Surface-Enhanced Raman Spectroscopy Biosensing: In Vivo

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Perspective

SERS biosensing: in vivo diagnostics and multimodal imaging Anne-Isabelle Henry, Bhavya Sharma, M. Fernanda Cardinal, Dmitry Kurouski, and Richard P. Van Duyne Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b01597 • Publication Date (Web): 06 Jun 2016 Downloaded from http://pubs.acs.org on June 8, 2016

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Analytical Chemistry

SERS biosensing: in vivo diagnostics and multimodal imaging. Anne-Isabelle Henry, Bhavya Sharma†, M. Fernanda Cardinal, Dmitry Kurouskiffi, and Richard P. Van Duyne* Northwestern University, Department of Chemistry, 2145 Sheridan Road, Evanston, Illinois 60208, United States KEYWORDS Surface-enhanced Raman spectroscopy; SERS; biosensing; bioimaging; nanomedicine; plasmonics. ABSTRACT: This perspective presents recent developments in the application of surface-enhanced Raman spectroscopy (SERS) to biosensing, with a focus on in vivo diagnostics. We describe the concepts and methodologies developed to date and the target analytes that can be detected. We also discuss how SERS has evolved from a „point-and-shoot‟ stand-alone technique in an analytical chemistry laboratory to an integrated quantitative analytical tool for multimodal imaging diagnostics. Finally, we offer a guide to the future of SERS in the context of clinical diagnostics.

The detection, identification, and quantitative analysis of biomarkers at very low concentrations for presymptomatic diagnosis represents a new frontier in biomedical research, enabled by nanomedicine1-5 and powered by (bio)analytical chemistry.6,7 Detecting diseases earlier simply means saving or improving patients‟ lives. This is especially critical in the case of cancers, for which the efficiency of already existing treatments is significantly improved when administered in the early stages of the disease. It is therefore straightforward to understand that highly sensitive, quantitative diagnostics are critical and vital. Additionally, the management of chronic diseases such as diabetes can also tremendously benefit from sensitive, accurate and quick diagnostic solutions for continuous glucose levels measurements. Yet, few molecular events and hence low quantities of markers available make diagnosis challenging. Biosensors with nanoscale dimensions, such as nanoparticles (NPs) and nanostructured surfaces, offer exciting prospects as they can act as signal transducers8 and ultimately offer a means to probe and quantify bioanalytes, such as disease-related biomarkers, in very small concentrations (nano- to zeptomolar).9 Plasmonic nanoplatforms such as gold - the material of choice in biomedical nanotechnology10 - and silver NPs and nanostructured surfaces, exhibit unique optical properties due to their intrinsic dielectric function, nanoscale dimensions and the oscillating nature of light. In this size regime (~10-100s nm), surface effects become prominent, such that electrons from the metal and photons from the incident light couple into a quasi-particle called a surface polariton. This quasi-particle oscillates at a frequency that is referred to as the localized surface plasmon resonance (LSPR), with two important conse-

quences on the properties of the nanoplatform: the wavelength dependence and the enhanced electromagnetic field. First, the LSPR wavelength-dependence in the visible-NIR region of the electromagnetic spectrum dictates that the plasmonic nanoplatform intrinsically possesses a specific color. This is the rationale for colorimetric LSPR-based assays, which can be qualitative or quantitative. Second, the high electromagnetic field at a nanoplatform surface provides the opportunity to couple the molecular vibrations of an analyte nearby (on or up to ~ 3 nm away from the surface)11 to the LSPR, resulting in a massive increase (106-108) of the molecular signal intensity. This phenomenon – called surfaceenhanced Raman scattering – enables the use of plasmonic nanoplatforms as a Raman signal amplifier. Raman scattering, which arises from the inelastic interaction of light with matter, is intrinsically a molecularly specific tool. However, Raman spectroscopy has poor sensitivity, as only 1 in 108 photons is Raman scattered. By using plasmonic nanoplatforms as Raman signal amplifiers, surface-enhanced Raman spectroscopy (SERS) is an ultrasensitive spectroscopic tool. The focus of this perspective is on the contribution of surface-enhanced Raman spectroscopy (SERS) towards the goals outlined above using molecular sensing for in vivo diagnostics. Since its inception as a spectroelectrochemistry analytical tool in the late 1970s,12-14 SERS has evolved as the technique of choice for any scientist seeking to access both molecular specificity and sensitivity.15 The two intrinsic and distinctive advantages of SERS compared to other (bio)analytical techniques are i) the bar-code like reading that comes from unique and narrow vibrational bands in the Raman spectrum, and ii) its exquisite sensitivity, down to single molecules16,17 in the

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most ideal case (resonant molecules with large Raman cross-sections). The experimental set-up for a SERS experiment and SERS-based diagnostics is the same as a „normal‟ Raman experiment, as the sensitivity brought by SERS is provided by the nanoplatform to which the target analyte(s) bind(s).15 As such, technical developments in Raman spectroscopy have directly benefited the field of SERS. For example, the range of accessible wavelengths now encompasses the whole visible spectrum (400-750 nm) to the NIR (750-1064 nm), which includes the biological optical window (~600-1200 nm) for throughtissue analysis. SERS has evolved into a field that includes a wealth of experiments at room temperature and inside living cells. The fast growth of SERS has resulted from its ability to probe either one NP at a time,18,19 or several, through imaging (both in vitro and in vivo) and remote sensing (through spatially-offset detection, described in section III). An abundant literature can be found on SERS substrates;20-22 SERS applied to biosensing,23-25 biomolecules,26 probing biofluids,27 medical problems28 and cancer detection29,30 in particular. This perspective summarizes the field of in vitro and in vivo molecular sensing for diagnostics by SERS. While we cover a significant portion of the literature on SERS biosensing we are not able to present here a fully comprehensive literature review of the topic within the mandated length of a perspective. Within this paper we point the readers to in-depth, topical and critical reviews and research articles recently published. With this in mind, we wish to expose the readers to the current most exciting developments in SERS biosensing and bring insight on where we envision this field to be headed, as discussed in the Outlook section. IN VIVO SERS FOR DIAGNOSTICS Origins of cancer detection by Raman spectroscopy. Before discussing the impact of SERS on in vivo detection of disease, we briefly introduce recent advances in the use of normal Raman spectroscopy for cancer detection as it importantly demonstrated the feasibility of Raman-enabled biosensing. Among the earliest applications of normal Raman spectroscopy for cancer detection is examining differences in the plasma membranes of normal and neoplastic lymphocytes transformed by simian virus 40.31,32 It was found that the neoplastic cells had a greater degree of amidation of the asparagine and glutamine residues of the membrane,31 and were more sensitive to changes in temperature32 than normal cells. Since then, normal Raman spectroscopy has been characterized as a tool for cancer detection that can quickly assess the presence of cancer biomarkers and aid in the evaluation of the different stages of cancer.33 In general, visible to NIR wavelengths are used both in vitro and particularly in vivo, since these wavelengths have a

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greater penetration depth in tissues. Cancers including brain,34,35 esophageal,36 skin,37 breast,38 epithelial39 and gynecological cancers, to name a few, have all been examined and characterized by Raman spectroscopy and reviewed in details elsewhere.33,40-42 Most studies have found that differences in the Raman peaks for macromolecules such as cholesterol, amino acids (phenylalanine, tyrosine, tryptophane, cysteine), DNA, RNA, phospholipids, proteins, and collagen are good diagnostic indicators when comparing normal versus neoplastic tissue.33-35,37,40,41,43-45 The differences between the normal and neoplastic Raman spectra are often slight and not easily visible by eye. Additionally, some Raman spectra are also complicated by fluorescence from the tissue samples, which creates a large amount of background noise. Since the normal Raman excitation of cellular components is not enhanced (through resonance or surface-enhancement), the fluorescence background can often overwhelm the Raman signals. The use of mathematical models and/or statistical analysis, such as Fourier transforms, principal component analysis, linear discriminant analysis, cluster analysis, as well as other algorithms, to treat Raman data with large fluorescence backgrounds and elucidate the small differences in peak position or intensity has revolutionized the use of Raman spectroscopy as biological and medical tool.34,39,43-45 Although normal Raman spectroscopy has proven useful for diagnosing cancer, it is an intrinsically weak scattering phenomenon. By employing methods of

Figure 1. In vivo SERS imaging. Map of the mouse liver following injections of four types of SERS nanotags (S420, S421, S440, S470): 1 hour post-injection (left) and 2 hours post-injection (right). Reproduced from Ref. 50 (Bohndiek et al. Proc. Natl. Acad. Sci. U. S. A. 2013, 110, 1240812413) with permission from the National Academy of Sciences.


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signal enhancement such as surface-enhancement, signal intensity is often increased between 106-108 times. As discussed in the introduction, SERS offers greater sensitivity (i.e., detect smaller concentrations) and provides a higher level of selectivity than normal Raman, which can result in the earlier detection of cancer and thus, improved prognoses for patients. In vivo SERS for cancer detection. In vivo SERS imaging has been used to image tumors, guide surgical removal of tumors (particularly for elucidating tumor margins), and is also used for its multiplexing capabilities. Multiplexed detection, i.e. the ability to simultaneously detecting several markers (analytes or Raman reporters) is an advantage of SERS, especially in the context of in vivo biosensing. Most in vivo SERS imaging involves the use of SERS nanotags, NP constructs with a strong Raman active molecule adsorbed on the surface of aggregated gold NPs, which are often encapsulated in silica, polyethylene glycol (PEG) or mixed bovine serum albumin-glutaraldehyde for stability and to protect the nanotags. There are several varieties of Raman active molecules available which can be injected simultaneously,46 resulting in multiplexing capabilities, as each molecule has a distinctive spectrum. Additionally, groups are working towards expanding the library of molecules available that have strong Raman crosssections and are Raman active in the near infrared wavelength range.47,48 The SERS nanotags are employed in one of two ways: they are either injected into the animal and passively accumulate in the body, or the surface of the nanotags is functionalized with a receptor or antibody which allows them to actively target specific cells or tumors. Several in vivo studies rely on passive targeting for accumulation of nanotags in tumors, but due to the size of the nanotags (>100 nm) these latter are often taken up by the Kupffer cells of the reticuloendothelial system and accumulate primarily in the liver, where they remain for extended periods of time.46,48-50 It was also found, however, that the signal of the nanotags in the tumors can remain elevated even when the concentration in blood return to pre-injection levels.51 Most passive targeting is now used in conjunction with the development of new instrumentation for SERS imaging of small animals. The Multimodality Molecular Imaging Lab at Stanford University has led the way in both the use of SERS nanotags for surgery, as well as the development of several new instruments to improve the imaging of nanotags in vivo. Zavaleta et al.46 demonstrated multiplexing of ten nanotags and separation of their Raman spectral signatures using a commercial point mapping Raman imaging system. Soon they realized that the accumulation time necessary for point mapping is not feasible for real-time imaging modalities. This led to the development of two separate Raman imaging systems: the small animal Raman imaging (SARI) system50 and a fiber

optic probe designed to be coupled with a clinical endoscope.52 The SARI system allows for rapid Raman imaging over a relatively large area (>6 cm22) with high spatial resolution. Instead of a point by point scan system, SARI utilizes a line scan that rasters along the x and y axes using two dimensional galvanometric mirrors, allowing the sample (here a small animal) to remain stationary, and can measure to a depth of ~ 4 cm. The SARI system is approximately 10 times faster in scan rates than the point mapping system, which required up to 5 hours of scan time to cover areas of a few cm.46,50 Passive accumulation of four nanotags in the liver can be imaged by the SARI system, with two nanotags clearly present 1 hour post-injection (Figure 1, left) and all four nanotags clearly evident 2 hours post-injection (Figure 1, right).50 Building on the SARI system, Zavaleta et al. coupled a non-contact fiber optic probe-based Raman system to an endoscope.52 This system incorporates the Raman fiber probe through a clinical endoscope to provide realtime, multiplexed SERS data from tumor-targeting nanotags. SERS nanotags were imaged in human tissue to test the multiplexing capabilities, and in porcine colon tissue in a “scavenger hunt”, where the nanotag location and concentration was unknown before the imaging measurements. Three “blind” testers achieved a sensitivity of 100%. The fiber optic Raman probe was approved for testing in human patients during routine colonoscopies, but due to the need of further regulatory approval, use of the nanotags was not implemented. However, the Raman device was used to collect Raman spectra in vivo when combined with the clinical endoscope. The development of this device is greatly promising for in vivo probing of several cancers, including colon, gynecologic, and esophageal. In vivo multiplexed SERS diagnostics. Another approach for in vivo SERS imaging utilized tunable bandpass filters to scan a window of 400-1500 cm-1 at 785 nm excitation, which allows for multiplexed wide-field imaging of nanotags.53,54 The nanotags used each have distinctive SERS spectra which are easily discernible even when overlapped in multiplexing experiments. The tunable filters are used to image narrow windows in the spectral range (~65 cm-1), resulting in isolation of specific peaks from the individual nanotags while minimizing the contribution from other tags. This single peak identification allowed the speed of the wide-field imaging to be significantly faster than point-by-point measures (5 seconds per bandpass image at low pM concentrations versus 50 hours by point measures). The SERS signal of four nanotags were easily detected and separated from background fluorescence from tissue samples, to depths of 5 mm. This technology has also been demonstrated with active targeting of lung cancer using nanotags coated with epidermal growth factor receptors (EGFR)-specific antibodies54 and is also being adapted for use with an endoscope.



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Active targeting is used more often because it allows for higher specificity in the target, with less non-specific accumulation of nanotags. Several groups have demonstrated the efficiency of actively targeting tumors and the decreased biodistribution of the nanotags in other tissues of the body.47,48,55,56 In one of the first active targeting studies by Qian et al.,5555 60 nm gold NPs with malachite green adsorbed on the surface as the Raman probe molecule were functionalized with a single chain variable fragment (ScFv) antibody. The nanotags were stabilized by the addition of thiolated PEG. The ScFv recognizes EGFR, which is overexpressed in many types of cancerous cells. The authors found that the functionalized nanotags accumulated in the tumor within 4-6 hours of injection into the tail veins of nude xenograft tumor model mice and remained localized in the tumor for 24-48 hours. Additionally, they compared active versus passive accumulation and found that the active target nanotags had higher signal intensity and accumulated 10x more in the tumor than the passive nanotags. Both active and passive nanotags accumulated in the liver and spleen, but the passive nanotags accumulated in both organs to a greater degree. Samanta et al.47 targeted human epidermal growth factor receptors 2 (Her2), which are involved with cell proliferation and are known to be upregulated in breast cancer. They conjugated 60 nm nanotags (gold NPs with CyNAMLA-381 dye and stabilized with BSA mixed with glutaraldehyde for cross-linked organic encapsulation) to a ScFv antiHer2 antibody, which was then injected into the tail vein of mice with xenograft tumor model of cells that overexpress Her2. In this case, the authors measured spectra of the nanotags at the tumor site only, and found no nonspecific binding of the nanotags. Moving towards multiplexed detection with active targeting, Dinish et al.56 demonstrated detection of three cancer biomarkers in a breast cancer cell line. They utilized xenograft mouse tumor models and targeted the following biomarkers: EGFR; CD44, which is a cell surface adhesion molecule; and TGF-βII, which down regulation of increases breast cancer. The SERS nanotags consisted of PEG-passivated gold NPs with three different Raman probe molecules adsorbed to the surface (Cyanine 5, malachite green isothiocyanate, and Rhodamine 6G) that were then conjugated to antibodies against the biomarkers. Instead of tail vein injection, the study here involved direct injection into the tumor itself, to test if the nanotags remained in the tumor with decreased accumulation in other organs. Comparing a control mouse versus the tumor model mouse, the signal intensity of the nanotags increased in the 1st hour, decreased significantly by the 6th hour and by 24 hours was no longer detected in the control mouse. While in the tumor model mouse, the signal increased, reaching a maximum at 6 hours, with the signal decreasing but still detectable at 48 hours and finally no signal at 72 hours.

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All of these studies demonstrate the positive contribution of combining nanotag-targeting strategies with new instrumentation to further develop SERS as a technique for in vivo tumor detection. Spatially-offset Raman spectroscopy (SORS). One disadvantage to the aforementioned detection methods is limited in vivo depth of penetration, which results from the high tissue scattering and autofluorescence from chromophores in the tissue. A Raman-based technique that allows for increased penetration depths and suppresses fluorescence is spatially-offset Raman spectroscopy (SORS).57 In SORS, light incident on the surface of a multi-layer sample, has the surface-generated Raman scattered light travel back along the same trajectory as the incident laser beam. Some of the incident photons travel deeper into the sample (into the lower layer) and migrate laterally away from the incident laser spot. The Raman scattered photons from this lower layer can then be detected at some distance that is offset from the inci

Figure 2. SESORS-enabled detection of nanotags through animal bone. (A) Waterfall plot of SESOR spectra (x-axis: Raman shift; z-axis: SERS intensity) through varying thicknesses of bone (y-axis), with red being most intense and light blue being least intense. (B) Representative SESOR spectra taken at bone thicknesses of 3, 5, and 8 mm. For all spectra, λex = 785 nm, t = 10 s, P = 50 mW. (Reprinted with permission from Ref. 63; Sharma et al J. Am. Chem. Soc. 2013, 135, 17290-17293. Copyright 2013 American Chemical Society).dent trajectory. SORS has

been used for several in vivo studies, including probing bone through the skin and calcifications in chicken breast tissue as a precursor to human breast cancer stud-


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ies.58,59 Additionally, to further improve signal intensity and targeting capabilities, SORS has been combined with SERS, resulting in a new methodology referred to as SESORS. With the use of SERS nanotags, SERS imaging and SORS have been demonstrated depths of penetration to ~5 mm in vivo; whereas SESORS imaging has demonstrated penetration depth of 15-25 mm in porcine tissue.60 Furthermore, SESORS multiplexed 3D imaging of nanotags has also been demonstrated to depths of 45-50 mm in porcine tissue.61 Hence, SESORS combines the sensitivity of SERS with unprecedented depth of penetration for imaging in tissue. Besides turbid media, SESORS has also been demonstrated to be effective for measuring SERS spectra of nanotags on and through bone. Xie et al.62 coated bone fragments with SERS nanotags functionalized with bisphosphonate, a type of drug used in the treatment of osteoporosis. They inserted these bones fragments into 20 mm thick porcine muscle tissue and demonstrated localization of the bone tissue through the obtained SESORS spectra of the bisphosphonate-coated SERS nanotags. The ability to localize bisphosphonate indicates the suitability of SESORS in future studies of metastatic breast cancer and for osteoporosis treatment. Sharma et al.63 injected SERS nanotags into ovine tissue and used SESORS to measure for the first time spectra of nanotags through an ovine shoulder bone. Through this study, our group demonstrated measurement of spectra through bone thicknesses from 3-8 mm, as shown in Figure 2. This result is significant in the context of developing a technique for noninvasive functional imaging of the brain through the skull; as the human skull can range from 3-14 mm in thickness. Moving from proof-of-concept experiments to demonstrated in vivo studies, our group demonstrated the first in vivo, transcutaneous glucose sensing in a rat with SESORS.64 The goal of this study was to develop an implantable sensor that could be used to measure interstitial glucose levels transcutaneously. Instead of SERS nanotags, we used a SERS sensor functionalized with a self-assembled mixed monolayer capture ligand for glucose. This sensor consisted in a silver nanostructured film over silica nanospheres deposited on a titania substrate (8 mm diameter disk). This nanoplatform was implanted in the subcutaneous space under the skin to probe the interstitial fluid. Upon data analysis, a correlation between SERS intensity and glucose concentration was developed using a partial least-squares method. By comparing the results with the gold standard of glucose sensing, an electrochemical blood glucometer, the SESORS concentrations were found to match well with the glucometer readings. Building on this study, our group continued to explore the use of SESORS for

Figure 3. In vivo SESORS-enabled quantitative detection of glucose. Transcutaneous SESOR spectra from days 6 through 20, with the functionalized SERS sensor (DT/MH AgFON), skin, and post-implant spectra shown at the top for reference. (Reprinted with permission from Reference 65: Ma et al Anal. Chem. 2011, 83, 9146-9152. Copyright 2010 American Chemical Society).

transcutaneous glucose sensing.65 Again using a functionalized SERS sensor implanted in 6 rats, the SERS sensor was measured to be stable and active for more than 17 days in vivo (Figure 3).64 The sensor was remarkably consistent over the 17 days, and it demonstrated high hypoglycemic accuracy, which was even greater than the current ISO standards. Both the proof-ofconcept and in vivo studies demonstrate that SESORS holds great promise for in vivo SERS detection of various diseases, including breast cancer, bone disease, diabetes, and diseases of the brain. MULTIMODAL SENSING AND IMAGING Imaging-based diagnosis of cancer or necrotic tissues can substantially increase the precision of surgical intervention and the survival outcome of the patient. An application of a multimodal rather than single imaging technique would therefore substantially decrease the chance of tumorigenic tissue left at the resection margins of cancerous tissues and result in more accurate delineation of the margins of tumors. In the past decades, numerous new multimodal imaging (MMI) technologies based on fluorescence imaging (FI), magnetic resonance imaging (MRI), photoacoustic (PA) imaging, photothermal therapy (PTT) imaging, computed X-ray tomog-



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raphy (CT), and SERS, have been introduced in clinical settings.66-69 The physical principles of these techniques differ, consequently driving the need for different contrast agents (CAs) for each. An ideal CA would be sequestered and retained by a tumor allowing visualization of its margins upon preoperative and intraoperative surgical resection.70,71 Yet, a drawback of any CA is the rapid accumulation in detoxification organs, such as liver and kidneys. As a result, the repeated administration of the same CAs or of various monomodal CAs (e.g., first for SERS, then for MRI) in sequence can be hazardous, thus limiting the repeated MMI of diseased tissues. Rather, using multiple contrast agents (MCA) would allow for imaging exactly the same area with multiple techniques while the total dose of CAs administered to a patient would be reduced. NP heterostructures with inorganic/organic or inorganic/inorganic components are ideal candidates for MMI by combining multiple functions72 in a single nanoplatform and as such could provide the basis for early detection and treatment of cancer.73 In recent years, several examples of these NP heterostructures have been reported.70,73,74 Of particular relevance, gold-coated iron oxide NPs with an interfacial organic fluorescent dyes were shown to be highly efficient CAs for optical and PA imaging, as well as MRI.75 In the following, we comment on some recent and significant progress in MMI and multimodal sensing involving SERS, with an emphasis on cancer detection. Fluorescence and SERS. The combination of SERS and fluorescence microcopy is a fairly popular MMI scheme, reported by several groups.76-78 The El-Sayed group studied the drug release and delivery of an anticancer drug, doxorubicin (DOX), carried by gold nanospheres in real time at a single living cell.78 DOX was conjugated to the surface of gold nanospheres via a pHsensitive hydrazone linkage that can be easily be monitored by SERS. At acidic pH such as the one found in lysosomes (pH~5.0), the hydrazone bond breaks, thus releasing DOX in the human oral squamous carcinoma cells. Upon DOX release, its SERS signal turns off, whereas its fluorescence turns on as it is unquenched by moving away from the gold NP surface. This 2-in-1 scheme enables both triggering and monitoring of the drug release in real time. Another pH-triggered DOX release was reported by Song et al.,79 using bioconjugated plasmonic vesicles carrying amphiphilic SERS-active gold NPs. Overall, both approaches show that the dual imaging of SERS and fluorescence is a powerful tool for precise drug delivery and related cellular response studies. Multimodal SERS and fluorescence studies have been also performed using multifunctional nanotags. For example, Cui et al. developed gold-organosilica core-shell NPs prepared by hydrolyzing of 3-

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Figure 4. Gold-organosilica SERS nanotags for multiplex detection and imaging. (a) SERS spectra of XRITC and MGITC functionalized Au-organosilica nanoparticles, and their 1:1 mixture. (b) Confocal microscopic fluorescence images showing the distribution of FITC-MGITC Auorganosilica nanoparticles and FITC-XRITC Auorganosilica nanoparticles in the cell. (c) Bright field microscopic image of a HeLa cell. SERS images produced by using the baseline corrected intensity of the 1613 cm-1 Raman band of MGITC (d) and the 1502 cm-1 Raman band of XRITC (e) respectively. (f) The merged figure of (d) and (e) to illustrate the distribution of two types of labelled multifunctional nanoparticles in the cell. (g) Typical SERS spectra obtained at different locations of (f). Reproduced from Ref. 80 (Cui et al. Chem. Sci. 2011, 2, 1463-1469) with permission from The Royal Society of Chemistry.

mercaptopropyltriethoxysilane (MPS) in an aqueous solution containing gold nanospheres cores.80 This sequential synthesis of multifunctional nanoplatforms enables to control i) the functionalization of the gold cores with the Raman reporter (malachite green isothiocyanate (MGITC) and X-rhodamine-5-(and-6)isothiocyanate (XRITC)), and ii) the covalent bonding of the fluorophores (fluorescein isothiocyanate (FITC)) to the shell. In particular, the authors chose a fluorescence probe that would ensure minimal spectral overlap between the 632.8 nm Raman excitation line, the MGITC absorption at ~ 629 nm, the fluorescence excitation line at 488nm, the FITC emission close to 520 nm and the emission collected from 500 to 600nm. After incubating HeLa cells with the gold-organosilica probes, the authors analyzed the samples by dark-field optical microscopy, SERS, fluorescence microscopy and confocal laser scanning microscopy (CLSM). Dark-field optical microscopy allowed for quick and straightforward localiza-


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tion within the cells before more accurately screening of the nanoplatforms with CLSM, which provided the very high spatial resolution in the vertical direction needed for subcellular distribution. As a result, by combining nucleus staining and CLSM, Cui et al. were able to study the subcellular distribution of the NPs and demonstrated continuous multiplexed detection with minimum signal degradation (Figure 4).80 Adding to the multifunctionality of dual SERSfluorescence nanoprobes,81 Wang et al. developed a magnetic core for use within their nanoprobes for targeted cancer cell separation.82 In this case, multi-layered nanoprobes were prepared starting with silica-coated magnetic nanobeads as the inner core, onto which goldsilver core-shell nanorods acting as SERS nanoplatforms were grafted. A silica layer was subsequently added to the whole nanoplatform enabling the anchoring of quantum dots (QDs), acting as fluorescent reporters, to the surface. Finally, cell-specific antibodies were covalently linked to the QDs for the multiplexed detection of four cancer cells lines (SKBR3, HeLa, Jurkat T, and LNCaP). With these multimodal nanoprobes, the feasibility of the simultaneous, multiple cancer cell separation from a large population of normal cells in human blood was confirmed. This result if of great significance for the detection of cancer cells with low abundance such as circulating tumor cells and cancer stem cells. MRI and SERS. In an effort to add molecular specificity to an existing, widely used imaging technique for medical diagnosis such as magnetic resonance imaging (MRI), MRI-SERS dual nanoprobes have been reported recently in the literature.83,84 Combining the high sensitivity, spatial resolution and intraoperative imaging capability of Raman with MRI is particularly interesting for functional medical imaging. Paramagnetic NPs used as MRI CAs have the potential advantage of longer circulation half-lives and better biocompatibility than conventional CAs such as Gd3+-based T1 (i.e., spin-lattice relaxation) complexes and iron-oxide nanoparticle-based T2 contrast agents with proven adverse effects.85 Motivated by this potential, Ju et al. designed a nanoprobe consisting of encapsulated Fe3+ ions as T1-weighted MRI contrast agent, and a hollow gold NP coated with a synthetic melanin shell.83 The synthetic melanin shell acted as the Raman reporter and as protective coating, which combined with thiolated PEG assured biological stability. These nanoprobes exhibited MRI relaxation values similar to commercial Gd3+-based complexes and high intensity SER signal which allowed mapping of their internalization in breast carcinoma (MDA-MB 231) cells in three-dimensional space. SERS imaging with photodynamic therapy (PDT) and photothermal treatment therapy (PTT). Integrating SERS sensing and/or imaging with not only diagnostic but therapeutic NP-based schemes is an ultimate

Figure 5. Multiplexed in vivo SERS imaging. Twodimensional axial MRI (top row), PA (middle row, green) and SERS (bottom row, red) images, before (left column) and after (right column) injection of Gd3+-decorated Ausilica nanotags. The post-injection images of all three modalities showed clear tumor visualization (dashed boxes outline the imaged area). Reproduced from Ref. 91 (Kircher et al. Nat. Med. 2012, 18, 829-834) with permission from the Nature Publishing Group.

goal, as both the diagnostics – by SERS – and the therapeutics – by photothermal treatment – could be embodied within the same so-called „theranostic‟ nanoplatform. Of the few successful attempts so far, the Vo-Dinh group, in the study by Fales et al.,86 used SERS nanotags as dual probes with photodynamic therapy (PDT) capabilities based on laser-triggered singlet-oxygen generation. In this work, gold nanostars with a maximal absorption in the NIR spectral region were functionalized with a NIR dye, 3,3‟-diethylthiatricarbocyanineiodide (DTTC), for diagnostic SERS imaging and a photosensitizer, and with methylene blue (MB), for therapeutic cellular treatment. Upon irradiation at 785 nm, MB becomes excited and can transfer its energy to the surrounding media, producing cytotoxic reactive oxygen species (ROS). Additionally, a mesoporous silica shell was used to encapsulate the bi-functionalized core while providing a mechanical protection towards MB reduction and



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particles aggregation in physiological conditions. These nanoplatforms were irradiated with 785 nm laser excitation to acquire DTTC SERRS spectra, and with 633 nm excitation to acquire MB fluorescence emission and generate increased ROS concentrations compared to control samples without MB. As a result, the authors were able to spatially activate and control the cellular death of BT 549 breast cancer cells using these „active‟ SERS nanotags for photothermal therapy (PTT). The Olivo group investigated similar multi-layered multimodal nanoprobes composed of DTTC-tagged gold nanostars functionalized with hypericin -a natural perylene-quinone dye - as the photosensitizer for PDT/PTT theranostic purposes. These nanoprobes were used in the study by Raghavan et al.87 for in vitro photothermal applications on the SCC9 human oral cancer cell line for PDT at 543 nm laser excitation. The nanoprobes cellular uptake was successfully imaged by both confocal fluorescence and dark field microscopies, revealing cytoplasmatic localization. These early developments of photodynamic therapy by SERS nanotags offer exciting perspectives for PTT and beyond. Other multimodal theranostic nanoprobes could be designed for optical coherence tomography PA imaging with great potential for early diagnosis and treatment of cancer and other diseases. In vivo multimodal imaging with SERS. Multimodal in vivo SERS imaging has been reported using PA imaging and MRI. PA imaging is based on the excitation of CA by light pulses causing slight heat production and thermal expansion. An ultrasound transducer records ultrasound waves produced as a result of this process.88 In PA tomography, this allows for obtaining a 3D image of the CAs distribution in living subjects.89 PA imaging and Raman spectroscopy present higher spatial resolution than optical imaging and do not exhibit autofluorescence background from the interrogated tissue. As such, both techniques are ideal for biomedical imaging.90,91 Furthermore, much lower quantities of CA and much shorter acquisition times are required compared to conventional techniques, owing to the high sensitivity of SERS. Gambhir and coworkers reported that MMI based on combining SERS, PA and MRI allowed visualization of brain tumor margins with very high precision.91 They developed MMI CAs based on nanotags and further functionalized with Gd organometallic complexes. The 60 nm-diameter gold NPs functionalized with trans-1,2bis(4-pyridyl)-ethylene (BPE) as a Raman and PA reporter, and 1,4,7,10-tetraazacyclododecane-1,4,7,10tetraacetic acid (DOTA)-Gd3+, as MRI reporter were ideal CAs for SERS, PA and MRI. Ultimately, the authors successfully demonstrated multiplexed imaging of a mouse brain tumor using the three combined techniques (Figure 5).91 OUTLOOK

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As outlined in throughout this Perspective, SERS has demonstrated to be both a powerful stand-alone bioanalytical technique for identifying and monitoring bioanalytes, and a versatile technique that can be integrated with existing biosensing assays and bioimaging techniques to quantify biomarkers levels and confirm a diagnosis or the efficacy of drug delivery. Owing to its sensitivity and the extensive ability to detect bioanalytes, its application has expanded beyond the analytical chemistry laboratory to the biomedical imaging facilities (e.g. MRI) and into clinics. The abundance and variety of the literature reported in this perspective attest that SERS is an ubiquitous, multimodal, analytical diagnostic tool. Tremendous technical progress has been made over the last five years toward portable nanoscale imaging and sensing tools,92 including SERS. The variety of SERS nanoplatforms has considerably adapted to the constraints of both in vitro and in vivo diagnostics through the development of NPs (aggregated and nonaggregated NPs such as nanospheres, nanorods and nanocubes), nanotags consisting of silica or polymer encapsulated NPs containing a Raman reporter, 2D sensing platforms (immobilized NPs, immobilized nanorod assembly such as the metal film over nanospheres) and 3D substrates such as NPs-impregnated paper that provide cost-effective and easy-to-fabricate substrates.93,94 Nanotags are of particular interest since, as emphasized through this article, they provide intense, multiplexed, stable Raman signal easily detectable by SERS and SESORS in cells, deep-tissue and even through bone. They have already been successfully used in in vivo SERS imaging of cancer imaging (vide supra). A translational study on the fate and toxicity showed no nanotag crossing of the gut lining into the body of mice, demonstrating potential for treatment of gut diseases.95 Technical portability outside the laboratory is now easier than ever, with Raman spectrometers the size of a smartphone providing high signal-to-noise ratio SER spectra in minutes.15,96 Commercial products based on SERS for ultrasensitive sensing are available. For example, the eSERSTM detection system from OndaVia, Inc. targets water pollutants detection in the ppm to ppb range for on-the-field chemical analysis; iFyber, LLC has developed SERS-active fibers for paper-based and textile-embedded molecular detection for medical diagnostics, trace analyte detection and authentication. Building upon such „smart‟, multifunctional fibers97, we can envision applications of SERS in wound care, textile-embedded biomarkers and tissue regeneration. Yet, the clinical expansion of in vivo SERS biosensing is intrinsically bound to the question of the biocompatibility and potential cytoxicity of the SERS nanoplatforms, as plasmonic (mostly gold) nanoprobes are an absolute requirement for this technique. NP dimensions, charge, possible aggregation and surface functional groups are important factors to account for when as-


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sessing cytoxicity and biocompatibility of SERS nanoplatforms.98,99 Owing to the spectacular progress in Raman-based diagnostics over the last five years, we are confident that SERS will continue its expansion as an ultrasensitive technique for in vivo diagnostics and as such, a routine technique in nanomedicine. Finally, in the context of personalized medicine, we can anticipate that the need for accurate, ultrasensitive, and biocompatible nanosensors will draw further interest and use of SERS and related techniques, e.g. SESORS for deep-tissue exploration. Recent advances in the development of implantable100,101 and wearable102 sensors for real-time monitoring of bioanalytes (e.g., glucose, lactate) or bioeletrolytes (e.g. Na+, K+) represent a dramatic advancement for individuals in managing their health through monitoring biomarkers. Integrating SERS nanoplatforms and corresponding reading systems (Raman/SORS) to portable, smart watch-like readers could be within reach in the future. CONCLUSIONS In this Perspective, we highlighted recent and significant literature results on SERS nanoplatforms and SERS-based biosensing schemes aimed at in vivo diagnostics and multimodal imaging. The reported studies demonstrate that the high sensitivity and specificity of SERS can be successfully utilized for detection of bioanalytes in low concentrations. This methodology has a high potential for the detection of cancer biomarkers in blood, biofluids and cells, and consequently for its early diagnostics. The combination of SERS with imaging techniques already established for clinical diagnostics and exploration, such as MRI, CT, and PA imaging creates a new, powerful modality. In particular, this substantially increases the precision of tumor removal surgery by providing analytical assistance in the operating room and opens the way to precise theranostics. AUTHOR INFORMATION Corresponding Author * E-mail: [email protected]. Present Addresses † University of Tennessee, Department of Chemistry, 1420 Circle Dr., Knoxville, TN 37996, United States. ffi Chemical Development Department, Boehringer Ingelheim Pharmaceuticals Inc., 900 Ridgebury Road, Ridgefield, CT 06877-0368, United States. Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes The authors declare no competing financial interest(s).

ACKNOWLEDGMENT The authors gratefully acknowledge financial support from: DARPA under SSC Pacific grant HR0011-13-2-002 and the National Science Foundation under NSF MRSEC grant DMR-1121262. This material is also based on research sponsored by the Air Force Research Laboratory under agreement FA8650-15-2-5518. The U.S. Government is authorized to reproduce and distribute reprints for Governmental purposes notwithstanding any copyright notation thereon. The views and conclusions contained herein are those of the authors and should not be interpreted as necessarily representing the official policies or endorsements, either expressed or implied, of Air Force Research Laboratory or the U.S. Government.

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Surface-Enhanced Raman Spectroscopy Biosensing: In Vivo

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