the stereo structures of biomolecules expressed in the cancerous samples. Aydin et al., on the other hand, studied SERS spectra measured from liquefied brain-tissue samples prepared within 2–3 h of surgery (Aydin et al., 2009). By mixing 50-nm Ag-NP colloidal suspension with the sample, and drying the mixture on a CaF2, SERS spectra could be obtained. In this way, Aydin et al. was able to observe significant variations in the acquired spectra corresponding to different tissue types. Particularly, the ratio of the Raman-peak intensity at 723 to that at 655 cm1 increased from healthy/peripheral brain tissue to tumor. Although encouraging, the study is unfortunately inconclusive as the statistical number of patients involved is not sufficient. Thus, whether the technique will eventually be suitable for brain-tumor diagnosis remains to be seen.

5.6.3.2 Acute renal failure Serum creatinine has been associated with acute renal failure, or ARF, but the lack of a rapid and reliable technique for detecting creatinine has severely hindered progress in ARF management. Recently, Wang et al. have shown that urinal creatinine can be used as a biomarker for ARF diagnosis. Using a new type of SERS substrate (based on Ag-coated poly(chloro-p-xylylene) nanostructured films) that is stable against the

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high ionic strengths in urine samples, it was revealed that the two peaks appearing at 840 and 900 cm1 of the urinal SERS spectra are assignable to creatinine and suitable for quantifying the urinal concentration of creatinine. On the basis of 13 clinical urine samples (including two control samples), Wang et al. concluded the SERS sensitivity to urinal creatinine to be comparable to that of the enzyme-based method (Wang et al., 2010).

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Respiratory disease

ELISA-based detection of Mycoplasma pneumoniae, a major cause of respiratory disease in humans, which accounts for 20% of all community-acquired pneumonia, has been the most common serologic test for M. pneumoniae, but is usually limited by the time-consuming antibody response. While PCR is a more rapid alternative, it is plagued by issues with reliability, standardization, and cost (Waltes et al., 2008). Hennigan et al., recently, carried out a study into the SERS analysis of clinical throat swab samples (Hennigan et al., 2010) as a new technique for M. pneumoniae detection. Here, SERS was provided by Ag nanorod arrays onto which 1-mL Mycoplasma samples were applied. Up to 10 spectra were collected and subsequently analyzed using a combination of PCA and hierarchal cluster analyses. The results, estimated from 10 clinical throat swab samples, showed that three M. pneumoniae strains in

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solution are differentiable with this technique with an attainable specificity and sensitivity of 95–100%, and 94–100%, respectively, and >97% accuracy in classifying M. pneumoniae-negative and positive samples.

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Prospects of the technology

Whereas the schemes employed for biosensing with SERS are many and varied, the central concept has been to seek some sort of correlation between a set of SERS peaks and the targeted molecular markers. A straightforward approach would be to identify biomarkers adsorbed on an SERS-active substrate by their unique intrinsic Raman signatures. However, despite the promising results from several proof-of-concept studies, the practice would require some fundamental understanding of the technique. This is because disease-related biomarkers often belong to the same molecular species (e.g., proteins) that cannot be easily distinguished simply based on their SERS spectra. This is evident when comparing studies carried out by Feng et al. (2011) and Aydin et al. (2009), in which the same Raman bands (725 and 635 cm1) were used as the diagnostic marker, even though these studies described the diagnosis of two different diseases. To bestow SERS with the necessary disease specificity, the use of antibodyconjugated Raman-dye labeled SERS nanoparticles has been proposed. Recently, such an SERS nanotag has been exploited to specifically target HER 2 expressed on cancerous cell membranes (Maiti et al., 2010). One added advantage of SERS nanotags is their amenability to operate at NIR, making them suitable for deep-tissue imaging because light in the NIR can penetrate into tissue layers as deep as 5 mm due to low absorption and low scattering (Deliolanis et al., 2008). This would, therefore, greatly facilitate the investigation into metastasis and tumor localization, cell migration, and embryogenesis. Another advantage of SERS nanotags is the possibility to use prudently engineered Raman-dye labelers that could confer the tags with many nonoverlapping spectral “flavors” needed for multiplex detection. As further demonstrated by Gambhir and coworkers, a simultaneous detection of as many as 10 different SERS-labeled bioanalytes is possible (Zavaleta et al., 2009). This is certainly in contrast to quantum dots, which possess only limited distinguishable spectral characteristics within the NIR window. However, despite the potential superiority of NIR SERS nanotags to their fluorescent counterparts, in terms of multiplexity, issues relating to their toxicity and clearance efficiency must first be properly addressed before they can be routinely used in a clinical setting. Although the problem of long spectral collection time required at each pixel position may appear to further hinder NIR SERS imaging, the fact that only one wavelength is needed to excite multiple SERS nanotags and the continuous reduction in the cost of hyper-spectral imaging systems would encourage persevering efforts in developing a high-resolution deep-tissue imaging system with SERS nanotags. Aside from imaging, SERS nanotags have also been used as a labeling agent in a sandwich immunoassay platform as demonstrated by Van Duyne et al. This not only

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substantially improves detection sensitivity, but also retains SERS’s advantages of being highly multiplexed as well as photo-stable (Sun et al., 2007). While such an SERS-based immunoassay can substantially improve detection sensitivity and multiplexity, it does not eliminate the issues of material wastages (since at least two antibodies are still required to detect each targeted antigen), multiple washing steps, and long analysis time as encountered in a conventional immunoassay (Ni et al., 1999). For biosensing applications, in order to minimize the total assay time and material consumptions, one could exploit certain SERS-active molecular-constructs that are responsive to chemical changes. Graham et al. achieved this by developing a means in which an SERS-active colloidal substrate was conjugated with an aptamer. Binding of the antigen to the aptamer then triggered the occurrence of Raman intensities, due to the antigen now being immobilized closer to the plasmonic near-fields on the SERS-active Au surface (Graham et al., 2011). Binding-induced changes in the local microenvironment of a capturing antibody may also be exploited for single-step biodetection. A plausible scheme would be to incorporate, at the proximity of the binding site of a capturing antibody, an SERS-active beacon or transducer that can sense local chemical changes following a binding event as demonstrated by Kho et al. (2012). While Graham et al.’s aptamer-based SERS sensor has eliminated the use of a secondary/tertiary antibody (Graham et al., 2011), it is expected that a label-free antibodybased approach, such as that developed by Kho et al., would be more advantageous as it is not restricted by the limited availability of aptamers. Other issues relating to SERS biosensing include the substrate’s non-negligible irreproducibility (10%) caused by inevitable fabrication errors (Dinish et al., 2011). One may circumvent this issue by making use of a 3D plasmonic porous substrate that allows signal variations between plasmonic hot spots within the probe volume to be adequately averaged out. Assuming the abovementioned difficulties can be sufficiently overcome, the availability of low-cost and miniaturized spectrometers in the near future may lead to the realization of a portable SERS-based diagnostics tool with high sensitivity.

References Ackermann, K.R., Henkel, T., Popp, J., 2007. Quantitative online detection of low-concentrated drugs via a SERS microfluidic system. ChemPhysChem 8, 2665–2670. Adams, J.M., Cory, S., 2007. The Bcl-2-regulated apoptosis switch: mechanism and therapeutic potential. Curr. Opin. Immunol. 19, 488–496. Adrian, F.J., 1982. Charge transfer effects in surface-enhanced Raman scattering. J. Chem. Phys. 77, 5302–5314. Agarwal, N.R., Fazio, E., Neri, F., Trusso, S., Castiglioni, C., Lucotti, A., Santo, N., Ossi, P.M., 2011. Ag and Au nanoparticles for SERS substrates produced by pulsed laser ablation. Cryst. Res. Technol. 46, 836–840. Aizpurua, J., Hanarp, P., Sutherland, D.S., Ka¨ll, M., Bryant, G.W., Garcı´a De Abajo, F.J., 2003. Optical properties of gold nanorings. Phys. Rev. Lett. 90, 057401. Albrecht, M.G., Creighton, J.A., 1977. Anomalously intense Raman spectra of pyridine at a silver electrode. J. Am. Chem. Soc. 99, 5215–5217.

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