Advances in Biofunctional SERS-Active Nanoparticles for Future

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Chapter 7

Advances in Biofunctional SERS-Active Nanoparticles for Future Clinical Diagnostics and Therapeutics Steven Asiala,† Lee Barrett,† Samuel Mabbott,† and Duncan Graham* Centre for Molecular Nanometrology, WestCHEM, Pure and Applied Chemistry, University of Strathclyde, Technology and Innovation Centre, 99 George Street, Glasgow, United Kingdom, G1 1RD †These authors contributed equally to the manuscript. *E-mail: [email protected]

The synergy afforded by the combination of biofunctionalised nanoparticles and surface enhanced Raman scattering (SERS) has expanded the analytical toolbox for clinical diagnostics and therapeutics. Since their inception, SERS-active nanoparticles have been developed into biofunctional nanoparticles (BFNPs) using a variety of methods to attach biomolecules and pacification layers to nanoparticles to enable detection of various diseases or cancers in vitro and in vivo. However, while there are many reports of the use of BFNPs for diagnostic or therapeutic applications, very few are implemented in a “real” clinical setting, for example, detection of disease biomarkers in tissue or the delivery of drugs to affected cells. This review covers recent advances made in the development of BFNPs for SERS-based detection of clinical samples using in vitro and in vivo methods.

© 2016 American Chemical Society Ozaki et al.; Frontiers of Plasmon Enhanced Spectroscopy Volume 1 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


Introduction In the ongoing fight against diseases, the need for novel, rapid tools for diagnosis to improve treatment is omnipresent. Much attention is being paid to developing methods and technologies aimed at providing clear and accurate information to clinicians, with the larger goal of improving patient outcomes through stratified medicine. One area of research that has been mined for its potential to aid in clinical settings is vibrational spectroscopy (1). Vibrational spectroscopies have been historically used to identify functional groups present in a given molecule due to their “fingerprint” spectra, but more recently to detect and identify biomarkers linked to a number of diseased states. The most commonly used forms of vibrational spectroscopy are infrared (IR) (2, 3) and Raman (4–6), with each applied to a wide range of biomedical applications (1, 7). Furthermore, nonlinear vibrational spectroscopies that utilize multi-photon, pump-probe arrangements, such as coherent anti-Stokes Raman (CARS) (8) and stimulated Raman (SRS) (9, 10) have been developed with an eye toward biomedical imaging and clinical application (11, 12). Concurrent with advances in vibrational spectroscopy methods has been the advancement of nanomaterials for biomedical applications, including imaging, photothermal therapy (PT), drug delivery, and sensing (13). Nanomaterials, such as metallic nanoparticles, quantum dots, and carbon-based materials have physical and chemical properties that differ from the bulk and can be exploited for a specific biomedical or clinical task. More specifically, plasmonic metal nanoparticles have been of great service in the realms of vibrational spectroscopy and imaging due to their ability to drastically increase the sensitivity of Raman spectroscopy. Surfaceenhanced Raman Spectroscopy (SERS) (14) has brought Raman into the realm of ultrasensitive techniques, with reports of single-molecule detection (15, 16). The prospects of using SERS for detection in clinical applications (17–19), including cancer detection and imaging (20) have been the subject of recent reviews (21). In addition to increased Raman sensitivity, another important asset of nanomaterials is the ability to tailor their surface chemistry (22). Nanoparticles can be functionalised with beacons, or reporter molecules (23–25), for tracking and detection, encapsulated for anti-fouling, stabilization and pacification in biological environments (26, 27), and targeted for specific biomarkers with the addition of antibodies (28), DNA (29), peptides (30), and aptamers (31). When each of these four elements is present— a plasmonic metal nanoparticle, reporter, pacification layer, and biomolecule—a biofunctional nanoprobe (BFNP) is created as depicted in Figure 1. This strict definition of a BFNP will be used as a guide for the work reviewed herein. The goal of this review is to highlight the most recent progress made toward the utilization of BFNPs for vibrational spectroscopy in clinical applications. This will include a brief review of especially relevant plasmonic nanoparticle properties and functionalization strategies, followed by a survey of the use of BFNPs for in vitro and in vivo diagnostics and therapeutics.

132 Ozaki et al.; Frontiers of Plasmon Enhanced Spectroscopy Volume 1 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


Figure 1. A cartoon representation of the criteria for biofunctional nanoparticles (BFNP) for clinical applications. BFNPs consist of a plasmonic metal nanoparticle, a reporter molecule, an encapsulation or pacification layer, and a biomolecule for specific targeting. Shown are common particles, reporters, encapsulation materials, and biomolecules. Bovine serum albumin structure from PDB reference 4F5S (32).

Plasmonic Nanoparticles Nanoparticles bridge the gap between bulk material and molecular structure (33) offering unique properties which make them appealing to the biosciences, especially in the area of diagnostics and therapeutics. Nanoparticles are often defined as having dimensions ranging from 1 to 100 nm. Particles synthesised on this scale have found use across both physical and biological sciences due to properties such as increased surface area and the ability to exchange energy from light to heat. However, one phenomenon that makes plasmonic nanoparticles appealing to physicists and spectroscopists alike is the confinement of electrons on their surface. The sinusoidal oscillation of electrons on the surface of the single metallic particles is termed a localised surface plasmon. To take advantage of increased field effects, nanoparticles have to be subjected to a resonant frequency. UVVis spectrophotometry is one analytical technique that can identify λmax of the particles at which both absorption and scattering is at its maximum; this frequency is often termed the local surface plasmon resonance (LSPR). It is the LSPR that is responsible for the enhancement seen in surface enhanced Raman scattering (SERS) when metallic nanoparticles are used. Many reviews have been written on SERS (34, 35) and its initial observation (36), therefore it is beyond the scope of this review to fully explain the intricacies of the technique. Instead, only a brief discussion of the mechanisms will be given with further sections, focusing on the use of nanoparticles to enable the method for in vitro and in vivo biological studies.

133 Ozaki et al.; Frontiers of Plasmon Enhanced Spectroscopy Volume 1 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


Electromagnetic Effect As mentioned previously, the tuning of light to the LSPR of nanoparticles causes an increase in electric field intensity as a consequence of surface plasmon excitation. This increase in the electric field is extended perpendicularly to the particle surface, causing it to interact with any molecules in close proximity. The molecules become bathed in a freely moving electron cloud, causing them to become polarized. It is the interaction of these electrons with the molecule that gives rise to intense Raman scattering effects. This mechanism contributes an enhancement factor of around 106, although this is estimated to be much less for a single particle than for a multi-nanoparticle system. Estimations of the increased electric field can be quantified using generalised Mie theory (GMT), and, although mathematically complex, the theory is explained exceptionally well by Stockman et al. (37) Chemical Effect Whilst electromagnetic enhancement is accountable for the greatest contribution to SERS enhancement, it alone cannot justify the high level of enhancement seen in many systems. The chemical mechanism of enhancement occurs when a physical bond is formed between the nanoparticle and the analyte. This effect is often interpreted as a HOMO and LUMO interaction by which charge can be transferred between the particles and analyte. SERS can also be facilitated on a range of two- and three-dimensional nanostructured metallic substrates (38–40), but the mobility of nanoparticles is paramount to their in vivo and in vitro application. Therefore, only nanoparticles will be discussed in this review and in particular gold and silver nanoparticles as these are the most commonly used in SERS.

General Nanoparticles Synthesis Gold nanoparticles have been used throughout history most commonly to adorn decorative objects such as stained glass. It was not until 1857 that the first scientific evaluation of colloidal gold was made by Michael Faraday. Synthesis of colloidal gold was achieved by reduction of ‘a moderately strong solution of chloride of gold’ using phosphorus (41). It was the development of the colloidal suspension that led to the discovery of the Faraday-Tyndall effect, whereby longer-wavelength light is transmitted whilst shorter wavelengths become more reflected via scattering. Most interesting, however, is that Faraday’s particles have remained stable for over 150 years, whilst modern researchers can often only maintain optical stability for a number of months. Two primary techniques can be used for the synthesis of gold and silver nanoparticles. Physical methods, which use processes such as thermal decomposition and laser ablation have been shown (42–49), but more often chemical methods are employed, where a chemical species is used to reduce metallic salts. It is a common theme that strong reducing agents produce small nanoparticles whilst nanoparticles reduced using weaker reagents are bigger. 134 Ozaki et al.; Frontiers of Plasmon Enhanced Spectroscopy Volume 1 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


Examples of reducing agents include NaBH4 (50, 51), hydroquinone (52, 53), ascorbic acid (54, 55) and, of course, the most commonly used citrate, which will be discussed in greater depth in future sections. In a move to incorporate the principles of green chemistry, it has also become increasingly popular to use natural products in the synthesis of nanoparticles, utilizing extracts from a variety of plant leaves, roots and tissues as reducing agents (56–58). Once synthesised, the maintenance of colloidal stability is paramount to ensure the longevity of the particles. Reagents used for stabilising the nanoparticles can be placed into two categories; electrostatic or steric. In electrostatic stabilisation, a charged species is often used to interact with the nanoparticle, whilst simultaneously repelling other nanoparticles. When steric stabilisation is carried out, bulky chemical species such as polyethylene glycol (PEG) (59–62), bovine serum albumin (63), or chitosan polysaccharide (64–67), are used but many more species are also available (27). It should be noted that some of the chemicals mentioned such as citrate and chitosan act as both a reducing and stabilising reagent. Nanoparticle: Elemental Composition Nanoparticles can and have been synthesised using a variety of metals, but it is important to remember that for SERS to occur, the LSPR of the metals must be excited across the UV-Vis-NIR region. A few papers report the use of Pt, Pd, Ru, Rh and transition metals such as Ni for SERS substrates, but many of these are non-nanoparticle based approaches, using mostly electrodes as the active interface (68–79). Copper nanoparticles (CuNPs) are, however, well mentioned in the literature. Whilst chemical reduction is the general route for nanoparticles synthesis, Muniz-Miranda et al. showed that CuNPs with a broad size range (majority 3-9 nm) and a LSPR band around 588 nm could be synthesised via laser ablation using a 1064 nm laser (80). SERS enhancement of both Phen and Bipy were achieved using by coupling the CuNPs to lasers with 532 nm and 785 nm excitations. Silicon-Hydrogen bond assisted assemblies of copper nanoparticles have also been proven capable of enhancements of 2.29 x107 and a RSD 25 UmL-1) with a commercially available ELISA kit. In the lower concentration range ( 25 UmL-1). However, it was found that the SERS immunoassay showed greater reproducibility in the negative anti-CCP group (n=43, 48 h) than controls (

Advances in Biofunctional SERS-Active Nanoparticles for Future

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