Molecular Affinity Agents for Intrinsic Surface-Enhanced Raman

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Review

Molecular Affinity Agents for Intrinsic Surfaceenhanced Raman Scattering (SERS) Sensors Victoria M Szlag, Rebeca Sarahi Rodriguez, JIAYI HE, Natalie V. HudsonSmith, Hyunho Kang, Ngoc Le, Theresa M. Reineke, and Christy L. Haynes ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b10303 • Publication Date (Web): 22 Aug 2018 Downloaded from http://pubs.acs.org on August 24, 2018

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ACS Applied Materials & Interfaces

Molecular Affinity Agents for Intrinsic Surfaceenhanced Raman Scattering (SERS) Sensors Victoria M. Szlag, Rebeca S. Rodriguez, Jiayi He†, Natalie Hudson-Smith†, Hyunho Kang†, Ngoc Le†, Theresa M. Reineke*, Christy L. Haynes* Department of Chemistry, University of Minnesota, Minneapolis, Minnesota 55455, United States †These authors contributed equally to this work. KEYWORDS: surface-enhanced Raman spectroscopy, affinity agent, intrinsic SERS, aptamer, antibody, small molecule, polymer

ABSTRACT: Materials, synthetic, and bio- chemistry convert analytical techniques into sensing platforms for applications across many research communities. Herein we review the materials used as affinity agents to create surface-enhanced Raman spectroscopy (SERS) sensors. Our scope includes those affinity agents (antibody, aptamer, small molecule, and polymer) that facilitate the intrinsic detection of targets relevant to biology, medicine, national security, environmental protection, and food safety.

We begin with an overview of the analytical

technique (SERS) and considerations for its application as a sensor. We subsequently describe four classes of affinity agents, giving a brief overview on affinity, production, attachment

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chemistry, first uses with SERS, characteristic SERS features of the affinity agent, and a review of the analytes detected by intrinsic SERS with that affinity agent class. We conclude with remarks on affinity agent selection for intrinsic SERS sensing platforms.

Introduction to Surface-enhanced Raman Spectroscopy (SERS) For Intrinsic Sensing:

In the 1970s, through the study of pyridine monolayers on roughened silver substrates, unprecedentedly high Raman intensities were observed1 and found to be the result of a newly apparent interaction of light and matter.2–4 This phenomenon of surface-enhanced Raman scattering has, to date, been mentioned in nearly 30,000 papers and is a title subject for over 9,000 papers.5 The unique, but weak, Raman scattering patterns from the excitation or relaxation of molecules’ vibrational modes6 can be used to identify and characterize molecular systems. As roughened/nanoscale surfaces provide large orders of enhancement, this spectroscopic technique is no longer confined to strong scattering targets or high concentration systems. A 104-1012 increase in the Raman scattering of a molecule,7 achieved simply by locating the molecule on or near particular nanoscale surface features, is the primary attraction of SERS as an analytical technique and a physical chemistry tool. The widely excepted mechanisms for enhancement include chemical effects, such as resonances within the molecule itself or chargetransfer between the molecule and the conduction band of the metal substrate, and electromagnetic (EM) effects originating from the surface plasmon resonance in the metal substrate.8,9 The EM mechanism is able to produce large enhancements ubiquitously among chemically diverse targets and is expounded upon here. SERS-viable surfaces, made of materials with negative real and near-zero imaginary dielectric constants, support a localized surface plasmon resonance (LSPR). The LSPR is a coherent oscillation of conductive surface electrons


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upon excitation by electromagnetic radiation (such as laser light).10 A strong electromagnetic (EM) field is generated upon LSPR excitation, and in this EM field a molecular target’s induced dipole, and resulting Raman scattering, increases. An LSPR EM field extends 5-30 nm from the surface, and decays (roughly) exponentially.11,12 Thus, the surface-dependence of this Raman technique is prominent and drives the need to localize the target within the EM field. Another way to understand the EM enhancement of SERS is through the Purcell effect. This is the idea that a cavity can radiate light, and the quality of said cavity dictates the radiation. This quantitative value is inversely proportional to volume, where the amount of radiation benefits from a small sensing volume. While the details of the Purcell effect are out of the scope of this review paper, we have listed a few papers that delve deeper into the theory behind this phenomenon.13–17 Within the field of SERS, research is typically focused in three areas: the physics and expansion of the technique, the use of the technique in physical chemistry studies, and the use of the technique as an analytical platform. While this review will focus on the last, it is important to note recent, often coupled, advances in the other two areas. Advances in the ultrafast,18,19 single molecule (SM),20,21 tip-enhanced (TERS),22 and shell-isolated nanoparticle-enhanced (SHINERS)23,24 branches of SERS are contributing to the exciting future of the field. The utility and versatility of SERS is recognized broadly and has promoted its extensive use in many research communities (e.g. biomedical25 and food safety26,27). Continuous improvement of enhancing substrates drives the expansion of SERS sensing applications in these communities.28 The considerations for what qualifies as a SERS sensor, what makes a good sensor, and what adds value to a sensing platform are depicted in Figure 1. Affinity agents can help achieve many of the requirements: they can provide a tunable degree of selectivity; they can


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concentrate the analyte at the SERS surface to improve detection limits; they can simplify methodology by removing the need to purify or concentrate samples; they can decrease cost by simplifying the method or enabling the use of less expensive SERS substrates; they can enable direct sensing of targets with poor attachment to bare plasmonic substrates; and lastly, again by tuning specificity, they can bind multiple targets, enabling sensor multiplexing.

Figure 1. Attributes and considerations for SERS sensing platforms, LOD stands for ‘limit of detection’. Affinity agents are used in both extrinsic and intrinsic sensing platforms (Figure 2). Extrinsic SERS sensors introduce a Raman label (sometimes called reporter or probe) to indirectly monitor an analyte. The labels, which must use affinity agents with high analyte specificity, have much stronger Raman signals than the analyte, and thus allow for lower limits of detection and/or detection in very complex samples. This increased sensitivity is accompanied by the loss of vibrational information about the analyte itself, and potentially, its interactions with the SERS substrate and its environment. Extrinsic SERS sensing can enable multiplex detection with multiple labels bound to specific affinity agents, and it is important to note the accuracy of extrinsic sensing is dependent on the exclusive binding of the label to the target. In


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contrast, intrinsic platforms are label-free and rely on detection of the SERS signal of either the analyte or the target-analyte complex. With intrinsic SERS, more detailed chemical information about the analytes can be investigated and obtained. Thus, multiplex detection is only dependent on multiple analytes having access to the SERS surface and presenting distinct Raman bands. Therefore, intrinsic SERS is more appropriate for the detection of unknowns and the understanding of intra- and intermolecular interactions in a system. Intrinsic SERS detection of a molecule can be performed directly from a SERS substrate if the analyte is confined near the surface through its own chemisorption or physisorption to the substrate.29 For analytes that are incapable of remaining in the enhancing EM of the substrate by these means, or in the cases of low analyte concentration, a surface bound affinity agent can be employed.

Figure 2. Examples of general SERS sensing schemes: (A) Chemisorption of the target to the SERS substrate for intrinsic detection; (B) Capture of the target by an affinity agent SERS substrate coating for intrinsic detection; (C) Capture of the target by an affinity agent grafted on a SERS substrate for intrinsic detection; (D) Use of affinity agent to attach a Raman reporter to a


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target for extrinsic SERS detection, which can be coupled with target capture agents similar to those shown in A-C. Affinity agents used for schemes like B and C will be covered in this review. In intrinsic SERS, an affinity agent captures and concentrates an analyte near the SERS surface enabling direct analyte detection (Figure 2, B and C). Affinity agents can have a wide range of target specificities, sizes, costs, and stabilities. Employing the best affinity agent for a given sensing scheme must take into consideration the target, the target concentration in samples, the sample matrix, and the SERS substrate. Inclusion of an affinity agent to a SERS sensing scheme requires the application of materials and surface science. Affinity agents also contribute their own spectral features, which can potentially mask target signals, and thus thorough SERS characterization of an affinity agent is important. Herein, as seen in Figure 3, we review these features for four classes of affinity agents (antibodies, aptamers, small molecules, and polymers) used in a wide range of intrinsic SERS detection schemes. Here, polymer affinity agents are defined (and differentiated from small molecule affinity agents) as those macromolecules produced from the intentional polymerization of a desirable repeat unit to a specific length and/or degree of crosslinking. These four classes represent the majority of affinity agents used in SERS sensors and create a spectrum of target specificity and SERS signal complexity. Our review of these classes of affinity agent includes a summary of each type’s affinity, production, attachment chemistry, SERS features, applicability to diverse target types (e.g. small molecules, proteins, and cells). In particular, we highlight the lowest target concentration distinguishable from a blank sample, aka the limit of detection (LOD), for each affinity agent/target pair review as a metric for comparison across affinity agent classes.


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Figure 3. The four major classes of affinity agents, discussed in this review of intrinsic SERS sensing schemes.

Antibody Affinity Agents

Antibody Structure, Affinity, Production, Attachment and a Note on Antibody Fragments Antibodies are immunoproteins (~150 kDa), expressed by B-cells, which contain molecular recognition sites to bind a specific target, known as an antigen.30 The molecular regions of the antibody and antigen that bind each other are referred to as the paratope and the epitope (represented in Figure 4), respectively. Generally, the binding of an epitope by a paratope is dependent on their complementary geometries, which enable regions of similar polarity to overlap.31,32 The dissociation constants, KD, for antibody/antigen interactions are often quantified between 10-8M and 10-10 M, demonstrating strong affinity.33,34 The specificity conferred by paratope geometry, and the high affinity of interactions it enables, was quickly realized for its diagnostic potential. In 1959, Yalow et al. reported the first use of an antibody as an affinity agent to detect an antigen, insulin, by radio-immunoassay.35 Further scientific use of these powerful biomacromolecules required numerous advances in antibody production, screening, and purification.


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Figure 4. An illustration of an antibody binding an antigen, showing the binding regions of both. The antibodies produced by an immune system are typically polyclonal; there are multiple antibodies for the same antigen with differing paratopes derived from distinct B-cells.36 Monoclonal antibodies, or antibodies derived from a single B-cell line, have identical paratopes and are thus preferred for therapeutic, diagnostic, or research purposes. In 1975, Kӧhler & Milstein reported a method that ensured a reliable source of monoclonal antibodies.37 The fusion of a mouse myeloma and a mouse spleen cell from an antigen-infected donor, creates an immortalized cell line for the production of a specific antibody. For known antibodies, production in mammalian cell lines is used commercially and has seen significant advances driven by the scale and product quality demands of the biotherapeutic industry.38,39 In these systems, antibody production is initiated by the transfection of the chosen mammalian cell system with plasmids for the desired antibody chains.38 Development of a highly homogenous and productive mammalian cell line can be a multi-month process with high capital and labor demands.39 New cell-free antibody production aims to avoid these culture-based drawbacks and uses extracted cellular translational machinery. Antibody production by this system is no longer dependent on cell viability and reaction conditions are more controlled without the plasma


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membrane barrier. However, it is still a developing technology and not widely used for commercial purposes.40 In addition to their means of production (and corresponding availability, reliability, and cost), the mode of antibody immobilization on a substrate is an important consideration when using antibodies as a SERS affinity agent. Numerous methods of antibody immobilization were developed for their application in affinity chromatography.41 Though the substrates differ considerably between chromatography and SERS platforms, the same categories of immobilization have been used: physical adsorption, chemical coupling of the antibody and substrate, and antibody site-specific covalent attachment. Physical adsorption was more prominent in early work42 and should be used cautiously due to the number of factors that influence antibody adsorption, the limited control over antibody orientation on the surface, and the possibility for desorption.43 To prevent loss of the antibody affinity agent, chemical coupling between the antibody and the surface is often preferred. As with any bioconjugation reaction, correct pairing of the reactive functionalities present on the antibody44,45 and on the surface must be well matched. Generally, coupling of an antibody to a metal surface (as is typically done with SERS) is accomplished in three facile steps: (1) functionalization of the metal surface with an alkylthiol or alkylamine; (2) activation of the surface groups with a coupling agent; (3) reaction between antibody functional groups and the activated surface groups. Some of the coupling strategies include the use of carbodiimides, succinimides, bis-aldehydes and anhydrides.46 Additionally, CuI has been employed to catalyze terminal alkyne-azide cycloaddition (click chemistry).47 Chemical coupling strategies can lead to stronger, more permanent antibody immobilization on a surface than physical adsorption. However, like physical adsorption, antibody orientation is not controlled on the surface by coupling strategies. The potential to couple with many of the antibody’s amine or carboxylic


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groups (depending on the coupling strategy) can lead to numerous and non-optimal orientations of the antibodies on the surface. Ideally, antibody immobilization on the surface would orient the two paratopes away from the surface, leaving them free to bind antigen.48 Single stranded DNA49 or synthetic peptides50 show great promise to direct antibody orientation on gold; however, these strategies are more complicated, more expensive, and less-versatile than coupling strategies. One cost-effective method to control antibody orientation is the reduction of an antibody’s disulfide bond, located in the hinge region away from the paratopes, to a thiol. This thiol can then easily bind the antibody half fragment to gold with some control over orientation.51,52 It is worth noting that antibody fragments, especially those generated from recombinant DNA, are becoming popular alternatives to full monoclonal antibodies. The attraction of recombinant antibody fragments (rAf) stems from their smaller size, more economic production, and ability to include affinity peptide sequences for easy purification and immobilization. rAfs are commonly produced by in vitro phage display technologies and in vivo microorganisms as hosts. Specifically relevant to SERS, it is important to note that the smaller molecular size of rAfs could result in an increased antibody density on a substrate, and thus more binding sites.53 Capture of the antigen by the smaller rAfs would also draw the antigen into more intense EM fields to achieve larger Raman signals. For this reason, the small size of rAfs could improve intrinsic SERS; however, the two have yet to be combined in literature.

SERS with Antibody Affinity Agents

First Uses


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The first report of antibodies used as affinity agents for SERS was an extrinsic, surfaceenhanced resonance Raman scattering (SERRS) experiment in 1989 to detect anti-thyroid stimulating hormone. In SERRS, the magnitude of enhancement is improved by selecting an excitation light with a frequency in resonance with a major absorption band of the target molecule, in this case the extrinsic Raman reporter, 2-[4’-hydroxyphenylazo]benzoic acid.42 The authors’ choice to detect extrinsically is a common one among research combining SERS and antibodies. This is for a variety of reasons, such as the large size of the antibodies excluding the target from the strongest regions of the enhancing EM field and the small scattering cross-section of some targets. A review of SERS platforms using antibody affinity agents, including extrinsic detection scheme, from the last five years is available.54 Herein, we will review the use of antibodies in intrinsic sensing platforms.

Assignments of Antibody Affinity Agent Spectra As will be reiterated multiple times throughout this review, thorough SERS characterization of an affinity agent is critical due to their inherent, sometimes strong, signals. For antibodies, which are composed of amino acids, it is important to note that a large amount of signal can come from the side chains (R groups) of amino acids. This is especially the case for the conjugated side chains of phenylalanine, tyrosine, and tryptophan, whose normal Raman spectra were collected by Gelder et al.55 Affinity agents occupy the volume just above the surface of the substrate, and thus are in the strongest region of the enhancing EM field. This is illustrated in the work of Drachev et al. which detected the formation of antibody/antigen complexes by SERS, for anti-FLAG M2 monoclonal antibody and a bacterial alkaline phosphatase-C-terminal FLAG-peptide fusion, respectively. This work compared SERS spectra


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from substrates exposed to antigen first and antibody second, and to those from antibody first and antigen second, respectively. In both the experiments, the signal from the protein exposed to the substrate first dominated the SERS spectra. Both experiments also detected the binding of the second proteins via reproducible changes in the SERS spectrum of the first protein.56 Additional SERS characterization of an antibody affinity agent is seen in the work of Sanles-Sobrido et al. (Figure 5). The authors used silver-coated carbon nanotubes (CNT@Ag) decorated with an antibody for a cocaine metabolite. They noted that the SERS spectrum of the antibody was dominated by C–H deformation, amide III vibrations, C–C stretching, C–C bending, and C–H bending at 1343, 1307, 1085 and 924, 764 and 693 cm-1 shift,57 respectively, referencing the work of Tuma for assignment.58

Figure 5. Adapted and reprinted from Ref 50 (permission requested). SERS spectra of (A) small molecule cocaine metabolite BCG, panel D, on silver-coated carbon nanotubes (CNT@Ag), (B) the anti-BCG antibody coupled to CNT@Ag, (C) antibody-BCG system complex on CNT@Ag.

Antibody-Enabled Detection of Small Molecule Targets by Intrinsic SERS


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The work of Sanles-Sobrido et al. (discussed above) demonstrated that benzoylecgonine (BCG), a small molecule cocaine metabolite, could be detected by comparing the SERS of the antibody-BCG complex spectrum with the spectrum of the antibody alone. The antibody-BCG complex SERS spectrum shows significant enhancement at 693 cm-1 shift (C-H bending).57 However, few similar reports exist of intrinsic small molecule SERS sensors that employ antibody affinity agents. In contrast, antibody-enabled intrinsic SERS detection of larger molecular targets is more popular and will be discussed further in detail. This is likely due to the large size discrepancy between antibody and small molecule targets. The large antibodies fill the high enhancing region of the SERS substrate, and spectral changes from associated small molecules can only result from: (1) structural changes to the antibody or (2) a large scattering cross-section from the target. The first case would be the result of flexible paratopes and could be accompanied by low target specificity. In the second case, small molecule targets with large scattering cross-sections can be captured by affinity agents that are less expensive and have lower signals to achieve more target SERS signal.

Antibody-Enabled Detection of Protein Targets by Intrinsic SERS The main peanut allergen protein, Ara h1, has been detected by antibody-enabled SERS; however, the antibody spectra significantly overlapped with that of the target protein, which make visual differentiation hard. Using the statistical clustering technique, principal component analysis (PCA), various spiked concentration of Ara h1 in samples could be distinguished from each other and the LOD was found to be 0.14 mg/ml.59 In 2014, a magnetic iron oxide/silvernanocomposite (Fe3O4@Ag@streptavidin@anti-IgG) was fabricated to selectively detect human immunoglobulin G (IgG) in blood samples via SERS. The magnetic property of


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the nanocomposite enabled separation of the composite from a blood sample after the anti-IgG antibody had bound the target. Spectral differences in the amide I (1650 cm-1 shift) and amide II (1539 cm-1 shift) bands were observed between just the antibody nanocomposite and the nanocomposite with captured IgG. The ratio of these peaks was used for the quantification of IgG target. An LOD of 0.6 ng/L was demonstrated, which is ~1000 times lower than similar nonSERS immunoassays of the time.60 A more recent paper also used core-shell iron oxide/silver (Fe3O4@Ag) nanoparticles (NP) for protein detection by SERS but further coupled the core-shell NP to 2D graphene oxide for additional chemical enhancement of the SERS signal. The platform was then conjugated with antibodies for tau and β-amyloid proteins, biomarkers for Alzheimer’s disease. In blood samples without the biomarkers, only graphene oxide SERS peaks were observed. After exposure to the targets, a SERS spectrum with protein vibrations were observed. The nanoplatform was reported to have an LOD for β-amyloid and tau proteins in blood of 100 fg/mL.61 Magnetic separation was also employed for the SERS detection of proteins isolated from pathogenic bacteria lysates. Two bacterial species commonly associated with prosthetic joint infection (PJI), Staphylococcus aureus and Streptococcus pyogenes, were identified. Antibodies were used as affinity agents against protein A, a surface protein from the S. aureus cell wall and group-A streptococci polysaccharide. Target bacteria were detected from model and real knee joint fluid matricies.62

Antibody-Enabled Detection of Microbe Targets by Intrinsic SERS Detection by intrinsic SERS has been reported for whole viruses for the mosquito-borne illnesses, Dengue and West Nile, of the family Flaviviridae. The antiflavivirus 4G2 antibody was used as an affinity agent and conjugated with gold nanoparticle (Au NP) SERS substrates. When


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incubated with the viruses, the antigen−antibody interaction formed assembled Au NP structures on the surface of the virus and resulted in high enhancing “hot spot” regions, highly localized areas of strong enhancement which are discussed in depth elsewhere,16,63 between Au NP. Without the virus, and the resulting hot spots, negligible SERS signals from the antibodies were observed. Upon assembly with the viruses, SERS of the virus-antibody-substrate complex was used as a fingerprint for the identification of each virus.64 While antibody/antigen interactions are commonly recognized as the epitome of specificity, their large size has limited their use in intrinsic SERS sensors. Additionally, high cost, low thermal stability, and antigen cross reactivity36 should be considered when employing antibodies as affinity agents. Importantly, as with any technique employing antibodies as affinity agents (i.e. immunoassays, immunohistochemical staining, immunoaffinity chromatography), SERS sensors relying on antibodies should include thorough controls exploring antibody crossreactivity with sample components.65

Aptamer Affinity Agents

Aptamer Structure, Affinity, Production, Attachment and Comparison to Antibodies An aptamer is a single stranded piece of DNA (ssDNA) or RNA that has a specific affinity for a target molecule. Aptamers are about 10 times smaller than antibodies (5-20 kDa, or 15-60 nucleotides).66 RNA aptamers occur naturally as a component of riboswitches—mRNA that binds a molecular target and induces a change in protein production encoded by that mRNA.67 Synthetic aptamers engineered for specific targets are primarily utilized in biotechnology research. Aptamers’ binding specificity and affinity is the result of the 3D


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structure an aptamer assumes upon associating with its target (Figure 6). The aptamer folds around the target promoting intermolecular interactions, exploiting van der Waals forces, hydrogen bonding and electrostatic interactions to form a stable target-aptamer complex.68 Binding dissociation constants (KD) represent a greater range in aptamers than antibodies, 10-310-12 M, and the higher affinity demonstrated is the result of a more rigorous, directed selection process.69

Figure 6. An illustration of an aptamer binding a target, emphasizing the conformation the aptamer assumers upon interaction with the target. The selection processes responsible for the synthesis of aptamers were introduced in the 1990s.70,71 Dubbed systematic evolution of ligands by exponential enrichment, or SELEX, this method begins with a random ssDNA library. Conventionally for DNA aptamers, the library is first exposed to just the matrix used for target immobilization. In this way ssDNAs that nonspecifically bind to the matrix are removed. The remaining sequences are exposed to targets that are immobilized on the matrix, and ssDNA that do not bind are removed. The bound ssDNA is eluted and amplified by PCR. Additional selection rounds, usually about 20, are performed until a small pool of best-performing ssDNA remains. These potential aptamers are sequenced and evaluated by binding kinetic studies for the final selection of an aptamer. For RNA aptamers, the


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process is considerably the same but with transcription and reverse transcription steps on both sides of PCR amplification. RNA aptamers thus require more time and labor, which results in higher cost. Method advances, to decrease the time and labor of the original SELEX method, have leveraged techniques such as microfluidics,72 capillary electrophoresis and surface plasmon resonance to improve efficient separation of ssDNAs and monitor ssDNA/target kinetics, respectively, and are reviewed elsewhere.69 Once selected, an aptamer affinity agent needs to be immobilized on a detection platform.73 The most popular means of attachment is the use of thiolated aptamers on gold surfaces. A disulfide can be incorporated to the 5’ end of an ssDNA through phophoramidite chemistry,74 which is available commercially for many aptamers, and this stable, asymmetric disulfide can be reduced to a thiol at the time of use. In another method, discussed above with antibodies, both surface and aptamer can be functionalized with groups amenable to coupling chemistry. Unlike antibodies, for aptamers, this scheme does not result in random orientation: the unique 5’ and 3’ ends of the ssDNA provide a more directed attachment approach. In addition to the more controlled attachment aptamers present over antibodies, the ssDNA affinity agents have several other benefits compared to their immunoprotein counterparts75,76 that have been alluded to above. The aptamer SELEX process is simpler and more cost effective than the commercially used mammalian cell production of antibodies, though antibody technology is currently more scalable. The ‘chemical’ synthesis of aptamers by DNA sequencing and PCR cheaper and more convenient than antibody production in cell lines and microorganisms.69 A high number of iterative selection steps can lead to lower aptamer dissociation constants (10-12 M) than those of antibodies (10-10 M), and more selectivity based on good aptamer folding around the target. Aptamers can accomplish this level binding as smaller


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molecules compared to full antibodies, ~15 kDa versus ~150 kDa. Their smaller size can enable more binding sites per area of substrate. For SERS, a smaller affinity agent can lead to better localization of affinity agent/target complex in the enhancing EM field, and thus more target signal. Finally, in consideration of the many protein targets of interest, an aptamer’s DNA SERS signal will have less overlap with protein targets’ spectra than that observed with antibodies.

SERS with Aptamer Affinity Agents

First Uses The use of aptamer affinity agents for SERS detection platforms began just over ten years ago, and several extrinsic sensing platforms were reported over those first several years; for small molecules, such as adenosine and cocaine77,78 and the protein target thrombin.79–81 The first report to use intrinsic SERS of the aptamer/target complex was published in 2009 and investigated both a small molecule and protein system, cocaine and platelet-derived growth factor, respectively. SERS spectral changes were observed in the single aptamer monolayers (SAM) upon target incubation. Interactions between SAMs and non-specific control molecules were measured as well. A calculated correlation function was used to quantify the conformational changes observed in the aptamers’ spectra upon target binding. The authors posited that changes in SERS spectra were due largely to changes in aptamer confirmation upon target binding and quantification of spectral heterogenicity (referencing the differences of spectra from different spots) was used to assess aptamer specificity.82

Assignments of Aptamer Affinity Agent Spectra


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In the work discussed above, the authors noted that the adenine ring breathing mode at 735 cm-1 shift was the largest feature in the SERS spectra of both aptamers.82 Assignment of an aptamer affinity agent’s SERS spectrum is relatively simple using SERS literature for DNA and DNA bases, and is useful for rationalizing the spectral changes observed upon target capture, as exemplified by Cottat et al. In this 2015 work, aptamer-based SERS sensors were reported for manganese superoxide dismutase (MnSOD), a biomarker for liver diseases and cancer. Strong aptamer vibrations of the C−O stretch in deoxyribose (1000 cm-1 shift), thymine ring-CH3 stretching (1234 cm-1 shift), and thymine/guanine (1380 cm-1 shift) were assigned. Less intense peaks were also assigned: adenine (715, 1556 and 1580 cm-1 shift), sugar vibration (850 cm-1 shift), PO2 backbone (1075 and 1125 cm-1 shift), sugar vibration (850 cm-1 shift), thymine (1234 and 1450 cm-1 shift), cytosine (1301 and 1450 cm-1 shift), and guanine (1580 cm-1 shift). Using these assignments, a nM LOD for the protein MnSOD was determined by identifying changes in the 1490-1679 cm-1 shift region from aromatic amino acids (Tyr, Phe), amide II, and amide I (Figure 7). MnSOD was detected in both serum and saliva, and the method was validated against a bovine serum albumin (BSA) negative control. 83


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Figure 7. (A) Adapted and reprinted from Ref 75 (permission requested). Bottom SERS spectrum shows the spectral features arising from the functionalization layer, predominantly DNA, with little input from the blocking agent 6-mercapto-1-hexanol (MOH). Top spectrum shows after MnSOD (10−7 M) exposure, new peaks are observed due to the protein presence (highlighted by the dashed boxes). (B) Structure of MnSOD (human manganese superoxide dismutase); image from the RCSB PDB (www.rcsb.org) of PDB ID 2ADQ.84 Aptamer-Enabled Detection of Small Molecule Targets by Intrinsic SERS High affinity from ssDNA enfolding and easy spectral assignment of the aptamer affinity agents have led to the aptamer-enabled SERS detection of many small molecules relevant to health and safety. Herein, we review those papers that detect small molecules using the intrinsic target signal or that from target/aptamer complex. For example, using an aptamer’s conformational changes upon drug exposure, a SERS aptasensor selective for the anti-cancer drug coralyne was developed. Coralyne was detected at concentrations as low as 0.1 µM with a dynamic range of 0.1-100 µM via an adenine vibration at 1376 cm-1 shift that appeared upon


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incubation with the drug. Specificity was validated with negative controls of similarly structured molecules berberine, pamatine, and neomycin.85 In addition to the monitoring of helpful therapeutics, SERS can be used to monitor harmful pollutants. Polychlorinated biphenyls (PCBs) are a varied class of pollutants that require monitoring due to their high toxicity and bioaccumulation. PCB-77 (3,3′,4,4′tetrachlorobiphenyl) has been the target of several aptamer-enabled SERS studies.86–88 One report demonstrated that use of a polythymine (poly T) base can enhance sensitivity, due to poly T’s low affinity for the metal surface of the core-shell silica/gold (SiO2@Au) substrate and decreased SERS signal compared to adenine and guanine.86 More recent work has decreased the LOD of aptamer-enabled SERS of PCB-77 to 1.0x10-8 M87 and demonstrated detection in complex matrices (real lake water, 3.3x10-7 M PCB-77).88 All three studies demonstrated good selectivity of the aptamer for PCB-77 over other PCBs. Another class of molecules that are considered pollutants and food contaminants are pesticides. In the 2012 report of aptamerenabled SERS detection of the pesticide malathion, characteristic peaks of malathion (e.g. 495 cm- 1 shift assigned to P-S stretching) were used for detection, though peaks of DNA aptamer were present as well. Without the aptamer, no signal was observed from SERS substrates exposed to malathion.89 Two years later, four other pesticides (isocarbophos, omethoate, phorate, and profenofos) were detected by SERS using specific aptamers. This work demonstrated optimization of the aptamer layer, and target exposure conditions. As each pesticide had its own aptamer, multiplexed discrimination of the four was possible.90 Besides pesticides, other small molecule contaminants in food require monitoring. Mycotoxins, such as ochratoxin A (OTA), are toxic small molecules released from fungi that can infect food and feedstocks. Two studies on the aptamer-enabled SERS detection of OTA


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reported OTA-induced aptamer spectral changes.91,92 Using orthogonal projection on latent structure analyses (OPLS), a processing method for multivariate data such as SERS spectra, OTA was detected at concentrations as low as the pM range. OPLS was also able to distinguish BSA protein and vomitoxin, another mycotoxin, from OTA.92 The toxic small molecule food adulterant melamine, which can be added to food to increase its apparent protein content, has also been detected by SERS via an aptamer that was stabilizing silver nanoparticles (Ag NP). Upon incubation with melamine, the aptamer released the Ag NP, bound melamine, and aggregation of Ag NP occurred and scattering at 240 cm-1 shift was increased. The SERS signal was weak in absence of melamine, even from clusters caused by other aggregation agents, and this method led to the detection of 25.5 µg/L melamine spiked in milk.93

Aptamer-Enabled Detection of Protein & RNA Targets by Intrinsic SERS The importance of the enzymatic protein thrombin to the regulation of blood clot formation has made it one of the most studied protein targets. The thrombin binding aptamer (TBA) is a well-studied aptamer that can be used therapeutically to prevent blood clots or as an affinity agent in sensing platforms. Two reports of unlabeled thrombin detected by SERS used TBA and demonstrated new protein vibrations (~1085, 1140, and 1550 cm-1 shift, ± 10 cm-1 between the two reports, both which showed specificity over BSA).94,95 Thrombin was detected as low at 0.1 fM, and sensor regeneration was possible with several washing and melting cycles to allow detection of fresh thrombin.94 Another protein of clinical importance detected using SERS and aptamers is an influenza viral nucleoprotein. Both aptamer peaks (changing conformation) as well as peaks from the nucleoprotein itself were detected, analyzed by dendrogram generations and PCA, and were found to be different from a virus control.96 A


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similar aptasensor was later developed by the authors to bind viral RNA strains encoding for a protein mutation associated with increased virulence. Multivariate analysis could distinguish between RNA with and without the mutation, without amplification of the RNA target.97 The protein ricin, a bioterror agent, has been the target in multiple aptamer-enabled SERS reports.98–100 The earliest report used an aptamer (SSRA1) developed for the B-chain of ricin and characterized the aptamer’s thermal stability, as well as the ability of the aptamer to bin ricin Bchain (RBC). The LOD of the SERS aptasenor was reported to be 25 ng/mL for ricin in phosphate buffered saline (PBS), and RBC was successfully detected in apple juice, orange juice, lemonade, and milk.98 A second report lowered the LOD of RBC in PBS to 10 ng/mL and increased the speed of analysis, enabling RBC “yes/no” detection in PBS, orange juice, and milk in less than 40 minutes.99 The third platform to study ricin intrinsically investigated its detection in blood. The LOD was significantly higher than the previous platforms (1 µg/mL) but the aptamer was stable and sensitive to RBC after 10 days in blood.100 All three studies demonstrated spectral changes between 550-640 and 930-1000 cm-1 shift upon aptasenor incubation with RBC.98–100

Aptamer-Enabled Detection of Microorganism Targets by Intrinsic SERS Like ricin protein (discussed above), Bacillus anthracis is a bacterium that has be classified as a bioterror agent based on its high toxicity. The bacteria spores can survive food processing steps and are thus a concern to food safety. Using a SERS aptasensor, spores were detected as low as 104 colony forming units (CFU) (estimated lethal dose by ingestion: 106 CFU). The aptamer also captured spores from a different bacteria, B. mycoides, but the spores’ SERS spectra were distinguishable by PCA so differentiation was possible.101


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Full bacteria have also been detected by SERS with aptamer affinity agents. Aptamers for the bacteria Salmonella typhimurium102 and Staphylococcus aureusis103 have enabled their detection and differentiation from negative control bacteria. For S. typhimurium, PCA showed good separation between S. typhimurium and all controls, and an increase at 725 cm-1 shift was quantifiably different from other bacteria at 108 colony forming units (CFU) of S. typhimurium.102 In the platform used to detect S. aureusis, a bacteria sample was first incubated with S. aureusis-specific aptamer to form bacteria-bound aptamers, which were then attached to Ag NPs. The SERS signal of the aptamer is greatly enhanced because one bacteria bound many aptamers and Ag NPs. A linear relationship between S. aureusis concentration and SERS intensity at 735 cm-1 shift was observed between 101 to 107 CFU/mL, and the LOD was reported to be 1.5 CFU/mL. From the spectra, one could visually distinguish S. aureusis from a mixture that also contained Listeria monocytogenes, Shigella flexneri, and to Escherichia coli O157:H7, eliminating the need for PCA or other similar analyses.103 For intrinsic SERS sensors, aptamers have been used more than antibodies as affinity agents for small molecules, biomacromolecules, and microorganisms. Aptamers’ smaller size, DNA Raman fingerprint, and tunable affinity are advantages that enable better direct detection of the targets. It should be noted that aptamer affinity for small molecule targets may be decreased compared to other targets. This is because covalent attachment of these targets to SELEX matrix can sterically hinder aptamer folding around the target.104 Small molecule targets may also be detected more easily using affinity agents with smaller or sharper SERS signals.

Small Molecule Affinity Agents


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Partition Layers, Functional Monolayers, Attachment, Affinity and Benefits Two types of small molecule affinity agents will be discussed below: partition layers and functional monolayers. In most of the papers discussed herein, both types are used to form selfassembled monolayers (SAMs) on SERS substrates prior to target exposure. These SAMs are formed by chemisorption of the monolayer molecules via a functional group with surface affinity. The most common surface “binding” moieties in this collection are thiols, disulfides, and other sulfur species to gold or silver substrates. SAM formation is driven by the strength of the surface binding moiety and the lower free energy associated with tight packing, enabled by long assembly times. Control of these factors can result in ordered monolayers and good surface coverage, if affinity agent concentration and time for monolayer formation are adequate.105 All target/affinity agent complexes exist on a continuum of molecular interactions, and here we distinguish between small molecule affinity agents that are governed primarily by solution driving forces (partition layers) and those that result from specific interactions such as covalent bonding, H-bonding, ionic, polar, nonpolar, and sterics (functional monolayers), as illustrated in Figure 8. In a qualitative sense, the “affinity” of a partition layer could be defined by the partition coefficient, or the propensity of the analyte to be in one layer of a biphasic system. A partition layer need not always be a perfect polarity match for the analyte of interest. Because the two phases are connected, diffusion will enable the transport of some analyte into any partition layer in which it is at all “soluble”. For the functional monolayers, the affinity is a function of the type of interaction between the target and affinity agent. The metal/functional monolayer systems used for intrinsic SERS have moderate conditional formation constants (Kf’), ~10-105 pH 3-6, compared to Cd/EDTA, 105-1011 over the same pH range. For comparison, bisboronic


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acids, small molecules that bind glucose, have KD of 10-5-10-4 M,106 demonstrating weaker affinity than those seen with bio-based affinity agents.

Figure 8. Comparing the capture of an analyte (green star) by the two most common types of small molecule affinity agents: partition layers and functional monolayers. The first localizes target near the surface solution driving forces and the latter binds the target from more specific interactions such as covalent bonding, H-bonding, ionic, polar, nonpolar, and sterics. Though the affinity for small molecule capture agents is moderate compared to their biobased counter parts, this class of affinity agents presents some distinct advantages. Partition layers have low specificity and can therefore easy be employed in multiplex detection. The small molecular size of both partition and functional monolayer affinity agents keeps targets within the more enhancing regions of SERS substrates. Large, analyte-specific signals are often observed. Because of the uniformity of the small molecule affinity agent monolayers on the SERS surfaces, the affinity agents’ spectra have sharp, easy-to-assign peaks, and changes due to analyte binding are easily observed. This sensitivity of the affinity agent spectra enables the detection of organic and metal ions that are not easily detected with other affinity agents. Lastly, many of these small molecule affinity agents are significantly cheaper than their bio-based counter parts.

SERS with Small Molecule Affinity Agents


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First Uses The first report of modifying a SERS substrate with an analyte-specific partition layer was published in 1992 and reported a coating of roughened silver with hydrophobic octadecylthiol to enable the capture of organics from water. The authors denoted the coating was used to (1) preconcentrate the sample at the surface, (2) serve as an internal standard. The authors motivate the use of partition layers over their previously reported ion indicators because of the observed increased stability in water samples. The partition layer system was able to distinguish between isomers of xylene and detect aromatic organic contaminants in water achieving a LODbenzene = 7.5 ppm, and a LODnaphthalene = 2.3 ppm.107 Three years later, the first “functional” monolayer was used to enable specific target detection. A disulfide-modified 4- (2-pyridylazo) resorcinol (PARD) was hypothesized to coordinate differently with various heavy metals, and thus, metal specific-spectra could be obtained. The effect of pH on complex formation was studied, and high pH was found to be more favorable for complex formation. The LODs of Pb2+, Cd2+, and Cu2+ at the highest pH accessible before the precipitation of metal hydroxides (pH 6) were 522, 50.3, and 1.49 ppb, respectively.108 It should be noted that both types of small molecule affinity agents were used for multiplexed detection from the first experiments. Compared to those of antibodies and aptamers, the lower order of the molecular interactions between small molecule and target decreases target specificity, which enables multiple target interactions. In these systems, there are ways to tune the small molecule affinity agents, such as pH adjustment, to optimize the signal from a target. Thus, due to the subtle changes seen from target-to-target or condition-to-condition, it is extremely important to thoroughly characterize the inherent SERS spectra of the affinity agent.


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Assignments of Small Molecule Affinity Agent Spectra The assignment of the octadecylthiol layer that formed the partition layer in the work of Carron et al. focused on the C-C stretches and methylene twist. Strong sharp peaks from 10501150 cm-1 shift are seen from C-C stretches that correspond to all-trans configuration, denoting the high degree of order. Upon introduction of benzene into the layer, a shoulder indicating some gauche character (1063 cm-1 shift) was observed in addition to the strong benzene vibration near ~990 cm-1 shift (Figure 9, A & B). This change, combined with the changes seen in the methylene region of the spectrum, led the authors to conclude that the spectrum demonstrated a lower degree of order in the partition layer when target was introduced.107 The simple, welloriented molecules of the partition layer can provide a lot of spectral information and are sensitive to target intercalation. Later studies used the easy assignment of the alkylthiol partition layers to determine the integrity and stability of the partition layer, the effect of alkylthiol chain length on SERS target sensitivity, and the impact of analyte solution polarity (and thus target partitioning) on signal.109,110 Full affinity agent spectral characterization is vital for the functional monolayers used to capture small molecule targets with low SERS scattering, like glucose, and targets that do not generate SERS spectra at all, like metal ions. Complete assignment, as tabulated in the work of Crane et al., enables differentiation of chemical species captured by small molecule functional monolayers. Crane et al. could discern that Pb ions interact most strongly with the pyridyl nitrogen lone pair based on Pb-induced shifts from 951 and 1005 cm-1 shift to 969 and 1023 cm-1 shift, respectively (Figure 9, C & D). Thus, Pb could be differentiated from Cd which effected the peak from C-N-N=C stretch + N-H bend (1286 cm-1 shift), and Cu which effected the peak from (C-C)res stretch + C-N-N=C stretch (1380 cm-1 shift).108


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Figure 9. Adapted and reprinted from Ref 99 (A&B) and 100 (C&D) (permission requested), SERS spectrum of (A) an octadecylthiol partition layer on a roughened silver surface, the center band at 1101 cm-1 shift (arrow) was used as an internal reference; (B) benzene adsorbed into an octadecylthiol partition layer on silver, the shoulder at 1063 cm-1 shift (arrow, trans C-C stretch) corresponds to the formation of a gauche component of the monolayer upon introduction of benzene; (C) a PARDS monolayer on silver in a pH 5.0 solution, 1023 cm-1 shift corresponds to the pyridyl vibration (arrow); (D) a PARDS monolayer on silver in a pH 5.0 solution with 0.01 M Pb2+, the 1023 cm-1 shift (right arrow) peak has shifted to 1005 cm-1 shift (left arrow). Below we review work that uses small molecule affinity agents as partition layers or functional monolayers. Special targets of interest in these systems are those that cannot be detected by larger affinity due to the relatively broad affinity agent signals obscuring spectral


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features of the targets. Therefore, targets probed by small molecule affinity agents include metal ions and small molecules.

Small molecule-Enabled Detection of Small Molecule Targets by Intrinsic SERS Partition layer affinity agents have been used to concentrate organic pollutants for low level detection and enable the detection of glucose at physiologically relevant concentrations. In addition to the smaller aromatic hydrocarbons discussed above,107,109–111 polychlorinated biphenyls (PCBs)112 and polycyclic aromatic hydrocarbons (PAHs),113,114 have been detected using partition layer-functionalized SERS substrates. For PCBs112 and PAHs113 captured by decanethiol partitions layers on silver substrates, the platform proved fast ( benzene.152 In 2015 a novel strategy utilized the self-assembling block copolymers as an affinity agent in proof-of-concept work detailing the SERS detection of dye molecules. The assembly of poly(styrene)-block-poly(acrylic acid) (PS-b-PAA) was oriented around gold nanostars which had been previously functionalized with thiol-terminated PS. During self-assembly, magnetic iron oxide nanocrystals, stabilized by oleic acid, were present and incorporated into the polymer shell. Clustering of the assembled particles was induced by external magnetic stimuli and resulted in a large SERS signal increase, likely due to formation of EM hot spots. The PAA groups on the nanostars’ outer shell demonstrated affinity for oppositely charged dyes, and repulsed dyes with a negative charge. Detection limits of dyes studied were between 5-10 nM.150


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Polymer-coated nanostars were also used to detect PAHs. A crosslinked polymer affinity agent was used to stabilize the polymer layer around the core153 instead of relying on hydrophobic interactions as seen with PS-b-PAA.150 Unlike MIPs, no target templates were used to create binding sites. Functioning much like the small molecule partition layers previously discussed, crosslinked poly(N-isopropylacrylamide) (pNIPAM) successfully captured PAH molecules from the gas phase, facilitating their detection on dry SERS arrays.153 For non-imprinted polymer (NIP) affinity agents, low affinity and potential for large polymer signals, have limited their use in the detection of small molecules. However, as noted in several systems,150–153,159 the low affinity enables multiplex detection and differentiation of targets via SERS. Despite the attraction of multiplexibility, a larger number of small molecule targets have been detected using platforms with the more selective polymer affinity agent, MIPs. MIP affinity agents have been used to detect small molecule targets such as pharmaceutical drugs, nicotine, TNT, and BPA. While most of the studies focused on the how MIP could improve SERS detection of analytes, the work of Kantraovich et al. emphasized how SERS could be employed to monitor MIP preparation. The SERS signal of the MIP and that of the template/target propranolol (a pharmaceutical β-blocker) were used to characterize template elution, template rebinding, and MIP array performance.161,162 Using similar MIP precursors (methacrylic acid (MAA) monomer, and an ethylene glycol (EG) difunctional crosslinker) and the same propranolol target, later work explored the system as a sensor. Employing Au NPs@MIP nanocomposites, Bompart et al. were able to their platform to detect the β-blocker propranolol at 10-7 M, showed specificity for the β-blocker over caffeine and acetylsalicylic acid, and was stable in equine serum.163 The detection of antibiotics in food matrices (milk, honey,164 and pig serum165) has utilized MIP-target affinity in two-step processes. First the target antibiotic


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is extracted from the complex samples and eluted into clean solvent. This “clean” solution was then deposited on a SERS substrate and measured. Chloramphenicol (CAP) was detected as low as 0.1 ppm in whole milk in

Molecular Affinity Agents for Intrinsic Surface-Enhanced Raman

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