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Cite this: J.Mater. Chem. B, 2019,

7, 815

Surface-enhanced Raman spectroscopy (SERS)nanoprobes for ratiometric detection of cancercells†

Linhu Li, Mengling Liao, Yingfan Chen, Beibei Shan and Ming Li *

We report a ratiometric strategy for detection of different types of breast cancer cells by surface-enhanced

Raman spectroscopy (SERS), which simultaneously quantifies the levels of dual biomarkers distinctly

expressed on cancer cells to consequently achieve their expression ratio. Two SERS nanoprobes that are

encoded with distinct SERS signatures are conjugated with urokinase plasminogen activation receptor

(uPAR)- and epidermal growth factor receptor (EGFR)-targeting peptides. The SERS imaging of single live

cells can accurately quantify the cellular biomarker expression difference from the SERS intensity ratio and

is further employed for cancer cell screening. The results show that MDA-MB-231 and MCF-7 exhibit

distinct expression of the uPAR and EGFR and they can be respectively discriminated by the intensity ratio

of SERS signals from uPAR- and EGFR-targeting SERS nanoprobes. The ratiometric strategy permits

background-free SERS detection of cancer cells and dramatically improves the signal-to-noise ratio of

targeted cellular SERS imaging, thus enabling accurate cancer cell screening without the need for

additional references. It is believed that the present ratiometric method should be a promising avenue

for breast cancer diagnostics and screening, which can be easily extended for detection of other cancer

cell types.

1. Introduction

Current efforts in several cancer types have shown that distinctsubtypes of cancer cells have significant implications of cancerprogression, prognostics and therapeutic efficacy.1–5 Rapididentification and analysis of cancer cells in tissues or bodyfluids can transform our understanding of cancer biology, andpermit a potential route for cancer diagnostics and real-timemonitoring of therapeutic efficacy, eventually benefiting theclinical management of cancer. However, the clinical methodfor detection of cancer cells as cancer biomarkers is yet to beestablished partly due to the biological complexity of cancer.Considerable studies have shown that each cancer cell line hasunique cellular biomarker (i.e., genomic and proteomic) expression,distinguishing them from each other.6 The utility of the exclusivefeatures of these biomarkers is a quite effective route toidentification of cancer cells. However, accurate identificationof cancer cells requires development of methods that enable

sensitive and selective cancer cell detection through precisemolecular recognition of their expressed biomarkers.7,8

Endogenous proteomic biomarkers have been extensivelyexploited for distinguishing and screening various cancer cells.9,10

Conventional approaches for cancer cell detection include reversetranscription-polymerase chain reaction (RT-PCR), optical methods,negative selection, cell-size filtration, CellSearcht and microfluidictechniques.11 Fluorescence-assisted cell sorting (FACS) is the mostcommonly used method for separating and identifying cancer cells.Cancer cells can be made visible using fluorescent labels such asorganic dye molecules and tiny quantum dots. The most advancedflow cytometers using fluorescent labels can accommodate a flowrate up to 50 000 cells per second.12 Despite the high sensitivity andspatial resolution, fluorescence-based approaches usually sufferfrom drawbacks in terms of limited multiplexing, photobleaching,phototoxicity, and autofluorescence interference from a biologicalbackground.13–21 In addition, detection of only a single biomarkerper assay is usually performed, limiting the accuracy of cancercell identification.

Herein, we introduce a ratiometric approach to discriminatebetween cancer cells using surface-enhanced Raman spectroscopy(SERS) through targeting extracellular biomarkers, characteristicof cancer cells. SERS has recently become the most sensitivetechnique among optical sensing/imaging modalities as the signalintensity of molecular vibration is enhanced by 108–1014 fold

School of Materials Science and Engineering, State Key Laboratory for Power

Metallurgy, Central South University, Changsha, Hunan 410083, China.

E-mail: liming0823@gmail.com, liming0823@csu.edu.cn;

Web: https://www.ming-group.com/

† Electronic supplementary information (ESI) available: Fig. S1–S7 and Table S1.See DOI: 10.1039/c8tb02828a

Received 26th October 2018,Accepted 18th December 2018

DOI: 10.1039/c8tb02828a

rsc.li/materials-b

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compared to spontaneous Raman signals.22–28 The SERSenhancement is mainly due to the strong localized surfaceplasmon resonance (LSPR) whenever Raman molecules comein close proximity to the surface of metal nanostructures.22,29

Benefits of SERS nanoprobes over existing methods include thegreat spectral multiplexing capability for simultaneous detectionof multiple targets owing to narrow vibrational Raman bands,quantification capacity with the help of molecular signatures, therequirement of only a single laser source having a single excitationwavelength, and high photostability.30–33 SERS has been demon-strated to be valuable for live-cell imaging based on the specifictargeting of endogenous biomarkers.34,35 Furthermore, we havepreviously developed a novel class of SERS nanoprobes whereRaman-active molecules were incorporated between a near-infrared (NIR) plasmonic gold nanostar (GNS) and a thin silicaprotective layer.22,36–38 This SERS nanoprobe has multiple meritsfor chemical analysis and biological imaging, including (i) highSERS signals due to encapsulation of a large number of Ramanmolecules, (ii) flexible encoding capacity with desirable SERSsignatures, (iii) powerful multiplexing capability, (iv) easymodification with biological functionalities, (v) high stability inrobust biological media, etc.31,36,39,40 Our previous studies havedemonstrated the drastically high, both in vitro and in vivodetection sensitivity of SERS nanoprobes compared to conven-tional fluorophores (i.e., organic dyes and quantum dots).

To exemplify the ratiometric detection of cancer cells ofdifferent types, two breast cancer cell lines, MDA-MB-231 andMCF-7, were chosen. MDA-MB-231 is a highly aggressive breastcancer cell line belonging to the Basal-like subtype with asignificantly over-expressing urokinase plasminogen activationreceptor (uPAR), and MCF-7 is a breast cancer cell line of theLuminal A subtype with low uPAR expression.41,42 In addition,both MDA-MB-231 and MCF-7 cell lines possess similar expressionlevels of the epidermal growth factor receptor (EGFR).42 Thus, weare in principle able to distinguish MDA-MB-231 cells from MCF-7cells by quantifying the uPAR : EGFR ratio. Based on the syntheticmethodology for SERS nanoprobes developed in our previouswork, two SERS nanoprobes encoded with different Ramansignatures were created and then conjugated with uPAR- andEGFR-specific peptides to obtain uPAR- and EGFR-targetingSERS nanoprobes, respectively. Targeting peptides are employedas the targeting elements in place of conventional antibodies totarget uPAR (or EGFR)-extracellular biomarkers.43–45 In addition,peptides as targeting elements permit flexible design and low-costin comparison to antibodies. This SERS approach permits theidentification of cancer cells by the SERS intensity ratio from SERSnanoprobes, representing the uPAR : EGFR expression ratio, andprovides facile, background-free, highly accurate identification ofcancer cells without the need for additional references.

2. Experimental section2.1 Chemicals and materials

Chloroauric acid (HAuCl4�xH2O, 99.999% trace metals basis),trisodium citrate dihydrate (HOC(COONa)(CH2COONa)2�2H2O,

Z99%), poly(vinylpyrrolidone) (PVP, (C6H9NO)n, molecularweight = 10 kg mol�1), sodium borohydride (NaBH4, Z99%),N,N-dimethylformamide (DMF, anhydrous 99.8%), sodiumhydroxide (pellets, 99.99% trace metals basis), (3-aminopropyl)-trimethoxysilane (APTMS, 97%), sodium silicate (Na2O(SiO2)x�xH2O,reagent grade), 4-nitrothiophenol (NTP, technical grade 80%),N-hydroxysuccinimide (NHS, 98%), N-ethyl-N0-(3-dimethylamino-propyl)carbodiimide (EDC, Z97.0%), RPMI 1640, fetal bovineserum (FBS), penicillin–streptomycin, and WST-1 reagent werepurchased from Sigma-Aldrich (St. Louis, MO). Diamino-1,3,5-triazine-2-thiol (DATT, 95%) was purchased from Enamine LLC(Cincinnati, OH). MDA-MB-231 and MCF-7 breast cancer celllines were purchased from the American Type Culture Collection(ATCC) (Manassas, VA). Methoxy-poly(ethylene glycol)-silane(mPEG-silane, molecular weight = 2 kg mol�1) was obtained fromLaysan Bio Inc. (Arab, AL). Phosphate buffered saline (1 � PBS,pH 7.4) solution was purchased from Quality Biology (Gaithersburg,MD). PYREXs Petri dish was purchased from Corning Incorporated(Corning, NY), and quartz coverslips were purchased from AlfaAesar (Ward Hill, MA). (3-Triethoxysilyl)propylsuccinic anhydride(TEPSA, C13H25O6Si, 495%) was purchased from Gelest Inc.(Morrisville, PA). Both uPAR-targeting peptide (VSNKYFSNIHWGC)and EGFR-targeting peptide (VRPMPLQ) with C-terminal amidationwere obtained from GenScript USA Inc. (Piscataway, NJ). All otherreagents or solvents used in this study were of analytical grade andused without further purification.

2.2 Preparation of SERS nanoprobes

Gold nanostars (GNSs) were synthesized by a seed-mediatedmethod detailed in our previous work.31,36,46 To create SERSnanoprobes encoded with NTP or DATT, a freshly preparedsolution of Raman-active molecules (NTP or DATT, 10 mM) wasadded to the GNS colloids and kept stirring for 30 min followedby addition of 5 mM freshly prepared APTMS in ethanol. Afteranother 30 min of continuous stirring, the pH value of thereaction solution was adjusted to around 9–10 by additionof NaOH aqueous solution. Then, 200 mL of freshly prepared0.54 wt% sodium silicate solution was added dropwise within30 min, and the reaction continued for one day with magneticstirring. 5 mL anhydrous ethanol was subsequently added, andthe reaction solution was allowed to stand for one more day togenerate a condensed silica layer. The reaction solution wasthen centrifuged and washed with anhydrous ethanol anddeionized water, respectively. The pellets were SERS nano-probes encoded with NTP or DATT, and re-dispersed in 1 �PBS for further use.

2.3 Functionalization of SERS nanoprobes with peptides

uPAR or EGFR targeting-SERS nanoprobes were prepared usingan established method with a slight modification.47,48 First,SERS nanoprobes were co-modified with mPEG-silane andTEPSA to improve the biocompatibility, and reduce aggregationand nonspecific binding of proteins when SERS nanoprobeswere used in biological settings.48–50 Typically, a mixture of5.65 mM mPEG-silane and 11.3 mM TEPSA was added to anethanolic solution of 50 pM SERS nanoprobes. After the reaction

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continued for 12 h with magnetic stirring, the solution wassuccessively centrifuged and washed with ethanol and deionizedwater, respectively. The resulting solids were carboxyl-terminated,PEGylated SERS nanoprobes and diluted to a concentration of50 pM with 1� PBS.

After successful modification with PEG and TEPSA, themodified SERS nanoprobes were functionalized with uPAR- andEGFR-targeting peptides by the EDC/NHS coupling chemistry. Bothpeptides were customized with C-terminal amidation. To prepareuPAR- or EGFR-SERS nanoprobes, the carboxyl-terminated, PEGy-lated SERS nanoprobes were first activated by NHS/EDC.47,48 Briefly,the co-modified SERS nanoprobes were diluted to 5 pM with1� PBS followed by addition of an aqueous solution of 25 mMNHS and 100 mM EDC. After kept stirring for 30 min, uPAR- orEGFR-targeting peptides were added to reach a final concentrationof 6 mM, and the solution was allowed to incubate for 20 h undermagnetic stirring at room temperature. After washing at least4 times with 1 � PBS, the SERS nanoprobes were cleaned viasuccessive centrifugation and washing with 1 � PBS. Finally,the uPAR- or EGFR-SERS nanoprobes were re-dispersed in1 � PBS with a concentration of 50 pM.

2.4 Cell culture

Human breast cancer cell lines, MDA-MB-231 and MCF-7, wereacquired from ATCC and incubated in RPMI 1640 supplementedwith 10% v/v FBS and 1% penicillin–streptomycin in a humidifiedincubator at 37 1C/5% CO2.

For the SERS study, the cells (1 � 106 cells per mL) weregrown on a 60 mm Petri dish with complete culture medium at37 1C for 24 h. The culture medium contains uPAR- and (or)EGFR-targeting SERS nanoprobes, and the cells were incubatedfor 1 h followed by replacement of culture medium with freshlyprepared RPMI 1640 culture medium and allowed to stand for12 h. After centrifugation, the pellets were added to a quartz-bottomed Petri dish containing new RPMI 1640 culture mediumsupplemented with 10% v/v FBS and 1% penicillin–streptomycin,and then SERS measurements were subsequently performed.

2.5 Cell viability measurement

The cellular cytotoxicity of the SERS nanoprobes was evaluatedby a WST-1 assay. MDA-MB-231 cells or MCF-7 cells were seededinto a 96-well plate at a density of 1 � 104 cells per well andincubated for 24 h in RPMI 1640 supplemented with 10% PBSand 1% penicillin–streptomycin, followed by addition of 50 pMuPAR- or EGFR-SERS nanoprobes. After incubation for 4 days,the cells were subject to the WST-1 assay.

2.6 Characterization

Transmission electron microscopy (TEM) images were collectedusing a FEI Tecnai G2 Spirit TWIN transmission electronmicroscope at an accelerating voltage of 120 kV. The samplewas added dropwise onto ultrathin Formvar-coated 200 meshcopper grids (Ted Pella, Inc.) and allowed to dry in air. Extinctionspectra were recorded on an Aviv Model 14DS UV-vis spectro-photometer (Aviv Biomedical, Lakewood, NJ).

2.7 SERS measurements

All SERS measurements were performed using a home-built,inverted high-speed confocal Raman microscope in ourlaboratory.28,32,46,48 A compact LM series solid laser of 785 nmemission wavelength (Ondax) mounted in front of a filter (LL01-785-12.5, Semrock) was used as the excitation source. High-speed XYscanning was controlled using galvanometer mirrors (GVS112,Thorlabs). A 0.65–1.25 NA, 60� oil immersion objective lens(RMS60X-PFOD, Olympus) was used to focus the laser beam andcollect the Raman-scattering photons from the sample. Thebackscattered photons were collected using a 50 mm multimodefiber (M14L01, Thorlabs), delivered to a HoloSpec f/1.8 spectro-graph (Kaiser Optical Systems, Andor) and the dispersed lightwas finally detected using an iDus CCD camera (DU420A-BEX2-DD, Andor). LabView 2013 (National Instruments) and MATLAB2013 (Mathworks) were used to control the system, acquire the data,and analyze the data. Raman and SERS spectra were recorded usinga laser power of 5 mW and an integration time of 1 s.

2.8 Statistical analysis

Statistical analyses of the experimental values were conveyed asmean � standard errors of at least three independent experiments.Statistical significance was determined using the two-tailed t-test.A difference is considered significant at p o 0.05.

3. Results and discussion3.1 SERS nanoprobes and their functionalization withpeptides

SERS nanoprobes were prepared using highly SERS-active GNSswith a near-infrared (NIR) LSPR maximum of 738 nm, and showoptimal SERS enhancement under the NIR 785 nm laser excita-tion, as demonstrated in our previous work.32,46 The overall size(core plus protruding tips) of GNSs is 41.4� 5.8 nm (Fig. S1, ESI†).NTP and DATT were respectively used as Raman molecules toencode SERS nanoprobes of unique spectral-molecular signaturesbecause of their non-overlapping Raman signatures and well-understood Au–S chemistry (Fig. 1 and Fig. S2, ESI†). In SERSnanoprobes, Raman-active (NTP or DATT) molecules were sand-wiched between the GNS core and the silica outer layer (Fig. S3,ESI†). To incorporate the targeting functionality into SERSnanoprobes and improve the biocompatibility, SERS nanoprobeswere first co-modified with TEPSA and mPEG-silane at a molarratio of 2 : 1. PEGylation of the SERS nanoprobes can improvethe biocompatibility, minimize the cytotoxicity in biologicalenvironments and inhibit nonspecific protein adsorption totheir surfaces (Fig. 1b).49–51 It has been shown that both the pHvalue and NaCl concentration have negligible effects on theSERS intensity, indicating excellent stability against variousrobust environments (Fig. S4, ESI†).

It has been reported that uPAR is significantly over-expressedin MDA-MB-231, which promotes an aggressive phenotype inbreast cancer. Thus, uPAR is of biological significance as amolecular target for breast cancer because of its accessibility onthe surface of cancer cells.41,42,52 In contrast, MCF-7 has a low

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expression level of uPAR while both MDA-MB-231 and MCF-7have similar expression to the EGFR. To target uPAR and EGFRbiomarkers, the uPAR-targeting peptide (VSNKYFSNIHWGC)and the EGFR-targeting peptide (VRPMPLQ) were exploited fortargeting molecules and conjugated onto the SERS nanoprobesthrough the well-established EDC/NHS coupling chemistry(Fig. 1a).43,44 These results in the uPAR- and EGFR-targetingSERS nanoprobes characteristic of SERS signatures of NTP andDATT, respectively (Fig. 1b). The SERS bands from NTP andDATT can be clearly distinguished due to the lack of over-lapping, as shown in the SERS spectrum of the 1 : 1 mixed SERSnanoprobes. In the following, we employed for the analysis thestrongest SERS bands at 1340 cm�1 and 913 cm�1 that areassigned to the stretching vibration of N–O of NTP and the ringbreathing mode I of the triazine ring of DATT, respectively.More interestingly, we observed that the SERS bands of DATTchanged in relative intensity at 633, 913, 945 and 981 cm�1

compared with its spontaneous Raman bands of the DATTpowder, which may be attributed to the changes in the localsymmetry on the SERS substrate surface (Fig. 1b and Fig. S2,ESI†).53 Detailed assignments of the SERS bands can be foundin Table S1 (ESI†). TEM studies further confirm a 3–5 nm silicalayer in SERS nanoprobes (Fig. 1d and Fig. S5, ESI†). Extinctionspectra show a ca. 18–22 nm red-shift of LSPR bands in bothSERS nanoprobes after silica coating, which is attributed tothe higher refractive index of silica compared with the watermedium (Fig. 1c).52 Thus, we have successfully prepared uPAR-and EGFR-SERS nanoprobes of distinct SERS signatures.

3.2 Ratiometric SERS response of mixed SERS nanoprobes

The SERS spectra of the mixed SERS nanoprobes of various molarratios were collected at a constant total (uPAR-plus EGFR-SERSnanoprobes) concentration (Fig. 2a). It can be clearly seen thatthe SERS intensity of the uPAR-SERS nanoprobes (i.e., 1340 cm�1)increases and the SERS intensity (i.e., 633 cm�1 and 913 cm�1) ofthe EGFR-SERS nanoprobes decreases with the increasing uPAR-nanoprobe : EGFR-nanoprobe ratio. We plot the intensity ratio(I@1340/I@913) as a function of the concentration percentage ofSERS nanoprobes, and the calibration curve is determined to bey = 3.00 (�0.29)�x � 0.056 (�0.16) through linear fitting (R2 = 0.96),where y is the SERS intensity ratio (I@1340/I@913), and x is the uPARconcentration percentage (Fig. 2b).

3.3 Targeted cellular SERS imaging of live cells

We first performed the in vitro SERS imaging of live cells usinguPAR- or EGFR-SERS nanoprobes to examine their targetingability. The cells investigated are breast cancer cell lines, MDA-MB-231 and MCF-7, with distinct expression of the uPAR andEGFR. Rapid, accurate identification of the two cell lines is ableto uncover the breast cancer progression and cancer biology. Ithas been demonstrated that there is no observable cytotoxicitytoward both cancer cell lines in the presence of SERS probeseven when exceeding the concentration used here (Fig. S6,ESI†). Since the SERS intensity ratio only relies on the biomarkerexpression ratio, a total SERS probe concentration of 50 pM wasused to avoid the complete binding of biomarkers as well as

Fig. 1 Encoded uPAR and EGFR targeting SERS nanoprobes. (a) Scheme of the preparation of uPAR- and EGFR-targeting SERS nanoprobes. Raman-activemolecules (NTP or DATT) are sandwiched between a highly SERS-active gold nanostar and a thin silica protective layer. The uPAR-targeting peptide(VSNKYFSNIHWGC) and the EGFR-targeting peptide (VRPMPLQ) are conjugated onto NTP- and DATT-encoded SERS nanoprobes by the EDC/NHScoupling chemistry, respectively. (b) SERS spectra of uPAR- and EGFR-SERS nanoprobes (50 pM), and their mixture at a 1 : 1 molar ratio (total concentrationof uPAR- and EGFR-SERS nanoprobes: 50 pM). The molecular structures of NTP and DATT as well as the schematic geometry of the SERS nanoprobes arealso shown (left). (c) Extinction spectra of gold nanostars and uPAR-/EGFR-targeting SERS nanoprobes. (d) Representative TEM image of DATT-encodedSERS nanoprobes.

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mitigate the cytotoxicity. Fig. 3a illustrates the detection principleof both SERS nanoprobes for the targeted cellular imaging forsimultaneous detection of the uPAR and EGFR. When the cellsare incubated with a mixture of uPAR- and EGFR-SERS nano-probes, the two SERS nanoprobes are respectively bound to theuPAR and EGFR expressed on the cell surface through a specificpeptide–biomarker interaction, similar to the antibody–antigeninteraction. Ideally, the SERS intensity ratio from the SERSnanoprobes rationally reflects the relative amount of uPAR andEGFR biomarkers on the cell. Thus, we are able to quantifythe relative expression of these two biomarkers on cells byperforming the SERS imaging after incubation with both SERSnanoprobes. Due to the multiplexing capability at the excitationof a single laser wavelength, simultaneous SERS imaging ofboth biomarkers can be achieved at a single test. As seen inFig. 3b, MDA-MB-231 exhibits a much brighter SERS image createdwith the 1340 cm�1 SERS peak in the presence of uPAR-SERSnanoprobes while the SERS image from the 913 cm�1 SERS peak isless bright in the presence of EGFR-SERS nanoprobes. As expected,MCF-7 exhibits a weak 1340 cm�1 SERS signal in the SERS image

created with the 1340 cm�1 peak in the presence of uPAR-SERSnanoprobes while the SERS image created with the 913 cm�1

peak is much brighter after incubation with the EGFR-SERSnanoprobes. However, we observed the insignificant uptake ofunconjugated SERS nanoprobes in both MDA-MB-231 cells andMCF-7 cells pretreated with uPAR-SERS nanoprobes and EGFR-SERS nanoprobes, respectively (Fig. S7, ESI†), which confirmedthe high specificity of the present SERS nanoprobes. It issuggested that distinct expressions of the uPAR and EGFR onMDA-MB-231 and MCF-7 cells are responsible for these observations.Thus, we demonstrate the targeted imaging of MDA-MB-231 andMCF-7 breast cancer cells to distinguish between the expressionof the uPAR and EGFR on the cell surfaces.

3.4 Ratiometric detection of live cells

Taking great advantages of high sensitivity and powerful multi-plexing capability using a single laser source, we employedSERS to perform simultaneous imaging detection of uPAR andEGFR biomarkers on live cells (Fig. 4). As expected, the uPAR-SERS nanoprobes bind to the uPAR expressed on the cellsurface while the EGFR-SERS nanoprobes to the EGFR throughtheir respective targeting peptides. There are more uPAR-SERSnanoprobes appearing around the MDA-MB-231 cells thanEGFR-SERS nanoprobes. On the other hand, we can see a fewEGFR-SERS nanoprobes but negligible uPAR-SERS nanoprobesappearing around MCF-7 cells. MDA-MB-231 expresses moreuPAR receptors than MCF-7 while they have a similar EGFRexpression, so that both cell lines have significantly differentuPAR : EGFR ratios. The percentage of individual SERS nano-probes around the cells can be derived from the SERS intensityratio over the whole cell based on the calibration curve (Fig. 2b).It is reasoned that the amount of SERS nanoprobes targeted tothe cells is proportional to the expression levels of the uPARand EGFR. The statistical results obtained from 19 cells showthat the IuPAR/IEGFR ratio of the MDA-MB-231 cells is a muchhigher middle value around 1.8 while it is around 0.48 forMCF-7 cells (Fig. 4b). Thus, we are able to quantitativelydiscriminate between MDA-MB-231 and MCF-7 by quantifying theuPAR : EGFR expression ratio using the encoded SERS nanoprobeswhich target to the uPAR and EGFR biomarkers on the cell surface.

Identification of different types of breast cancer cells hasprognostic and therapeutic implications for patients with breastcancer. Numerous efforts have been made to distinguish betweendifferent types of cancer cells or between cancer cells and normalcells based on cell surface biomarker expression. The present SERSapproach exploits the difference in expression levels of the uPARand EGFR to ratiometrically identify MDA-MB-231 and MCF-7cells. The uPAR/EGFR expression ratio is derived from the SERSintensity ratio of uPAR- to EGFR-targeting nanoprobes. Theratiometric strategy excludes the background interference andimproves the detection accuracy of cancer cells with highsensitivity and excellent specificity. Unlike the conventionalfluorescence method, SERS has no photobleaching, less auto-fluorescence interference, and requires only a single laserwavelength for multiplexing. The unique features associatedwith SERS make it ideal as a screening tool for live cancer cells.

Fig. 2 SERS spectra of the mixed SERS nanoprobes and the correspondingcalibration curve. (a) SERS spectra of the mixed uPAR- and EGFR-SERSnanoprobes (total concentration: 50 pM) with various molar ratios (0 : 5,1 : 4, 2 : 3, 1 : 1, 3 : 2, 4 : 1 and 5 : 0 for uPAR- : EGFR-SERS nanoprobes).(b) Plot of I@1340/I@913 as a function of their concentration percentage.The SERS intensity of the 1340 cm�1 band is assigned to the stretchingvibration of N–O of NTP in the uPAR-SERS nanoprobe, and the 913 cm�1

band is from the ring breathing mode I of the triazine ring of DATT in theEGFR-SERS nanoprobe. Their intensity ratio is linearly correlated with theuPAR- : EGFR-SERS nanoprobe ratio. The calibration curve is determinedto be y = 3.00 (�0.29)�x � 0.056 (�0.16) (R2 = 0.96), where y is the SERSintensity ratio (I@1340/I@913) and x is the uPAR concentration percentage.

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Our SERS approach utilizes the intrinsic cell-resident marker,EGFR, as the internal reference, which would be particularlyadvantageous for accurate identification of cancer cells because

expression of a biomarker may occur in both cancer cells andnon-cancer cells to a different degree. The ratiometric strategyexploits the ratio of biomarker expression levels to reduce theinterference from other cells. In addition, the ratiometricstrategy developed in this work permits identification of cancercells, independent of the variations in cell concentration,measurement conditions and nanoprobe concentration, andis superior to conventional approaches for cancer cell detectionbased on the evaluation of the absolute expression level ofbiomarkers. Few studies have focused on the developmentof ratiometric SERS biosensors for detection of biomarkers ofpathological significance,54–56 but our current work further movesforward the ratiometric SERS strategy for cell discrimination,taking advantages of plasmonic GNSs with highly SERS activity,unique design of SERS nanoprobes and intrinsic extracellularbiomarker ratios.

4. Conclusions

In summary, we have reported a SERS-based strategy forquantitative ratiometric discrimination of two different types ofbreast cancer cell lines, MDA-MB-231 and MCF-7. SERS nanoprobesare encoded with unique SERS signatures and conjugated withuPAR- or EGFR-targeting peptides, which target cell-surface proteins,uPAR and EGFR, distinctly expressed on MDA-MB-231 and MCF-7.We demonstrated that simultaneous quantitative detection ofboth protein biomarkers on single live cells enables ratiometricdiscrimination between MDA-MB-231 and MCF-7. MDA-MB-231

Fig. 3 In vitro SERS imaging of live cells. (a) Schematic illustration of simultaneous SERS imaging of uPAR and EGFR biomarkers expressed on cells usinguPAR- and EGFR-SERS nanoprobes encoded with distinct Raman signatures. When a mixture of uPAR- and EGFR-SERS nanoprobes are incubated withcells, the uPAR and EGFR biomarkers on the cell surface are bound with their respective SERS nanoprobes through specific peptide–biomarkerinteractions. The relative amount of the two biomarkers can be determined by the SERS intensity ratio of SERS nanoprobes over the whole cell. Overlaidimages of the bright-field image of a single cell with the uPAR and EGFR alone or both of them are also shown to illustrate the concept of simultaneousimaging of both biomarkers. (b) Bright-field images and overlaid images of MDA-MB-231 cells and MCF-7 breast cancer cell lines in the presence ofuPAR- or EGFR-SERS nanoprobes. The concentration of the SERS probes is 50 pM for both MDA-MB-231 and MCF-7 cell lines. The SERS images areconstructed using the SERS intensity of 1340 cm�1 for uPAR-SERS nanoprobes and (or) 913 cm�1 for EGFR-SERS nanoprobes, respectively. The SERSimaging is performed with 50 � 50 pixels over an 80 mm � 80 mm area (5 mW power and 1 s integration time). The images are in false color: redrepresents the 913 cm�1 SERS peak intensity and blue represents the 1340 cm�1 SERS peak intensity.

Fig. 4 Ratiometric detection of live cells by SERS imaging. (a) Bright-fieldimages (top) and overlaid SERS images (bottom) of MDA-MB-231 andMCF-7 cells in the presence of both uPAR- and EGFR-SERS nanoprobes(1 : 1 m/m). The mixed SERS nanoprobe solution consists of uPAR- andEGFR-SERS nanoprobes (25 pM for each) in the RPMI 1640 incubationmedium. The images are in false color: red represents the 913 cm�1 SERSpeak intensity and blue represents the 1340 cm�1 SERS peak intensity. TheSERS intensity ratio can be approximately transformed into the expressionratio of biomarkers on the cell surface. The SERS measurement is per-formed with 50 � 50 pixels over an 80 mm � 80 mm area (5 mW power and1 s integration time). (b) Box chart of the expression ratio of the uPAR andEGFR on MDA-MB-231 and MCF-7 cells, quantified by SERS imaging.Statistical results are from 19 cells for both cell lines. All the results ofthe uPAR : EGFR expression ratio on both MDA-MB-231 and MCF-7 arealso shown. IuPAR and IEGFR are calculated based on the average SERSintensities at 1340 cm�1 and 913 cm�1 over a single cell, respectively.

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showed significant aggregation of uPAR-SERS nanoprobes butthe aggregation is low for MCF-7 cells. Both cell lines havesimilar and relatively weak SERS signals when EGFR-SERSnanoprobes were used. The two cell lines show an uPAR : EGFRexpression ratio of ca. 1.8 and 0.48 for MDA-MB-231 and MCF-7cells, respectively. In ratiometric detection, the EGFR biomarkeris used as the internal reference, and the SERS intensity ratioonly depends on the biomarker expression, regardless of thecell concentration, measurement conditions and nanoprobeconcentrations. The present method excludes the backgroundinterference from normal cells or other cancer cells that mayhave biomarker expressions as well. Taking into account themerits of high sensitivity and powerful multiplexing capabilityof SERS, and accuracy due to the ratiometric discriminationwithout any external reference, the ratiometric SERS methodholds great potential for cancer cell screening and diagnosticapplications of cancer along with the development of cellularbiology for biomarker expression profiling.

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

M. L. acknowledges financial support from the National ThousandYoung Talents Program of China, the National Natural ScienceFoundation of China (No. 51871246), the Innovation-DrivenProject of Central South University (No. 2018CX002) and theHunan Provincial Science & Technology Program (No. 2017XK2027).

References

1 S. S. Pinho and C. A. Reis, Nat. Rev. Cancer, 2015, 15, 540.2 M. Huang, A. Shen, J. Ding and M. Geng, Trends Pharmacol.

Sci., 2014, 35, 41–50.3 T. Li, H. J. Kung, P. C. Mack and D. R. Gandara, J. Clin.

Oncol., 2013, 31, 1039–1049.4 E. C. Dreaden and M. A. El-Sayed, Acc. Chem. Res., 2012, 45,

1854–1865.5 C. Alix-Panabieres and K. Pantel, Nat. Rev. Cancer, 2014, 14,

623–631.6 R. Rosell, T. G. Bivona and N. Karachaliou, Lancet, 2013,

382, 720–731.7 S. Mittal, H. Kaur, N. Gautam and A. K. Mantha, Biosens.

Bioelectron., 2017, 88, 217–231.8 A. M. Aravanis, M. Lee and R. D. Klausner, Cell, 2017, 168,

571–574.9 F. K. Lu, S. Basu, V. Igras, M. P. Hoang, M. Ji, D. Fu,

G. R. Holtoma, V. A. Neele, C. W. Freudigera, D. E. Fisher andX. S. Xie, Proc. Natl. Acad. Sci. U. S. A., 2015, 112, 11624–11629.

10 R. Downey, L. Murillo, T. McHale, T. Wallace, C. Seufert,A. Schetter, T. Dorsey, C. Johnson, R. Goldman, C. Loffredo,P. Yan, F. Sullivan, F. Giles, F. Wang-Johanning, S. Ambsand S. Glynn, Cancer Res., 2015, 75, 536.

11 Z. A. Nima, M. Mahmood, Y. Xu, T. Mustafa, F. Watanabe,D. A. Nedosekin, M. A. Juratli, T. Fahmi, E. I. Galanzha,J. P. Nolan and A. G. Basnakian, Sci. Rep., 2014, 4, 4752.

12 L. A. Sklar, M. B. Carter and B. S. Edwards, Curr. Opin.Pharmacol., 2007, 7, 527–534.

13 K. Abe, L. Zhao, A. Periasamy, X. Intes and M. Barroso, PLoSOne, 2013, 8, e80269.

14 E. Y. Lukianova-Hleb, X. Ren, J. A. Zasadzinski, X. Wu andD. O. Lapotko, Adv. Mater., 2012, 24, 3831–3837.

15 F. Fabbri, S. Carloni, W. Zoli, P. Ulivi, G. Gallerani, P. Fici,E. Chiadini, A. Passardi, G. L. Frassineti, A. Ragazzini andD. Amadori, Cancer Lett., 2013, 335, 225–231.

16 A. Pallaoro, M. R. Hoonejani, G. B. Braun, C. D. Meinhartand M. Moskovits, ACS Nano, 2015, 9, 4328–4336.

17 M. Colombo, S. Mazzucchelli, V. Collico, S. Avvakumova,L. Pandolfi, F. Corsi, F. Porta and D. Prosperi, Angew. Chem.,Int. Ed., 2012, 51, 9272–9275.

18 E. Q. Song, J. Hu, C. Y. Wen, Z. Q. Tian, X. Yu, Z. L. Zhang,Y. B. Shi and D. W. Pang, ACS Nano, 2011, 5, 761–770.

19 J. K. Herr, J. E. Smith, C. D. Medley, D. Shangguan andW. Tan, Anal. Chem., 2006, 78, 2918–2924.

20 X. Ji, F. Peng, Y. Zhong, Y. Su, X. Jiang, C. Song, L. Yang,B. Chu, S. T. Lee and Y. He, Adv. Mater., 2015, 27, 1029–1034.

21 M. Fritzsche and G. Charras, Nat. Protoc., 2015, 10, 660–680.22 B. Shan, Y. Pu, Y. Chen, M. Liao and M. Li, Coord. Chem.

Rev., 2018, 371, 11–37.23 M. Li, S. K. Cushing and N. Wu, Analyst, 2015, 140, 386–406.24 S. Nie and S. R. Emory, Science, 1997, 275, 1102–1106.25 K. Kneipp, Y. Wang, H. Kneipp, L. T. Perelman, I. Itzkan,

R. R. Dasari and M. S. Feld, Phys. Rev. Lett., 1997, 78, 1667.26 S. Lee, H. Chon, J. Lee, J. Ko, B. H. Chung, D. W. Lim and

J. Choo, Biosens. Bioelectron., 2014, 51, 238–243.27 Y. Li, X. Qi, C. Lei, Q. Yue and S. Zhang, Chem. Commun.,

2014, 50, 9907–9909.28 Q. Jin, M. Li, B. Polat, S. K. Paidi, A. Dai, A. Zhang,

J. V. Pagaduan, I. Barman and D. H. Gracias, Angew. Chem.,Int. Ed., 2017, 129, 3822–3826.

29 M. Rycenga, C. M. Cobley, J. Zeng, W. Li, C. H. Moran, Q. Zhang,D. Qin and Y. Xia, Chem. Rev., 2011, 111, 3669–3712.

30 Y. Wang, B. Yan and L. Chen, Chem. Rev., 2012, 113,1391–1428.

31 M. Li, J. Zhang, S. Suri, L. J. Sooter, D. Ma and N. Wu, Anal.Chem., 2012, 84, 2837–2842.

32 M. Li, S. R. Banerjee, C. Zheng, M. G. Pomper and I. Barman,Chem. Sci., 2016, 7, 6779–6785.

33 R. Liu, J. Zhao, G. Han, T. Zhao, R. Zhang, B. Liu, Z. Liu,C. Zhang, L. Yang and Z. Zhang, ACS Appl. Mater. Interfaces,2017, 9, 38222–38229.

34 M. Vendrell, K. K. Maiti, K. Dhaliwal and Y. T. Chang,Trends Biotechnol., 2013, 31, 249–257.

35 L. A. Lane, X. Qian and S. Nie, Chem. Rev., 2015, 115,10489–10529.

36 M. Li, S. K. Cushing, J. Zhang, J. Lankford, Z. P. Aguilar,D. Ma and N. Wu, Nanotechnology, 2012, 23, 115501.

37 M. Li, S. K. Cushing, H. Liang, S. Suri, D. Ma and N. Wu,Anal. Chem., 2013, 85, 2072–2078.

Journal of Materials Chemistry B Paper

822 | J. Mater. Chem. B, 2019, 7, 815--822 This journal is©The Royal Society of Chemistry 2019

38 M. Li, H. Gou, I. Al-Ogaidi and N. Wu, ACS SustainableChem. Eng., 2013, 1, 713–723.

39 Y. Lai, S. Sun, T. He, S. Schlucker and Y. Wang, RSC Adv.,2015, 5, 13762–13767.

40 S. Sun, D. Thompson, U. Schmidt, D. Graham and G. J. Leggett,Chem. Commun., 2010, 46, 5292–5294.

41 K. Subik, J. F. Lee, L. Baxter, T. Strzepek, D. Costello,P. Crowley, L. Xing and M.-C. Hung, Breast Cancer, 2010,4, 35–41.

42 A. M. LeBeau, S. Duriseti, S. T. Murphy, F. Pepin, B. Hann,J. W. Gray, H. F. VanBrocklin and C. S. Craik, Cancer Res.,2013, 73, 2070–2081.

43 R. Devulapally, N. M. Sekar, T. V. Sekar, K. Foygel, T. F. Massoud,J. K. Willmann and R. Paulmurugan, ACS Nano, 2015, 9,2290–2302.

44 A. S. Thakor, R. Luong, R. Paulmurugan, F. I. Lin, P. Kempen,C. Zavaleta, P. Chu, T. F. Massoud, R. Sinclair and S. S. Gambhir,Sci. Transl. Med., 2011, 3, 79ra33.

45 J. Zhou, B. P. Joshi, X. Duan, A. Pant, Z. Qiu, R. Kuick,S. R. Owens and T. D. Wang, Clin. Transl. Gastroenterol.,2015, 6, e101.

46 M. Li, J. W. Kang, R. R. Dasari and I. Barman, Angew. Chem.,Int. Ed., 2014, 53, 14115–14119.

47 M. Li, S. K. Cushing, J. Zhang, S. Suri, R. Evans, W. P. Petros,L. F. Gibson, D. Ma, Y. Liu and N. Wu, ACS Nano, 2013, 7,4967–4976.

48 M. Li, J. W. Kang, S. Sukumar, R. R. Dasari and I. Barman,Chem. Sci., 2015, 6, 3906–3914.

49 S. S. Lucky, N. Muhammad Idris, Z. Li, K. Huang, K. C. Sooand Y. Zhang, ACS Nano, 2015, 9, 191–205.

50 L. Cui, Q. Lin, C. S. Jin, W. Jiang, H. Huang, L. Ding, N. Muhanna,J. C. Irish, F. Wang, J. Chen and G. Zheng, ACS Nano, 2015, 9,4484–4495.

51 B. Kang, L. A. Austin and M. A. El-Sayed, ACS Nano, 2014, 8,4883–4892.

52 K. M. Mayer and J. H. Hafner, Chem. Rev., 2011, 111, 3828–3857.53 M. D. King, S. Khadka, G. A. Craig and M. D. Mason, J. Phys.

Chem. C, 2008, 112, 11751–11757.54 Q. Xu, W. Liu, L. Li, F. Zhou, J. Zhou and Y. Tian, Chem.

Commun., 2017, 53, 1880–1883.55 S. He, Y. M. E. Kyaw, E. K. M. Tan, L. Bekale, M. W. C. Kang,

S. S. Y. Kim, I. Tan, K. P. Lam and J. C. Y. Kah, Anal. Chem.,2018, 90, 6071–6080.

56 Y. W. Wang, A. Khan, M. Som, D. Wang, Y. Chen, S. Y. Leigh,D. Meza, P. Z. McVeigh, B. C. Wilson and J. T. Liu, Technology,2014, 2, 118–132.

Paper Journal of Materials Chemistry B

S1

Supporting Information

Surface-enhanced Raman Spectroscopy (SERS) Nanoprobes for Ratiometric Detection of Cancer Cells

Linhu Li, Mengling Liao, Yingfan Chen, Beibei Shan and Ming Li*

Electronic Supplementary Material (ESI) for Journal of Materials Chemistry B.This journal is © The Royal Society of Chemistry 2018

S2

Figure S1. TEM image of gold nanostars used for preparation of SERS nanoprobes and size

distribution statistically obtained from TEM image of GNSs using the ImageJ software. More than

150 GNSs were counted. The size of GNSs is called the overall size including the core and

protruding tips, the model of which is schematically illustrated above. It can be seen that the overall

size of GNSs is about 41.4±5.8 nm.

S3

Figure S2. Spontaneous Raman spectra of NTP and DATT powder. The spectra were collected

under the excitation of 785 nm with 5 mW power and 1 s integration time.

S4

Figure S3. Schematic illustration of NTP or DATT encoded SERS tags. (a) Molecular structures of

Raman reporters, 4-nitrothiophenol (NTP) and diamino-1,3,5-triazine-2-thiol (DATT), (b) Au-S

interaction to form NTP (or DATT)-GNS complexes, and (c) synthesis progress of NTP or DATT

encoded SERS tags. Raman reporters (NTP or DATT) are chemically bound onto the Au surface

through the strong Au-S bond and then a SiO2 layer are coated to encapsulate the encoded SERS

tags, forming the sandwich structure, Au@Ramam-reporters@SiO2. The SiO2 layer possesses

excellent biocompatible and can be used for flexible bioconjugation with biomolecules such as

proteins, peptides and nucleic acids.

S5

Figure S4. Stability of SERS nanoprobes without peptides conjugation. SERS spectra of SERS

nanoprobes in the solution of (A) pH=2.5 and pH=11.6, (B) 1⨯PBS solution with 1.0 mM and 1.0 M

NaCl. All results show that the present SERS nanoprobes have excellent stability against robust

environments (e.g., pH, NaCl concentration).

S6

Figure S5. TEM image of (A) uPAR-SERS nanoprobes and (B) EGFR SERS nanoprobes. It can be

seen that 3-5 nm silica layer was coated onto the GNS surface.

S7

Table S1. Peak assignments of normal Raman bands and SERS bands of NTP, DATT, NTP-

encoded SERS nanoprobes and DATT-encoded SERS nanoprobes 1-4

NTP NTP-encoded SERS nanoprobes

DATT DATT-encoded SERS nanoprobes

Peak (cm-1)

Assignment Peak (cm-1)

Assignment Peak (cm-1)

Assignment Peak (cm-1)

Assignment

727 Wagging vibrations of C-H, C-S, C-C

618 Ring breathing mode II of in-plane deformation of triazine ring

631 Ring breathing mode II of in-plane deformation of triazine ring

855 Wagging vibration of C-H

855 Wagging vibration of C-H

934 Ring breathing mode I of triazine ring

913 Ring breathing mode I of triazine ring

1080 Stretching vibration of C-S

1180 Stretching vibration of C-S

970 Ring breathing 992 Ring breathing

1100 Bending vibration of C-H

1100 Bending vibration of C-H

1516 C-N stretching;Ring C-N stretching; Symmetry NH2 bending; Asymmetry NH2 bending

1206-1242

H-N-H rocking

1332 Stretching vibration of N-O

1332 Stretching vibration of N-O

1496 Side chain C-N breathing

1575 Stretching vibration of phenyl ring

1575 Stretching vibration of phenyl ring

1 M. Li, J. W. Kang, R. R. Dasari and I. Barman, Angew. Chem. Int. Ed., 2014, 53, 14115 –14119.2 M. Li, J. W. Kang, S. Sukumar, R. R. Dasari and I. Barman, Chem. Sci., 2015, 6, 3906–3914.3 S. Gunasekaran, K. Srinivasan and S. X. J. Raja, Proc. Indian Natn. Sci. Acsd., 1993, 59, 347-352.4 E. Koglin, B. J. Kip and R. J. Meier, J. Phys. Chem. 1996, 100, 5078-5089.

S8

Figure S6. Cell viability of MDA-MB-231 cells and MCF-7 cells incubated with uPAR or EGFR-

SERS nanoprobes. MDA-MB-231 cells were incubated with uPAR-SERS nanoprobes (200 pM),

and MCF-7 cells were incubated with EGFR-SERS nanoprobes (200 pM). It can be clearly seen that

no significant cytotoxicity was observed for both SERS nanoprobes used in this work.

S9

Figure S7. SERS images overlaid with bright-field images of uPAR-SERS nanoprobe-pretreated

MDA-MB-231 cells and EGFR-SERS nanoprobe-pretreated MCF-7 cells incubated with

unconjugated NTP-encoded SERS nanoprobes (50 pM). It is clearly seen that there is no significant

SERS signal observed in both cell lines, indicating high specificity of both SERS nanoprobes toward

to uPAR and EGFR expressed on the cell surface.