surface-enhanced raman spectroscopy biosensing: in … raman spectroscopy biosensing: in vivo...

Surface-Enhanced Raman Spectroscopy Biosensing: In VivoDiagnostics and Multimodal ImagingAnne-Isabelle Henry, Bhavya Sharma,† M. Fernanda Cardinal, Dmitry Kurouski,‡

and Richard P. Van Duyne*

Northwestern University, Department of Chemistry, 2145 Sheridan Road, Evanston, Illinois 60208, United States

ABSTRACT: This perspective presents recent developments inthe application of surface-enhanced Raman spectroscopy (SERS) tobiosensing, with a focus on in vivo diagnostics. We describe theconcepts and methodologies developed to date and the targetanalytes that can be detected. We also discuss how SERS hasevolved from a “point-and-shoot” stand-alone technique in ananalytical chemistry laboratory to an integrated quantitativeanalytical tool for multimodal imaging diagnostics. Finally, weoffer a guide to the future of SERS in the context of clinicaldiagnostics.

The detection, identification, and quantitative analysis ofbiomarkers at very low concentrations for presympto-

matic diagnosis represents a new frontier in biomedicalresearch, enabled by nanomedicine1−5 and powered by(bio)analytical chemistry.6,7 Detecting diseases earlier simplymeans saving or improving patients’ lives. This is especiallycritical in the case of cancers, for which the efficiency of alreadyexisting treatments is significantly improved when administeredin the early stages of the disease. It is therefore straightforwardto understand that highly sensitive, quantitative diagnostics arecritical and vital. Additionally, the management of chronicdiseases such as diabetes can also tremendously benefit fromsensitive, accurate, and quick diagnostic solutions forcontinuous glucose levels measurements. Yet, few molecularevents and hence low quantities of markers available makediagnosis challenging. Biosensors with nanoscale dimensions,such as nanoparticles (NPs) and nanostructured surfaces, offerexciting prospects as they can act as signal transducers8 andultimately offer a means to probe and quantify bioanalytes, suchas disease-related biomarkers, in very small concentrations(nano- to zeptomolar).9

Plasmonic nanoplatforms such as gold, the material of choicein biomedical nanotechnology,10 and silver NPs and nano-structured surfaces exhibit unique optical properties due totheir intrinsic dielectric function, nanoscale dimensions, and theoscillating nature of light. In this size regime (∼tens tohundreds of nanometers), surface effects become prominent,such that electrons from the metal and photons from theincident light couple into a quasi-particle called a surfacepolariton. This quasi-particle oscillates at a frequency that isreferred to as the localized surface plasmon resonance (LSPR),with two important consequences on the properties of thenanoplatform: the wavelength dependence and the enhancedelectromagnetic field. First, the LSPR wavelength-dependence

in the visible-NIR region of the electromagnetic spectrumdictates that the plasmonic nanoplatform intrinsically possessesa 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 surfaceprovides the opportunity to couple the molecular vibrationsof an analyte nearby (on or up to ∼3 nm away from thesurface)11 to the LSPR, resulting in a massive increase (106−108) of the molecular signal intensity. This phenomenon, calledsurface-enhanced Raman scattering, enables the use ofplasmonic nanoplatforms as a Raman signal amplifier. Ramanscattering, which arises from the inelastic interaction of lightwith 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 nanoplatformsas Raman signal amplifiers, surface-enhanced Raman spectros-copy (SERS) is an ultrasensitive spectroscopic tool.The focus of this perspective is on the contribution of

surface-enhanced Raman spectroscopy (SERS) toward thegoals outlined above using molecular sensing for in vivodiagnostics. Since its inception as a spectroelectrochemistryanalytical tool in the late 1970s,12−14 SERS has evolved as thetechnique of choice for any scientist seeking to access bothmolecular specificity and sensitivity.15 The two intrinsic anddistinctive advantages of SERS compared to other (bio)-analytical techniques are (i) the bar-code like reading thatcomes from unique and narrow vibrational bands in the Ramanspectrum and (ii) its exquisite sensitivity, down to singlemolecules16,17 in the most ideal case (resonant molecules withlarge Raman cross sections).

Received: April 22, 2016Accepted: June 6, 2016

Perspective

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© XXXX American Chemical Society A DOI: 10.1021/acs.analchem.6b01597Anal. Chem. XXXX, XXX, XXX−XXX

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http://dx.doi.org/10.1021/acs.analchem.6b01597

The experimental setup 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 thenanoplatform to which the target analyte(s) bind(s).15 Assuch, technical developments in Raman spectroscopy havedirectly benefited the field of SERS. For example, the range ofaccessible wavelengths now encompasses the whole visiblespectrum (400−750 nm) to the NIR (750−1064 nm), whichincludes the biological optical window (∼600−1200 nm) forthrough-tissue analysis.SERS has evolved into a field that includes a wealth of

experiments at room temperature and inside living cells. Thefast growth of SERS has resulted from its ability to probe eitherone NP at a time,18,19 or several, through imaging (both in vitroand in vivo) and remote sensing (through spatially offsetdetection, described in Multimodal Sensing and Imaging). Anabundance of literature can be found on SERS substrates,20−22

SERS applied to biosensing,23−25 biomolecules,26 probingbiofluids,27 medical problems,28 and cancer detection29,30 inparticular.This Perspective summarizes the field of in vivo molecular

sensing for diagnostics by SERS. While we cover a significantportion of the literature on SERS biosensing, we are not able topresent here a fully comprehensive literature review of the topicwithin the mandated length of a perspective. Within this paperwe point the readers to in-depth, topical, and critical reviewsand research articles recently published. With this in mind, wewish to expose the readers to the current most excitingdevelopments in SERS biosensing and bring insight on wherewe envision this field to be headed, as discussed in the Outlooksection.

■ IN VIVO SERS FOR DIAGNOSTICSOrigins of Cancer Detection by Raman Spectroscopy.

Before discussing the impact of SERS on in vivo detection ofdisease, we briefly introduce recent advances in the use ofnormal Raman spectroscopy for cancer detection as itimportantly demonstrated the feasibility of Raman-enabledbiosensing. Among the earliest applications of normal Ramanspectroscopy for cancer detection is examining differences inthe plasma membranes of normal and neoplastic lymphocytestransformed by simian virus 40.31,32 It was found that theneoplastic cells had a greater degree of amidation of theasparagine and glutamine residues of the membrane31 and weremore sensitive to changes in temperature32 than normal cells.Since then, normal Raman spectroscopy has been characterizedas a tool for cancer detection that can quickly assess thepresence of cancer biomarkers and aid in the evaluation of thedifferent stages of cancer.33 In general, visible to NIRwavelengths are used both in vitro and particularly in vivo,since these wavelengths have a greater penetration depth intissues. Cancers including brain,34,35 esophageal,36 skin,37

breast,38 epithelial,39 and gynecological cancers, to name afew, have all been examined and characterized by Ramanspectroscopy and reviewed in detail elsewhere.33,40−42 Moststudies have found that differences in the Raman peaks formacromolecules such as cholesterol, amino acids (phenyl-alanine, tyrosine, tryptophane, cysteine), DNA, RNA, phos-pholipids, proteins, and collagen are good diagnostic indicatorswhen compar ing norma l ver sus neop la s t i c t i s -sue.33−35,37,40,41,43−45 The differences between the normal andneoplastic Raman spectra are often slight and not easily visibleby eye. Additionally, some Raman spectra are also complicated

by fluorescence from the tissue samples, which creates a largeamount of background noise. Since the normal Ramanexcitation of cellular components is not enhanced (throughresonance or surface-enhancement), the fluorescence back-ground can often overwhelm the Raman signals. The use ofmathematical models and/or statistical analysis, such as Fouriertransforms, principal component analysis, linear discriminantanalysis, cluster analysis, as well as other algorithms to treatRaman data with large fluorescence backgrounds and elucidatethe small differences in peak position or intensity hasrevolutionized the use of Raman spectroscopy as biologicaland medical tools.34,39,43−45

Although normal Raman spectroscopy has proven useful fordiagnosing cancer, it is an intrinsically weak scatteringphenomenon. By employing methods of signal enhancementsuch as surface-enhancement, signal intensity is often increasedbetween 106−108 times. As discussed in the introduction, SERSoffers greater sensitivity (i.e., detect smaller concentrations) andprovides a higher level of selectivity than normal Raman, whichcan result in the earlier detection of cancer, and thus improvedprognoses for patients.

In Vivo SERS for Cancer Detection. In vivo SERS imaginghas been used to image tumors, guide surgical removal oftumors (particularly for elucidating tumor margins), and alsoused for its multiplexing capabilities. Multiplexed detection, i.e.,the ability to simultaneously detecting several markers (analytesor Raman reporters) is an advantage of SERS, especially in thecontext of in vivo biosensing. Most in vivo SERS imaginginvolves the use of SERS nanotags, NP constructs with a strongRaman active molecule adsorbed on the surface of aggregatedgold NPs, which are often encapsulated in silica, polyethyleneglycol (PEG), or mixed bovine serum albumin-glutaraldehydefor stability and to protect the nanotags. There are severalvarieties of Raman active molecules available which can beinjected simultaneously,46 resulting in multiplexing capabilities,as each molecule has a distinctive spectrum. Additionally,groups are working toward expanding the library of moleculesavailable that have strong Raman cross sections and are Ramanactive in the near-infrared wavelength range.47,48

The SERS nanotags are employed in one of two ways: theyare either injected into the animal and passively accumulate inthe body or the surface of the nanotags is functionalized with areceptor or antibody which allows them to actively targetspecific cells or tumors. Several in vivo studies rely on passivetargeting for accumulation of nanotags in tumors, but due tothe size of the nanotags (>100 nm) these latter are often takenup by the Kupffer cells of the reticuloendothelial system andaccumulate primarily in the liver, where they remain forextended periods of time.46,48−50 It was also found, however,that the signal of the nanotags in the tumors can remainelevated even when the concentration in blood return topreinjection levels.51 Most passive targeting is now used inconjunction with the development of new instrumentation forSERS imaging of small animals.The Multimodality Molecular Imaging Lab at Stanford

University has led the way in both the use of SERS nanotags forsurgery as well as the development of several new instrumentsto improve the imaging of nanotags in vivo. Zavaleta et al.46

demonstrated multiplexing of 10 nanotags and separation oftheir Raman spectral signatures using a commercial pointmapping Raman imaging system. Soon they realized that theaccumulation time necessary for point mapping is not feasiblefor real-time imaging modalities. This led to the development

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of two separate Raman imaging systems: the small animalRaman imaging (SARI) system50 and a fiber optic probedesigned to be coupled with a clinical endoscope.52 The SARIsystem allows for rapid Raman imaging over a relatively largearea (>6 cm2) with high spatial resolution. Instead of a point bypoint scan system, SARI utilizes a line scan that rasters alongthe x and y axes using two-dimensional galvanometric mirrors,allowing the sample (here a small animal) to remain stationaryand can measure to a depth of ∼4 cm. The SARI system isapproximately 10 times faster in scan rates than the pointmapping system, which required up to 5 h of scan time to coverareas of a few centimeters.46,50 Passive accumulation of fournanotags in the liver can be imaged by the SARI system, withtwo nanotags clearly present 1 h postinjection (Figure 1, left)and all four nanotags clearly evident 2 h postinjection (Figure 1,right).50

Building on the SARI system, Zavaleta et al. coupled anoncontact fiber optic probe-based Raman system to anendoscope.52 This system incorporates the Raman fiber probethrough a clinical endoscope to provide real-time, multiplexedSERS data from tumor-targeting nanotags. SERS nanotags wereimaged in human tissue to test the multiplexing capabilities andin porcine colon tissue in a “scavenger hunt”, where thenanotag location and concentration were unknown before theimaging measurements. Three “blind” testers achieved asensitivity of 100%. The fiber optic Raman probe was approvedfor testing in human patients during routine colonoscopies, butdue to the need of further regulatory approval, use of thenanotags was not implemented. However, the Raman devicewas used to collect Raman spectra in vivo when combined withthe 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. Anotherapproach for in vivo SERS imaging utilized tunable bandpassfilters to scan a window of 400−1500 cm−1 at 785 nmexcitation, which allows for multiplexed wide-field imaging ofnanotags.53,54 The nanotags used each have distinctive SERSspectra which are easily discernible even when overlapped inmultiplexing experiments. The tunable filters are used to imagenarrow windows in the spectral range (∼65 cm−1), resulting inisolation of specific peaks from the individual nanotags whileminimizing the contribution from other tags. This single peakidentification allowed the speed of the wide-field imaging to besignificantly faster than point-by-point measures (5 s perbandpass image at low picomolar concentrations versus 50 h bypoint measures). The SERS signal of four nanotags were easilydetected and separated from background fluorescence fromtissue samples to depths of 5 mm. This technology has alsobeen demonstrated with active targeting of lung cancer usingnanotags coated with epidermal growth factor receptors(EGFR)-specific antibodies54 and is also being adapted foruse with an endoscope.Active targeting is used more often because it allows for

higher specificity in the target, with less nonspecificaccumulation of nanotags. Several groups have demonstratedthe efficiency of actively targeting tumors and the decreasedbiodistribution of the nanotags in other tissues of thebody.47,48,55,56 In one of the first active targeting studies byQian et al.,55 60 nm gold NPs with malachite green adsorbedon the surface as the Raman probe molecule were function-alized 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 manytypes of cancerous cells. The authors found that thefunctionalized nanotags accumulated in the tumor within 4−6h of injection into the tail veins of nude xenograft tumor modelmice and remained localized in the tumor for 24−48 h.Additionally, they compared active versus passive accumulationand found that the active target nanotags had higher signalintensity and accumulated 10× more in the tumor than thepassive nanotags. Both active and passive nanotags accumulatedin the liver and spleen, but the passive nanotags accumulated inboth organs to a greater degree. Samanta et al.47 targetedhuman epidermal growth factor receptors 2 (Her2), which areinvolved with cell proliferation and are known to beupregulated in breast cancer. They conjugated 60 nm nanotags(gold NPs with CyNAMLA-381 dye and stabilized with BSAmixed with glutaraldehyde for cross-linked organic encapsula-tion) to a ScFv anti-Her2 antibody, which was then injectedinto the tail vein of mice with xenograft tumor model of cellsthat overexpress Her2. In this case, the authors measuredspectra of the nanotags at the tumor site only and found nononspecific binding of the nanotags.Moving toward multiplexed detection with active targeting,

Dinish et al.56 demonstrated detection of three cancerbiomarkers in a breast cancer cell line. They utilized xenograftmouse tumor models and targeted the following biomarkers:EGFR; CD44, which is a cell surface adhesion molecule; andTGF-βII, which its down regulation of increases breast cancer.The SERS nanotags consisted of PEG-passivated gold NPs withthree different Raman probe molecules adsorbed to the surface(Cyanine 5, malachite green isothiocyanate, and Rhodamine6G) that were then conjugated to antibodies against the

Figure 1. In vivo SERS imaging. Map of the mouse liver followinginjections of four types of SERS nanotags (S420, S421, S440, S470): 1h postinjection (left) and 2 h postinjection (right). Reproduced fromref 50. (Bohndiek et al. Proc. Natl. Acad. Sci. U.S.A. 2013, 110, 12408−12413) with permission from the National Academy of Sciences.



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biomarkers. Instead of tail vein injection, the study hereinvolved direct injection into the tumor itself, to test if thenanotags remained in the tumor with decreased accumulationin other organs. Comparing a control mouse versus the tumormodel mouse, the signal intensity of the nanotags increased inthe first hour, decreased significantly by the sixth hour, and by24 h was no longer detected in the control mouse. While in thetumor model mouse, the signal increased, reaching a maximumat 6 h, with the signal decreasing but still detectable at 48 h andfinally no signal at 72 h.All of these studies demonstrate the positive contribution of

combining nanotag-targeting strategies with new instrumenta-tion to further develop SERS as a technique for in vivo tumordetection.Spatially Offset Raman Spectroscopy (SORS). One

disadvantage to the aforementioned detection methods islimited in vivo depth of penetration, which results from the hightissue scattering and autofluorescence from chromophores inthe tissue. A Raman-based technique that allows for increasedpenetration depths and suppresses fluorescence is spatiallyoffset Raman spectroscopy (SORS).57 In SORS, light incidenton the surface of a multilayer sample, has the surface-generatedRaman scattered light travel back along the same trajectory asthe incident laser beam. Some of the incident photons traveldeeper into the sample (into the lower layer) and migratelaterally away from the incident laser spot. The Ramanscattered photons from this lower layer can then be detectedat some distance that is offset from the incident trajectory.SORS has been used for several in vivo studies, includingprobing bone through the skin and calcifications in chickenbreast tissue as a precursor to human breast cancer studies.58,59

Additionally, to further improve signal intensity and targetingcapabilities, SORS has been combined with SERS, resulting in anew methodology referred to as SESORS.With the use of SERS nanotags, SERS imaging and SORS

have demonstrated depths of penetration to ∼5 mm in vivo;whereas SESORS imaging has demonstrated a penetrationdepth of 15−25 mm in porcine tissue.60 Furthermore, SESORSmultiplexed 3D imaging of nanotags has also been demon-strated to depths of 45−50 mm in porcine tissue.61 Hence,SESORS combines the sensitivity of SERS with unprecedenteddepth of penetration for imaging in tissue. Besides turbidmedia, SESORS has also been demonstrated to be effective formeasuring SERS spectra of nanotags on and through bone. Xieet al.62 coated bone fragments with SERS nanotags function-alized with bisphosphonate, a type of drug used in thetreatment of osteoporosis. They inserted these bones fragmentsinto 20 mm thick porcine muscle tissue and demonstratedlocalization of the bone tissue through the obtained SESORSspectra of the bisphosphonate-coated SERS nanotags. Theability to localize bisphosphonate indicates the suitability ofSESORS in future studies of metastatic breast cancer and forosteoporosis treatment. Sharma et al.63 injected SERS nanotagsinto ovine tissue and used SESORS to measure for the first timespectra of nanotags through an ovine shoulder bone. Throughthis study, our group demonstrated measurement of spectrathrough bone thicknesses from 3 to 8 mm, as shown in Figure2. This result is significant in the context of developing atechnique for noninvasive functional imaging of the brainthrough the skull, as the human skull can range from 3 to 14mm 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 Thegoal of this study was to develop an implantable sensor thatcould be used to measure interstitial glucose levels trans-cutaneously. Instead of SERS nanotags, we used a SERS sensorfunctionalized with a self-assembled mixed monolayer captureligand for glucose. This sensor consisted in a silver nano-structured film over silica nanospheres deposited on a titaniasubstrate (8 mm diameter disk). This nanoplatform wasimplanted in the subcutaneous space under the skin to probethe interstitial fluid. Upon data analysis, a correlation betweenSERS intensity and glucose concentration was developed usinga partial least-squares method. By comparing the results withthe gold standard of glucose sensing, an electrochemical bloodglucometer, the SESORS concentrations were found to matchwell with the glucometer readings. Building on this study, ourgroup continued to explore the use of SESORS for trans-cutaneous glucose sensing.65 Again using a functionalized SERSsensor implanted in 6 rats, the SERS sensor was measured to bestable and active for more than 17 days in vivo (Figure 3).64

The sensor was remarkably consistent over the 17 days, and itdemonstrated high hypoglycemic accuracy, which was evengreater than the current ISO standards. Both the proof-of-concept and in vivo studies demonstrate that SESORS holdsgreat promise for in vivo SERS detection of various diseases,

Figure 2. SESORS-enabled detection of nanotags through animalbone. (A) Waterfall plot of SESOR spectra (x-axis, Raman shift; z-axis,SERS intensity) through varying thicknesses of bone (y-axis), with redbeing 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 ChemicalSociety).



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including breast cancer, bone disease, diabetes, and diseases ofthe brain.

■ MULTIMODAL SENSING AND IMAGINGImaging-based diagnosis of cancer or necrotic tissues cansubstantially increase the precision of surgical intervention andthe survival outcome of the patient. An application of amultimodal rather than single imaging technique wouldtherefore substantially decrease the chance of tumorigenictissue left at the resection margins of cancerous tissues andresult in more accurate delineation of the margins of tumors. Inthe past decades, numerous new multimodal imaging (MMI)technologies based on fluorescence imaging (FI), magneticresonance imaging (MRI), photoacoustic (PA) imaging,photothermal therapy (PTT) imaging, computed X-raytomography (CT), and SERS have been introduced in clinicalsettings.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 retainedby a tumor allowing visualization of its margins uponpreoperative and intraoperative surgical resection.70,71 Yet, adrawback of any CA is the rapid accumulation in detoxificationorgans, such as liver and kidneys. As a result, the repeatedadministration of the same CAs or of various monomodal CAs(e.g., first for SERS, then for MRI) in sequence can behazardous, thus limiting the repeated MMI of diseased tissues.Rather, using multiple contrast agents (MCA) would allow forimaging exactly the same area with multiple techniques while

the total dose of CAs administered to a patient would bereduced.NP heterostructures with inorganic/organic or inorganic/

inorganic components are ideal candidates for MMI bycombining multiple functions72 in a single nanoplatform andas such could provide the basis for early detection andtreatment of cancer.73 In recent years, several examples of theseNP heterostructures have been reported.70,73,74 Of particularrelevance, gold-coated iron oxide NPs with an interfacialorganic fluorescent dyes were shown to be highly efficient CAsfor optical and PA imaging as well as MRI.75 In the following,we comment on some recent and significant progress in MMIand multimodal sensing involving SERS, with an emphasis oncancer detection.

Fluorescence and SERS. The combination of SERS andfluorescence microcopy is a fairly popular MMI scheme,reported by several groups.76−78 The El-Sayed group studiedthe drug release and delivery of an anticancer drug, doxorubicin(DOX), carried by gold nanospheres in real time at a singleliving cell.78 DOX was conjugated to the surface of goldnanospheres via a pH-sensitive hydrazone linkage that can beeasily monitored by SERS. At acidic pH such as the one foundin lysosomes (pH ∼ 5.0), the hydrazone bond breaks, thusreleasing DOX in the human oral squamous carcinoma cells.Upon DOX release, its SERS signal turns off, whereas itsfluorescence turns on as it is unquenched by moving away fromthe gold NP surface. This 2-in-1 scheme enables both triggeringand monitoring of the drug release in real time. Another pH-triggered DOX release was reported by Song et al.,79 usingbioconjugated plasmonic vesicles carrying amphiphilic SERS-active gold NPs. Overall, both approaches show that the dualimaging of SERS and fluorescence is a powerful tool for precisedrug delivery and related cellular response studies.Multimodal SERS and fluorescence studies have been also

performed using multifunctional nanotags. For example, Cui etal. developed gold-organosilica core−shell NPs prepared byhydrolyzing of 3-mercaptopropyltriethoxysilane (MPS) in anaqueous solution containing gold nanospheres cores.80 Thissequential synthesis of multifunctional nanoplatforms enablesone to control (i) the functionalization of the gold cores withthe Raman reporter (malachite green isothiocyanate (MGITC)and X-rhodamine-5-(and-6)-isothiocyanate (XRITC)) and (ii)the covalent bonding of the fluorophores (fluoresceinisothiocyanate (FITC)) to the shell. In particular, the authorschose a fluorescence probe that would ensure minimal spectraloverlap between the 632.8 nm Raman excitation line, theMGITC absorption at ∼629 nm, the fluorescence excitationline at 488 nm, the FITC emission close to 520 nm, and theemission collected from 500 to 600 nm. After incubating HeLacells with the gold-organosilica probes, the authors analyzed thesamples by dark-field optical microscopy, SERS, fluorescencemicroscopy, and confocal laser scanning microscopy (CLSM).Dark-field optical microscopy allowed for quick and straightfor-ward localization within the cells before more accuratelyscreening the nanoplatforms with CLSM, which provided thevery high spatial resolution in the vertical direction needed forsubcellular distribution. As a result, by combining nucleusstaining and CLSM, Cui et al. were able to study the subcellulardistribution of the NPs and demonstrated continuous multi-plexed detection with minimum signal degradation (Figure4).80

Adding to the multifunctionality of dual SERS-fluorescencenanoprobes,81 Wang et al. developed a magnetic core for use

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



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within their nanoprobes for targeted cancer cell separation.82 Inthis case, multilayered nanoprobes were prepared starting withsilica-coated magnetic nanobeads as the inner core, onto whichgold−silver core−shell nanorods acting as SERS nanoplatformswere grafted. A silica layer was subsequently added to the wholenanoplatform 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 themultiplexed detection of four cancer cells lines (SKBR3, HeLa,Jurkat T, and LNCaP). With these multimodal nanoprobes, thefeasibility of the simultaneous, multiple cancer cell separationfrom a large population of normal cells in human blood wasconfirmed. This result is of great significance for the detectionof cancer cells with low abundance such as circulating tumorcells and cancer stem cells.MRI and SERS. In an effort to add molecular specificity to

an existing, widely used imaging technique for medicaldiagnosis such as magnetic resonance imaging (MRI), MRI-SERS dual nanoprobes have been reported recently in theliterature.83,84 Combining the high sensitivity, spatial resolution,and intraoperative imaging capability of Raman with MRI isparticularly interesting for functional medical imaging. Para-magnetic NPs used as MRI CAs have the potential advantage oflonger circulation half-lives and better biocompatibility thanconventional CAs such as Gd3+-based T1 (i.e., spin−lattice

relaxation) complexes and iron-oxide nanoparticle-based T2contrast agents with proven adverse effects.85 Motivated by thispotential, Ju et al. designed a nanoprobe consisting ofencapsulated Fe3+ ions as T1-weighted MRI contrast agentand a hollow gold NP coated with a synthetic melanin shell.83

The synthetic melanin shell acted as the Raman reporter and asprotective coating, which combined with thiolated PEG assuredbiological stability. These nanoprobes exhibited MRI relaxationvalues similar to commercial Gd3+-based complexes and highintensity SER signal which allowed mapping of their internal-ization in breast carcinoma (MDA-MB 231) cells in three-dimensional space.

SERS Imaging with Photodynamic Therapy (PDT) andPhotothermal Treatment Therapy (PTT). Integrating SERSsensing and/or imaging with not only diagnostic buttherapeutic NP-based schemes is an ultimate goal, as boththe diagnostics, by SERS, and the therapeutics, by photo-thermal treatment, could be embodied within the same so-called “theranostic” nanoplatform. Of the few successfulattempts so far, the Vo-Dinh group, in the study by Fales etal.,86 used SERS nanotags as dual probes with photodynamictherapy (PDT) capabilities based on laser-triggered singlet-oxygen generation. In this work, gold nanostars with a maximalabsorption in the NIR spectral region were functionalized witha NIR dye, 3,3′-diethylthiatricarbocyanineiodide (DTTC), fordiagnostic SERS imaging and a photosensitizer, and withmethylene blue (MB), for therapeutic cellular treatment. Uponirradiation at 785 nm, MB becomes excited and can transfer itsenergy to the surrounding media, producing cytotoxic reactiveoxygen species (ROS). Additionally, a mesoporous silica shellwas used to encapsulate the bifunctionalized core whileproviding a mechanical protection toward MB reduction andparticles aggregation in physiological conditions. These nano-platforms were irradiated with 785 nm laser excitation toacquire DTTC SERRS spectra, and with 633 nm excitation toacquire MB fluorescence emission and generate increased ROSconcentrations compared to control samples without MB. As aresult, the authors were able to spatially activate and control thecellular death of BT 549 breast cancer cells using these “active”SERS nanotags for photothermal therapy (PTT). The Olivogroup investigated similar multilayered multimodal nanoprobescomposed of DTTC-tagged gold nanostars functionalized withhypericin, a natural perylene-quinone dye, as the photo-sensitizer for PDT/PTT theranostic purposes. These nanop-robes were used in the study by Raghavan et al.87 for in vitrophotothermal applications on the SCC9 human oral cancer cellline for PDT at 543 nm laser excitation. The nanoprobescellular uptake was successfully imaged by both confocalfluorescence and dark field microscopies, revealing cytoplas-matic localization. These early developments of photodynamictherapy by SERS nanotags offer exciting perspectives for PTTand beyond. Other multimodal theranostic nanoprobes couldbe designed for optical coherence tomography PA imaging withgreat potential for early diagnosis and treatment of cancer andother diseases.

In Vivo Multimodal Imaging with SERS. Multimodal invivo SERS imaging has been reported using PA imaging andMRI. PA imaging is based on the excitation of CA by lightpulses causing slight heat production and thermal expansion.An ultrasound transducer records ultrasound waves producedas a result of this process.88 In PA tomography, this allows forobtaining a 3D image of the CAs distribution in livingsubjects.89 PA imaging and Raman spectroscopy present higher

Figure 4. Gold-organosilica SERS nanotags for multiplex detectionand imaging. (a) SERS spectra of XRITC and MGITC functionalizedAu-organosilica nanoparticles and their 1:1 mixture. (b) Confocalmicroscopic fluorescence images showing the distribution of FITC-MGITC Au-organosilica nanoparticles and FITC-XRITC Au-organo-silica nanoparticles in the cell. (c) Bright field microscopic image of aHeLa cell. SERS images produced by using the baseline correctedintensity of the 1613 cm−1 Raman band of MGITC (d) and the 1502cm−1 Raman band of XRITC (e), respectively. (f) The merged figureof parts d and e to illustrate the distribution of two types of labeledmultifunctional nanoparticles in the cell. (g) Typical SERS spectraobtained at different locations of part f. Reproduced from ref 80. (Cuiet al. Chem. Sci. 2011, 2, 1463−1469) with permission from The RoyalSociety of Chemistry.



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spatial resolution than optical imaging and do not exhibitautofluorescence background from the interrogated tissue. Assuch, both techniques are ideal for biomedical imaging.90,91

Furthermore, much lower quantities of CA and much shorteracquisition times are required compared to conventionaltechniques, owing to the high sensitivity of SERS. Gambhirand co-workers reported that MMI based on combining SERS,PA, and MRI allowed visualization of brain tumor margins withvery high precision.91 They developed MMI CAs based onnanotags and further functionalized with Gd organometalliccomplexes. The 60 nm-diameter gold NPs functionalized withtrans-1,2-bis(4-pyridyl)-ethylene (BPE) as a Raman and PAreporter, and 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraace-tic acid (DOTA)-Gd3+, as the MRI reporter were ideal CAs forSERS, PA, and MRI. Ultimately, the authors successfullydemonstrated multiplexed imaging of a mouse brain tumorusing the three combined techniques (Figure 5).91

■ OUTLOOK

As outlined throughout this Perspective, SERS has demon-strated to be both a powerful stand-alone bioanalyticaltechnique for identifying and monitoring bioanalytes and a

versatile technique that can be integrated with existingbiosensing assays and bioimaging techniques to quantifybiomarkers levels and confirm a diagnosis or the efficacy ofdrug delivery. Owing to its sensitivity and the extensive abilityto detect bioanalytes, its application has expanded beyond theanalytical chemistry laboratory to the biomedical imagingfacilities (e.g., MRI) and into clinics. The abundance andvariety of the literature reported in this perspective attest thatSERS is an ubiquitous, multimodal, analytical diagnostic tool.Tremendous technical progress has been made over the last

5 years toward portable nanoscale imaging and sensing tools,92

including SERS. The variety of SERS nanoplatforms hasconsiderably adapted to the constraints of both in vitro and invivo diagnostics through the development of NPs (aggregatedand nonaggregated NPs such as nanospheres, nanorods, andnanocubes), nanotags consisting of silica or polymer encapsu-lated NPs containing a Raman reporter, 2D sensing platforms(immobilized NPs, immobilized nanorod assembly, such as themetal 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 interestsince, as emphasized through this Perspective, they provideintense, multiplexed, stable Raman signal easily detectable bySERS and SESORS in cells, deep-tissue, and even throughbone. They have already been successfully used in in vivo SERSimaging of cancer tumors (vide supra). A translational study onthe fate and toxicity showed no nanotag crossing of the gutlining into the body of mice, demonstrating potential fortreatment of gut diseases.95 Technical portability outside thelaboratory is now easier than ever, with Raman spectrometersthe size of a smartphone providing high signal-to-noise ratioSER spectra in minutes.15,96 Commercial products based onSERS for ultrasensitive sensing are available. For example, theeSERS detection system from OndaVia, Inc. targets waterpollutants detection in the ppm to ppb range for on-the-fieldchemical analysis; iFyber, LLC has developed SERS-activefibers for paper-based and textile-embedded moleculardetection for medical diagnostics, trace analyte detection, andauthentication. Building upon such “smart”, multifunctionalfibers,97 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 andpotential cytoxicity of the SERS nanoplatforms, as plasmonic(mostly gold) nanoprobes are an absolute requirement for thistechnique. NP dimensions, charge, possible aggregation, andsurface functional groups are important factors to account forwhen assessing cytoxicity and biocompatibility of SERSnanoplatforms.98,99

Owing to the spectacular progress in Raman-baseddiagnostics over the last 5 years, we are confident that SERSwill continue its expansion as an ultrasensitive technique for invivo diagnostics and as such, a routine technique in nano-medicine. Finally, in the context of personalized medicine, wecan anticipate that the need for accurate, ultrasensitive, andbiocompatible nanosensors will draw further interest and use ofSERS and related techniques, e.g., SESORS for deep-tissueexploration. Recent advances in the development of implant-able100,101 and wearable102 sensors for real-time monitoring ofbioanalytes (e.g., glucose, lactate) or bioeletrolytes (e.g., Na+,K+) represent a dramatic advancement for individuals inmanaging their health through monitoring biomarkers.Integrating SERS nanoplatforms and corresponding reading

Figure 5. Multiplexed in vivo SERS imaging. Two-dimensional axialMRI (top row), PA (middle row, green), and SERS (bottom row, red)images, before (left column) and after (right column) injection ofGd3+-decorated Au-silica nanotags. The postinjection images of allthree modalities showed clear tumor visualization (dashed boxesoutline the imaged area). Reproduced from ref 91. (Kircher et al. Nat.Med. 2012, 18, 829−834) with permission from the Nature PublishingGroup.



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systems (Raman/SORS) to portable, smart watch-like readerscould be within reach in the future.

■ CONCLUSIONSIn this Perspective, we highlighted recent and significantliterature results on SERS nanoplatforms and SERS-basedbiosensing schemes aimed at in vivo diagnostics and multimodalimaging. The reported studies demonstrate that the highsensitivity and specificity of SERS can be successfully utilizedfor detection of bioanalytes in low concentrations. Thismethodology has a high potential for the detection of cancerbiomarkers in blood, biofluids, and cells and consequently forits early diagnostics. The combination of SERS with imagingtechniques already established for clinical diagnostics andexploration, such as MRI, CT, and PA imaging creates a new,powerful modality. In particular, this substantially increases theprecision of tumor removal surgery by providing analyticalassistance in the operating room and opens the way to precisetheranostics.

■ AUTHOR INFORMATIONCorresponding Author*E-mail: [email protected] Addresses†B.S.: University of Tennessee, Department of Chemistry, 1420Circle Dr., Knoxville, TN 37996.‡D. K.: Chemical Development Department, BoehringerIngelheim Pharmaceuticals Inc., 900 Ridgebury Road, Ridge-field, CT 06877-0368.Author ContributionsThe manuscript was written through contributions of allauthors. All authors have given approval to the final version ofthe manuscript.NotesThe authors declare no competing financial interest.

■ ACKNOWLEDGMENTSThe authors gratefully acknowledge financial support fromDARPA under SSC Pacific Grant HR0011-13-2-002 and theNational Science Foundation under NSF MRSEC Grant DMR-1121262. This material is also based on research sponsored bythe Air Force Research Laboratory under Agreement FA8650-15-2-5518. The views and conclusions contained herein arethose of the authors and should not be interpreted asnecessarily representing the official policies or endorsements,either expressed or implied, of Air Force Research Laboratoryor the U.S. Government.

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