Open access peer-reviewed chapter

Plasmonic Nanostructures as Surface-Enhanced Raman Scattering (SERS) Substrate for Protein Biomarker Sensing

By Dan Li and Yuling Wang

Submitted: September 26th 2016Reviewed: February 27th 2017Published: June 21st 2017

DOI: 10.5772/intechopen.68164

Downloaded: 1111

Abstract

Plasmonic nanostructures have attracted considerable interest in biomarker sensing with the goal of rapid diagnostics and personalized nanomedicine. Surface‐enhanced Raman scattering (SERS) is a versatile technique for the characterization of the plasmonic effect of the metallic nanostructures as well as a sensitive read‐out approach for biomarkers detection. In this contribution, we will give a review on the key optical properties of plasmonic nanostructures as SERS substrate for protein biomarkers detection. As a consequence, two approaches, label‐free and SERS labels will be discussed in details for protein biomarkers sensing by using the plasmonic nanostructures as the substrate.

Keywords

  • plasmonic nanostructures
  • surface‐enhanced Raman scattering (SERS)
  • protein biomarker
  • label‐free
  • SERS labels
  • sensing

1. Introduction

In order to understand the fundamental of plasmonic nanostructures and the related application, it is important to start with “plasmon”, which was first named by Pines in the 1950s [12]. As the valence electron collective oscillations resemble closely the electronic plasma oscillations observed in gaseous discharges, the term “plasmon” was thus used to describe the quantum of elementary excitation associated with this high‐frequency collective motion [2]. Based on the definition, plasmon is very similar as photon, which is the quantum particle representing the elementary excitations, or modes, of the free electromagnetic field oscillations. To be simple, plasmon can be described in the classical picture as an oscillation of free electron density against the fixed positive ions in a metal [3, 4]. To visualize a plasma oscillation, imagine a cube of metal is placed in an external electric field pointing to the right. Electrons will then move to the left side (uncovering positive ions on the right side) until they cancel the field inside the metal [5]. When the electric field is switched off, and the electrons move to the right, repelled by each other and attracted to the positive ions left bare on the right side. They oscillate back and forth at the plasma frequency until the energy is lost in some kind of resistance or damping [610]. Plasmons are a quantization of this kind of oscillation, which plays a large role in the optical properties of metals. Light of frequency below the plasma frequency is reflected, because the electrons in the metal screen the electric field of the light. Light of frequency above the plasma frequency is transmitted, because the electrons cannot respond fast enough to screen it [5]. In most metals, the plasma frequency is in the ultraviolet, making them shiny (reflective) in the visible range [811]. Some metals such as copper and gold having electronic interband transitions in the visible range, whereby specific light energies (colors) are absorbed, yield distinct colors.

Surface plasmons are those plasmons that are confined to surfaces and that interact strongly with light resulting in a polariton. They occur at the interface of a vacuum or material with a positive dielectric constant and a negative dielectric constant (usually a metal or doped dielectric) [5]. Plasmonics is related to the localization, guiding and manipulation of electromagnetic waves beyond the diffraction limit and down to the nanometer‐length scale [12, 13]. The key component of plasmonics is a metal, which can support surface plasmon polariton (electromagnetic waves coupled to the collective oscillations of free electrons in the metal, plasmon coupled with photons). There are two types of surface plasmon resonances (SPRs) that can be generated [12, 13]. The first one is the surface plasmon polariton (SPP), which is generated when light becomes trapped at a metal‐dielectric interface. The second one is the localized surface plasmon resonance (LSPR), which happened when a surface plasmon is confined to a particle of size comparable to the wavelength of light, that is, a nanoparticle, the particle’s free electrons participate in the collective oscillation. For the full detailed description of the fundamentals of plasmon and plasmonic effect, several excellent reviews are recommended [1319].

Figure 1.

TEM image of gold/silver nanoshell (a) and the tunable LSPR of gold/silver nanoshells with the shell thickness (b) (cited from Ref. [33] with permission from the Royal Society of Chemistry).

Plasmonic nanostructures are thus the metallic nanostructures on which the electromagnetic field was generated by exciting the oscillation of surface plasmon in the metal‐light dielectric interface. The study on the plasmonic nanostructures has attracted more and more interest in the research areas from the fundamentals to applications in a variety of scientific disciplines. In this chapter, we will focus on the plasmonic nanostructures as the surface‐enhanced Raman scattering (SERS) substrate for protein biomarkers detection.

SERS was first discovered in the 1970s [20, 21] on electrochemical roughed silver electrode and dramatically developed with the advancement of nanotechnology. Currently, two basic principles that contribute the SERS effect are accepted, which are the chemical effect (CM) e.g., charge‐transfer and the electromagnetic effect (EM) [2228]. Basically, CM is the interaction of the adsorbate molecules and the metal surface, mostly from the first layer of the charge‐transfer resonance between molecules and the metal [2224]. Whereas EM mechanism is based on the interaction of the transition moment of an adsorbed molecule with the electric field of a surface plasmon induced by the incoming light at the metal, as discussed above [2528]. Thus, SERS has been utilized in different areas including the catalysis, energy, and biology, and, in particular, in biomedical application. For instance, SERS has been used to monitor the catalytic reaction, image the live cell and tumor, monitor the nanoparticle distribution in live body, and so on [2931]. Several excellent reviews have been published on SERS including the basic principle of SERS, SERS nanoparticles, SERS labels as well as SERS applications in biomedicine [3237]. In this chapter, we will mostly focus on the optical properties (plasmonic effect) of the metallic nanostructures for SERS, with the highlight on the application of plasmonic nanostructures for protein detection by using SERS as a read‐out technology.

2. Plasmonic nanostructures

In this section, optical property of plasmonic nanostructures (surface plasmon resonance) will be discussed in terms of the SERS‐activity. Plenty of methods have been reported for the synthesis of plasmonic nanostructures including the wet‐chemistry (seed‐mediated growth), template nanoscale lithography, thin‐film, and the template approach. Here in this chapter, we will mainly focus on the synthesis and characterization of plasmonic nanostructures that are widely used for SERS study. Based on the shape or geometry of the nanostructures, we will give a brief overview for the quasi‐spherical metallic nanostructures, anisotropic nanoparticles/structures as well as the plasmonic nanoassemblies with the highlight on the plasmonic effect for SERS activity. As Mirkin et al. have discussed in details of the template techniques for assembly of plasmonic nanostructures [13], we will not include this technique in this chapter.

2.1. Quasi‐spherical metallic nanoparticles

As gold and silver nanostructures show the most significant SERS effect due to the surface plasmon resonance generated on gold and silver surface (EM enhancement mechanism), other metals such as copper and the transit metal also show the SERS effect; however, the impact factor is very low, which depends on several factors, including the size and geometry of nanoparticles as well as the laser excitations. Numerous protocols are available for the synthesis of metallic spheres [3841]. The simplest and most common approach is the reduction of metal salts with a variety of reducing and capping agents. To improve the stability and the enhancement of plasmonic nanoparticles, the combination of the alloy metal is an option. Thus, gold/silver nanoshells are designed and synthesized as the composite metallic nanostructures [42, 43] that show higher SERS effect compared to single metal. More importantly, by tuning the shell thickness, the LSPR of gold/silver nanoshells can be tuned (Figure 1b). In regard to the enhancement factor (EF) on the metallic sphere nanoparticles for SERS, it has been reported that individual spherical nanoparticles generate very low EF for SERS based on the EM calculation [34, 44]. Single molecule detection [25, 27] was reported on silver nanoparticles with the enhancement factor (EF) around 1012–1014, which is due to the strong surface plasmon coupling effect (called “hot‐spot”). In order to get high order of EF, well‐defined plasmonic nanostructures with multiple “hot‐spots” are required, which will be discussed in the following session.

Figure 2.

Typical electronmicrographs of anisotropic nanostructures for efficient SERS. Nanorods (a), nanoflowers (b), nanoplates (c), nanoprisms (d), nanocubes (e), and nanostars (f). (cited from Ref. [33] with permission from the Royal Society of Chemistry).

2.2. Anisotropic nanoparticles

As it is hard to obtain the SERS signal in the individual metallic nanospheres, anisotropic nanoparticles such as rods, stars, cubes, prisms, and nanoplates became very important SERS substrates (Figure 2a), which exhibit significantly higher electromagnetic field enhancements at sharp edges (“lighting rod effect” or “plasmonic antenna effect”), making them attractive for use as plasmonic enhancers in SERS [4549]. For the synthesis of anisotropic nanoparticles, the most widely employed approach is seed‐mediated growth, which involves seed‐formation and growth. Typical example is the synthesis of gold nanorods (Figure 2a), which starts with the quasi‐spherical ∼4 nm gold seeds and subsequent reduction of more metal salt with a weak reducing agent such as ascorbic acid in the presence of structure‐directing additives e.g., CTAB (cetyltrimethylammonium bromide), leading to the formation of gold nanorods. The aspect ratio can be controlled by the relative concentrations of the reagents. By tuning the aspect ratio of the gold nanorods, their localized surface plasmon resonance (LSPR) can be tuned, which is an important aspect for the application of gold nanorods in SERS as well as phototherapy.

Figure 3.

L‐shape trimer nanoantenna (a), LSPR spectrum of the trimer obtained by dark‐field Raleigh scattering microscopy (b), SERS spectrum of the Raman molecule (PCEPE) (c), normal Raman spectrum of PCEPE (d), and Raman spectrum of PECEP calculated using DFT (e). (Reproduced with permission cited from Ref. [50]).

2.3. Plasmonic nanoassemblies

In order to generate the hot‐spots for the SERS enhancement, plasmonic clusters including the dimers, trimers, and high orders nanoaggregates have been prepared. As it is very important to understand the relationship between nanoparticle structure and the SERS activity, Van Duyne et al. have reported the SERS EF with the different nanostructures (mainly dimers and trimers) [50]. As indicated in Figure 3, individual trimers have been encapsulated with silica‐shell to avoid the interference from the environment as well as ensure the contribution solely from the individual nanoparticles. It was demonstrated that the creation of hot spots, where two nanoparticles are in sub‐nanometer proximity or have coalesced to form crevices, is the paramount to achieving maximum SERS EF. Specifically, L‐shaped trimer nanoantenna comprised of three Au cores showed the EF of 108–1010 (Figure 3a) and the dark‐field Rayleigh scattering spectrum of the L‐shaped trimer contains three peaks (Figure 3b), corresponding to dipolar and multipolar LSPRs [50]. Single‐particle SERS obtained from the trimer nanoantenna showed distinct peaks from the Raman molecule (PCEPE), which are correlated with the density functional theory (DFT) calculations (Figure 3ce).

Figure 4.

Raman‐flow system for label‐free protein detection (a) and the interaction of the protein with a silver nanoparticle monitored by SERS (b). (Reproduced with permission cited from Ref. [59]).

Plasmonic nanostructures with multiple hot‐spots have been reported with the satellite nanostructures by using either gold sphere or gold nanorods as the core [5153]. To obtain the high orders of nanoassemblies, the linker is the key to connect the nanoparticles. Chemicals with dual/multifunctional groups are often used such as cystamine and dithiol‐polymer. As silica surface is versatile, the functionalization of silica surface followed by the assembly of small nanoparticles is a very useful strategy. More importantly, the distance between the satellite nanoparticles with the core nanoparticles can be controlled by the silica‐thickness. As reported by Gellner et al. [51], gold nanoparticles with the diameter of 80 nm were incubated with Raman reporters and encapsulated with a very thin silica shell. The ultrathin glass shell was then functionalized with a binary mixture of silanes including an aminosilane, which can adsorb the negatively charged gold nanospheres to form the 3D structures. Correlated HR‐SEM/dark‐field/LSPR/SERS experiments on individual 3D SERS‐active superstructures together with finite element method (FEM) calculations confirmed the plasmonic coupling between the core and the satellite particles, with hot‐spots occurring between core and satellites as well as between satellites [51].

Figure 5.

Scheme for aggregating nanoparticles for directly label‐free SERS detection of proteins. (Reproduced with permission cited from Ref. [65]).

3. Label‐free protein biomarkers detection

SERS is a powerful vibrational spectroscopy, which can provide rich molecular information for the target, making it very useful for direct protein detection. The fingerprint information extracted from the SERS spectra of proteins can be used directly to identifying the protein confirmation, the structure as well as the component of the target protein [5456]. Label‐free SERS detection for protein originates from the chromophores such as hemoglobin, myoglobin, and cytochrome c, which showed strong SERS signals with good reproducibility due to the Raman resonance (RR) effect of the chromophore center of the proteins [57, 58]. The information related to the conformation and orientation of proteins as well as the charge‐transfer processes between protein and surface can be obtained. As reported by Feng and Tachikawa [59], to determine the factors that contribute to the difference of SER(R)S signals and RR signal of metmyoglobin, they designed a Raman flow system and found that both the degree of protein‐nanoparticles interaction and the laser irradiation contribute to the structural changes (Figure 4).

Figure 6.

Label‐free SERS for native protein detection on iodide‐modified silver nanoparticles. (Reproduced with permission cited from Ref. [66]).

As the majority of proteins have no chromophore, the detection of proteins become much harder since the SERS signal from the native protein is very weak and most of the signals are generated from the amino acid residues and amide backbones, which are very similar for most of the proteins [60, 61]. Therefore, the key to get robust and sensitive label‐free protein detection is the SERS substrate, which should have the high SERS‐activity as well as proper preparation of the reproducible surface. Gold and silver nanocolloids are the widely used plasmonic nanoparticles for the label‐free SERS detection [6264]. As the gold and silver nanoparticles have relatively lower SERS enhancement effect, the procedure that could induce the aggregation, thus generate “hot‐spot” nanostructures is required. Typically, the addition of aggregation agent such as salt is the simplest and easiest approach for the aggregation. As displayed in Figure 5, Han et al. utilized sulfate as an aggregation agent to induce strong SERS signal due to the weak binding of SO42– on silver surface, making it easier for protein binding [65]. With this scheme, proteins including lysozyme, ribonuclease B, avidin, catalase, and hemoglobin have been detected and analyzed [65]. As SERS signal from the protein can provide useful information in terms of the structure, constituents, conformation as well as the potential interaction of the protein with the surface, in recent years, several groups have put great effort to improve the method of SERS‐based label free identification of proteins. For instance, Zhao et al. have conducted serious study on the label‐free protein detection by controlling the plasmonic nanoparticles and optimizing the purification procedures of the proteins [6265]. A typical example is the Western‐blot SERS, which was based on the silver staining of the membrane after the protein separation with the gel electrophoresis, which will purify and separate the proteins on the surface, making the detection more simple and easy.

Figure 7.

Schematic illustration of SERS ERLS (a) and typical SERS sandwich immunoassay for protein biomarkers detection (b). (Reproduced with permission cited from Ref. [35]).

Furthermore, Ren et al. have proposed a facile method to enable reliable label‐free SERS detection of the native structures of a wider range of proteins by using the iodide‐modified silver colloids as illustrated in Figure 6 [66]. The colloidal state of Ag NPs will help to keep the native structures of proteins and promote the photostability of samples. The iodide modification affords a one‐atom‐thick monolayer on the surface without producing interfering signal. Therefore, they demonstrated that the iodide‐modified silver colloids could not only clean the surface but also avoid the strong chemical interaction between the metal surface and the proteins, and reduce the possibility of denaturation, thus make the detection reliable and reproducible.

Figure 8.

Dye labeled SERS nanoparticles (a) and protein array (b) for high throughput proteins detection by SERS. (Reproduced with permission cited from Ref. [83]).

Recently, label‐free SERS has been attempted for the serum protein detection, aiming for the cancer diagnosis. By inducing the aggregation of silver nanoparticles for high‐quality SERS spectra, colorectal cancer has been identified based on the principal component analysis combined with linear discriminant analysis. [6769]

4. SERS Labels for protein biomarkers sensing

Compared to label‐free SERS approach, SERS labels have attracted significant attention for protein detection because of its high sensitivity comparable to fluorescence. The high sensitivity is most due to the strong plasmonic effect of the SERS substrates, which could be AuNPs, AgNPs, anisotropic nanoparticles (gold nanorods), nanoshells, plasmonic nanoassemblies, as discussed in the early session of this chapter. More importantly, SERS labels have demonstrated their unique optical properties and potential for simultaneous and multiplexed detection [3337] owing to the advantages of SERS labels over fluorescent label including (i) the multiplexing capability for simultaneous target protein detection due to the narrow width of the vibrational Raman bands, (ii) quantification using the SERS fingerprint of the corresponding labels, (iii) the need for only a single laser excitation wavelength, and (iv) high photostability. In this section, the sensor platform using SERS labels for protein biomarker sensing will be discussed from the conventional sandwich immunoassay, dot‐blot semi‐sandwich immunoassay, and protein microarray to microfluidic protein assay, with the highlights on our recent works for the detection of breast cancer biomarker [7073], pathogen antigens [74], cytokines [75, 76], and related works.

SERS sandwich immunoassay is a conventional sensor platform for the protein biomarker detection with SERS labels. Porter et al. have reported a serial of works on SERS immunoassay for various protein biomarkers detection [7782]. The SERS labels were named as ERLs (external reporter label). Typically, ERLs were composed of metallic nanoparticles (e.g., widely used gold nanoparticles duo to the distinguished plasmonic effects) and Raman reporters (dyes or the small molecule) to indicate the presence of the target. As indicated in Figure 7a, detection antibodies were immobilized on the plasmonic nanoparticles by either electrostatic force or the covalent binding through the links such as DSP (dithiobis (succinimidyl propionate)) [79] and carboxyl‐PEG‐SH (poly (ethylene glycol) 2‐mercaptoethyl ether acetic acid) [30]. To fabricate the sandwich SERS platform, the substrate could be the glass or gold film (Figure 7b). Additional studies have shown the higher sensitivity with gold film as the substrate because of the plasmonic coupling effect between the gold film (SPP—surface plasmon polarition) and the SERS labels (LSP—localized surface plasmon) [81, 82]. Followed by the capture of the target antigen or probes by the antibody on the surface, ERLs are bound on the surface for the signal to indicate the presence of the target proteins. With the similar platform design, plenty of proteins have been detected with few from the clinic important biomarkers [7883].

Figure 9.

Scheme of the direct SERS dot-blot immunoassay platform for duplex cytokine detection (a), schematic illustration of hydrophilically stabilized Au -Ag nanoshells with Ra-MEG-OH/TEG-COOH (b). (cited from Ref. [75] with permission from the Royal Society of Chemistry).

Microassay is the platform that could detect proteins with high throughput by SERS labels using different dyes as the Raman reporters. As a typical example indicated in Figure 8, multiple protein targets have been immobilized on the surface, followed by the probe with the dye labeled SERS nanoparticles [83]. Upon the laser excitation, SERS signals from the dye will indicate the presence of the target proteins. Microfluidic assay is another platform with high‐throughput properties for protein detection as the design for the channels can separate each of the individual proteins with different channels as well as enhance the reaction by controlling the flow conditions [74].

Figure 10.

Two typical platforms of rotating capture substrate (a) and syringe pump (b) to enhance the assay time (Reproduced with permission cited from Refs. [80, 84]).

Although it is a high throughput, the requirement for the professional training, long‐time incubation as well as the labor intensive procedures has hindered the application of the platform in the clinic setting. To improve the efficiency of the platform, a dot‐bot assay was thus developed, which is targeting on the rapid and sensitive and simultaneous multiple protein detections with a simplified procedure [75]. As displayed in Figure 9, duplex cytokines (interleuckin‐6 and interleuckin‐8; IL‐6 and IL‐8) were detected simultaneously on the dot‐blot assay with femtogram (fg) sensitivity, which was achieved by using the rational designed Au/Ag nanoshells as the plasmonic substrate (Figure 9). As specificity is a key issue for this study because it is hard to test the real samples without purification. Therefore, much more works were reported in this area, the limitations in the long incubation time and the labor‐intensive procedures with this platform have become a big obstacle toward the application in real, in particular, for the point‐of‐care diagnosis.

Figure 11.

Scheme of the microfluidic device platform for protein biomarker sensing by using ac‐EHD to enhance the assay time and minimum the nonspecific binding by applying a potential on the unsymmetry electrode pair. Dual functional Au/Ag nanoshells were used as SERS nanotags for breast cancer biomarker, HER2 detection (a) and simultaneously detecting four biomarkers in a five‐channel device (b). (Reproduced with permission cited from Refs. [70, 71]).

To minimize the assay time and enable the rapid detection, several strategies have been employed [80, 84]. For instance, Driskell et al. prepared an approach to increase the flux of antigen and SERS particles to the solid‐phase surface by using a rotated capture substrate. As illustrated in Figure 10a, by controlling the rotating rate for the capture substrate, the reaction kinetics can be improved quickly [80]. Instead of gold nanoparticles, gold nanorods were used as plasmonic nanostructures for ERLs to improve the sensitivity. The assay time was reduced from 24 hours to 25 minutes, however, in a 10‐fold loss of sensitivity compared to the conventional SERS sandwich immunoassay. Further, to improve the simplicity of the assay, the syringe pump SERS immunoassay platform (Figure 10b) was developed to overcome diffusion‐limited binding kinetics that often impedes rapid analysis in conventional SERS immunoassay. The assay time was reduced from 24 hours to 10 minutes with a 10‐fold improvement in detection limit [84].

Despite these attempts being successful in reducing assay times, nonspecific adsorption of nontarget molecules still remains the biggest challenge in immunoassay for protein detection. To circumvent this problem, our group recently proposed an innovative platform that utilizes nanoscaled alternative current electrohydrodynamic (ac‐EHD)‐induced surface shear forces to enhance the capture efficiency as well as significantly reduce the nonspecific binding of the molecules on the surface (Figure 11a). Meanwhile, to improve the sensitivity, rational designed silica‐coated gold/silver nanoshells have been employed as the SERS labels. It was found that the detection limit can go down to 1 fg/mL. Further, to improve the design of the channel (Figure 11b), simultaneous detection of four biomarkers was achieved both from the serum and patient samples.

Due to the great advantages of SERS in the sensitivity, multiplexed capability with only one single laser excitation, and photostability, it is expected that SERS labels will have more applications in the point‐of‐care diagnosis platforms for protein biomarkers detection by using the rationally designed plasmonic nanostructures.

5. Conclusions and perspectives

Plasmonic nanostructures with various size and shapes have been utilized in different areas including the catalysis, energy, and biomedicine. In this chapter, the fundamentals of plasmonic effect as well as the plasmonic nanostructures have been reviewed. Surface‐enhanced Raman scattering (SERS) is an optical phenomena happened on the plasmonic nanostructures and have shown distinguished properties for protein biomarkers detection. With two typical approaches (label‐free and SERS label) developed, SERS with plasmonic nanostructures has shown great potentials for proteins detection further to understand the biological system that protein involved. Meanwhile, although SERS detection for the protein biomarkers has been reported since 1990s, the application of this technology toward the real clinical sample is limited due to the rich information on the Raman peaks from the proteins, which have similar chemical bonds, thus making it very hard to be identified quickly and easily. Thanks to the development of the approach for the statistical analysis, label‐free SERS detection for proteins has become feasible for clinical samples. On the contrast, SERS labels with the well‐designed plasmonic nanoparticles show great potential for sensitive, reproducible, and simultaneous multiplexed detection for critical protein biomarkers. By combining the label‐free SERS with SERS labels approach, we expect that SERS combined with the rational designed plasmonic nanostructures will greatly enhance the research and application in this field.

Acknowledgments

This work was supported by ARC DECRA (DE140101056) to Y.W. D. Li would like to thank the finance support from the China Scholarship Council (CSC) for her to work in Macquarie University.

© 2017 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution 3.0 License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Dan Li and Yuling Wang (June 21st 2017). Plasmonic Nanostructures as Surface-Enhanced Raman Scattering (SERS) Substrate for Protein Biomarker Sensing, Nanoplasmonics - Fundamentals and Applications, Gregory Barbillon, IntechOpen, DOI: 10.5772/intechopen.68164. Available from:

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