Improved SERS-Active Nanoparticles with Various Shapes for CTC

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Improved SERS-Active Nanoparticles with Various Shapes for CTC Detection without Enrichment Process with Supersensitivity and High Specificity Xiaoxia Wu, Yuanzhi Xia, Youju Huang, Juan Li, Huimin Ruan, Tianxiang Chen, Liqiang Luo, Zheyu Shen, and Aiguo Wu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b07205 • Publication Date (Web): 19 Jul 2016 Downloaded from http://pubs.acs.org on July 22, 2016

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ACS Applied Materials & Interfaces

Improved SERS-Active Nanoparticles with Various Shapes for CTC Detection without Enrichment Process with Supersensitivity and High Specificity

Xiaoxia Wu,a,b Yuanzhi Xia,a,b Youju Huang,a Juan Li,a Huimin Ruan,a Tianxiang Chen,a Liqiang Luo,b Zheyu Shen,a,* and Aiguo Wua,*

a

Key Laboratory of Magnetic Materials and Devices & Key Laboratory of Additive Manufacturing

Materials of Zhejiang Province & Division of Functional Materials and Nanodevices, Ningbo Institute of Materials Technology and Engineering, Chinese Academy of Sciences, Ningbo 315201, P. R. China b

College of Sciences, Shanghai University, 99 Shangda Road, Shanghai 200444, China

*Corresponding authors Email: [email protected], or [email protected] Tel: +86 574 87617278, or +86 574 86685039; Fax: +86-57486685163

1

ACS Paragon Plus Environment


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ABSTRACT Circulating tumor cells (CTCs) received more and more attention in medical biology and clinical practice, especially diagnosis, prognosis and cancer treatment monitoring. The detection of CTCs within the large number of healthy blood cells is a big challenge due to their rarity, which requires a detection method with supersensitivity and high specificity. In this study, we developed three kinds of new nanoparticles with function of surface-enhanced Raman scattering (SERS) based on spherical gold nanoparticles (AuNPs), gold nanorods (AuNRs) and gold nanostars (AuNSs) with similar particle size, similar modifications and different shapes for CTC detection without enrichment process from the blood. The nanoparticles possess strong SERS signal due to modification of 4-mercaptobenzoic acid (4-MBA) (i.e. Raman reporter molecule), possess excellent specificity due to stabilization of reductive bovine serum albumin (rBSA) to reduce the non-specific catching or uptake by healthy cells in blood, and possess high sensitivity due to conjugation of folic acid (FA) (i.e. a targeted ligand) to identify CTCs. Under the optimized experimental conditions, the results of detection demonstrate that these nanoparticles could all be utilized for CTC detection without enrichment process from the blood with high specificity, and the AuNS-MBA-rBSA-FA is the best one due to its supersensitivity, whose limit of detection (i.e. 1 cell/mL) is much lower than the currently reported lowest value (5 cells/mL).

KEYWORDS: SERS; gold nanoparticles with various shape; CTC detection; without enrichment process; blood samples

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1. INTRODUCTION Cancer cell diffusion by local invasion or metastasis to distant organs could result in death of patients, which is a severe world-wide medical problem and difficult to be solved.1-4 The cancer diagnosis at early stage is very significant for enhancing the survival rates of cancer patients, and the blood testing that is a non-invasive method and easy to perform is widely used for disease diagnosis.5,6 Hence, more and more attention has recently been focused on circulating tumor cells (CTCs), which are epithelial cancer cells fallen from solid tumors to blood vessels and the concentration is very low. CTCs are considered as a good indicator for the presence of a primary tumor and/or metastasis.7-10 CTCs play a central role in biomedical research and clinical practice, especially diagnosis, prognosis and cancer treatment monitoring.11-13 However, detection of CTCs within the large number of normal blood cells is a big challenge due to their rarity (just a few CTCs amongst around 10 million leukocytes and 5 billion erythrocytes in 1.0 mL of blood), which requires a detection method with supersensitivity and high specificity.14 Surface-enhanced Raman scattering (SERS) technique can amplify the spectroscopic signals of single molecules enormously based on the unique property of noble metallic nanoparticles, i.e. localized surface plasmon resonance (LSPR).15-19 It has been developed to be a supersensitive and universally analytical technology, which can provide detection limits even down to the single molecule level in lots of scientific fields.20-22

Therefore, SERS-based method can offer a strong

analytical tool with a variety of strategies for CTC detection.23-27 Wen et al. have successfully developed novel magnetic nanospheres, which have an ability of quick-response and can be used for CTC capturing and detection with a high sensitivity. The CTC capturing efficiency have been verified via real blood samples from cancer patients.28 Zhang et al. have developed a new simple strategy based on nitrocellulose membrane and SERS imaging method on a large scale, which can be used for enrichment and detection of CTCs.29 Currently, most of the reported CTC detection strategies based on SERS need an enrichment process of few CTCs from healthy cells in the blood, which enhances the detection cost and 3



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detection time. In our previous study, we developed new SERS-active nanoparticles based on spherical gold nanoparticles (AuNPs) for CTC detection in blood samples without enrichment process. The sensitivity is to some extent high with a 5 cells/mL of LOD (i.e. limit of detection), which is not higher than other reported LOD values.30 However, the sensitivity of the SERS-active nanoparticles for direct CTCs’ detection in blood samples needs to be further enhanced to realize the early diagnosis of cancer. The electromagnetic (EM) enhancement could be very high up to ~1010-1011, which is much higher than the chemical enhancement and should be main contribution to SERS.31 The morphology of the SERS-active nanoparticles can significantly affect the EM enhancement.32 Typically, asymmetric shape of the nanoparticles can induce new LSPR emerging in the Raman spectrum in a wide range.16,33,34 Therefore, the shape is a very important factor to enhance the LSPR and SERS signal of the noble metal nanoparticles. To enhance the sensitivity of SERS-active nanoparticles for CTCs’ detection, we developed three kinds of new SERS-active nanoparticles with similar particle size, similar modifications and different shapes for CTC detection without enrichment process in blood samples. The SERS-active nanoparticles with different shapes are respectively constructed based on AuNPs, gold nanorods (AuNRs) and gold nanostars (AuNSs) as shown in Scheme 1. All the three SERS-active nanoparticles possess strong SERS signal due to modification of 4-mercaptobenzoic acid (4-MBA) (i.e. Raman reporter molecule), possess excellent specificity due to stabilization of reductive bovine serum albumin (rBSA) to reduce the non-specific catching or uptake by healthy cells in blood, and possess high sensitivity due to conjugation of folic acid (FA) (i.e. a targeted ligand), which can recognize CTCs of various cancers including ovarian, brain, kidney, breast, lung, cervical and nasopharyngeal cancers due to their overexpressing of folate receptor alpha (FRα).35,36 However, it’s unclear that which shape of the SERS-active nanoparticles is best for detection of CTCs because the best dosage of 4-MBA, rBSA and FA may be different due to the different stability of the nanoparticles. In this study, the modification method and the dosage of 4-MBA, rBSA and FA are 4


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investigated in accordance with the nanoparticle stability and SERS signal intensity. The three optimized SERS-active nanoparticles with similar particle size, similar modifications and different shape (without aggregation) are utilized for detection of tumor cells from blood samples and the final LOD is enhanced to 1 cell/mL.

Scheme 1. Scheme of the construction of our SERS-active nanoparticles with various shapes for CTC detection without enrichment process in blood samples.

2. MATERIALS AND METHODS 2.1 Materials Gold (III) chloride trihydrate (HAuCl4·3H2O), sodium citrate dihydrate (Na3Ct·2H2O), hydroxylamine hydrochloride (NH2OH·HCl), silver nitrate (AgNO3), 30% hydrogen peroxide (30% H₂O₂) and L-ascorbic acid (C6H8O6) were purchased from Sinopharm Chemical Reagent Co. Ltd (Shanghai, China). Bovine serum albumin (BSA), folic acid (FA), 4-mercaptobenzoic acid (4-MBA), sodium borohydride (NaBH4), hexadecyl trimethyl ammonium Bromide (CTAB), sodium hydroxide

(NaOH),

polyvinyl

pyrrolidon

(PVP)

and

1-ethyl-3-(3-dimethylaminopropyl)

carbodiimide hydrochloride (EDC·HCl) were obtained from Aladdin Reagent Co. Ltd (Shanghai, China). N-Hydroxysuccinimide (NHS) was purchased from Sigma-Aldrich. Trypsin-EDTA (0.25%) 5



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and Dulbecco’s Modified Eagle Medium (DMEM) were purchased from Gibco ® Life Technologies. Peripheral blood lymphocyte separation medium were purchased from Beijing Slolarbio Science and Technology Co. Ltd (China). Other chemicals used in this study were all analytical grade and used without any purification.

2.2 Synthesis of the AuNPs, AuNRs and AuNSs The AuNPs were synthesized by an amended seed growth method.37,38 First, 3.0 mL of HAuCl4 solution (5.0 mM) was first mixed with 45 mL of MilliQ water and the mixture was heated to be boiling. 2.5 mL of Na3Ct aqueous solution (1.0%) was then immediately added to the above-mentioned mixtures during strong stirring, and the reaction continued for 3.0 min (the mixture color shifted to red from yellow). After that, the mixture was kept at room temperature for natural cooling. The obtained gold seeds were kept in the fridge at 4 oC for further use. Second, HAuCl4 aqueous solution (3.0 mL, 5.0 mM) and PVP aqueous solution (4.0 mL, 0.7 mM) were added into 60 mL MilliQ water and stirred for 10 min. Then, 3.2 mL of NH2OH·HCl (40 mM) was dropwise added into the mixture at room temperature. After 3.0 min, the previously-mentioned gold seed solution (3.2 mL) was poured into the above mixture. The reaction was continued for 5.0 minutes (the solution color changes to be deep red). The resulted AuNPs were centrifuged (6500 rpm, 10 min), washed twice, and stored at 4 °C. The AuNRs were also synthesized by a seed growth method, and the synthesis approach of gold seeds was close to a reported one with slight modifications.39 Typically, HAuCl4 (0.2 mL, 5.0 mM) was mixed with CTAB solution (4.0 mL, 0.1 M) and stirred for 3 min. 24 µL of NaBH4 (100 mM) was then charged to the mixture during stirring (the mixture color changed to brown). After 2.0 min, the stirring was stopped, and the mixture was aged at room temperature for over 2.0 h to obtain the gold seeds. Second, AgNO3 aqueous solution (30 µL, 0.1 M) and HAuCl4 aqueous solution (3.0 mL, 5.0 mM) were charged to CTAB stock solution (30 mL, 0.1 M) under stirring. After 10 min, NaOH (113 µL, 1.0 M) and H2O2 (0.3 mL, 30%) were charged to the above mixture. After 10 min, 30 µL 6


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of the above gold seed solution was poured into the above solution. After further 5.0 min of stirring, the solution was kept in the dark for at least 2.0 h. The resulted AuNRs were centrifuged (8500 rpm, 10 min), washed thrice, and stored at 4 °C. AuNSs were also synthesized by a seed-mediated growth protocol with minor modifications.40 First, CTAB aqueous solution (1.0 mL, 100 mM), HAuCl4 (2.75 mL, 5.0 mM) and AgNO3 (50 µL, 50 mM) were separately added into 45 mL of MilliQ water. After 20 min of stirring, 565 µL of ascorbic acid (10 mg/mL) was charged to the mixture. When the color of the mixture become transparent, the above-mentioned gold seed solution (50 µL), which was same with that used for the preparation of AuNPs, were added. After the color of the solution turned to blue/brown (i.e. the transmission light is blue and the scattering light is brown), it was centrifuged (7500 rpm, 10 min), washed twice, and stored at 4 °C.

2.3 Synthesis of rBSA-FA First, FA (40.0 mg), EDC (32.0 mg) and NHS (19.2 mg) were added into PBS (50 mL, pH7.4, 10 mM) and the mixture was kept at room temperature under stirring for 8.0 h of reaction to obtain the activated FA (i.e. FA-NHS). Second, 260 µL of NaBH4 (1.0 M) was added into 20 mL of BSA (20 mg/mL) under stirring at room temperature. After 1.0 h of reaction, the rBSA was obtained. The obtained rBSA solution (5.0 mL, 20 mg/mL) was then mixed with 50 mL of the above-mentioned FA-NHS solution. After 4.0 h of reaction at room temperature, the solution of rBSA-FA was purified via centrifugal ultrafiltration (Amicon Ultra-15, Millipore, 3000 MWCO). The purified rBSA-FA was finally dissolved in MilliQ water and kept in the fridge as stock solution.

2.4 Preparation of the SERS-active nanoparticles by a three-step method First, the AuNPs, AuNRs or AuNSs were modified with 4-MBA (a Raman reporter molecule). Typically, 4-MBA in THF (10.0 µL) with different concentrations (Table S1) was separately mixed with the solution of AuNPs, AuNRs or AuNSs (2.0 mL, 0.02 mg/mL) under stirring at room 7



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temperature. After 2.0 min of reaction, the 4-MBA-encoded AuNPs, AuNRs or AuNSs (i.e. AuNP-MBA, AuNR-MBA or AuNS-MBA) were obtained. Second, the obtained AuNP-MBA, AuNR-MBA or AuNS-MBA was stabilized using rBSA. 20 µL of the rBSA aqueous solutions with different concentrations (Table S2, or S3) were respectively mixed with the solution of AuNP-MBA, AuNR-MBA or AuNS-MBA solution (2.0 mL) under stirring at room temperature for 5.0 min. Thirdly, FA was connected to the obtained AuNP-MBA-rBSA, AuNR-MBA-rBSA or AuNS-MBA-rBSA. 0.1~1.0 mL of the above-mentioned FA-NHS (0.8 mg/mL) were charged to 8.0 mL of the AuNP-MBA-rBSA, AuNR-MBA-rBSA or AuNS-MBA-rBSA solution. The final solution was adjusted to be 10.0 mL using pure water and stirred at room temperature for 4 h of reaction.

2.5 Synthesis of the SERS-active nanoparticles by a two-step method First, the AuNPs, AuNRs or AuNSs were modified with 4-MBA in the light of a reported approach.30 4-MBA in THF (5.0 µL) with different concentrations (Table 1-3) was separately mixed with the AuNPs, AuNRs or AuNS (4.0 mL, 0.02 mg/mL) under stirring at room temperature. After 2.0 min of reaction, the 4-MBA-encoded AuNPs, AuNRs or AuNSs (i.e. AuNP-MBA, AuNR-MBA or AuNS-MBA) were obtained for next use. Second, the AuNP-MBA, AuNR-MBA or AuNS-MBA was stabilized with rBSA-FA to reduce the non-specific catching or uptake by healthy cells in blood and enhance the specificity toward CTCs. The rBSA-FA aqueous solutions (0.1 mL) with different concentrations (Table S4, Table 1-3) were respectively mixed with the solution of AuNP-MBA, AuNR-MBA or AuNS-MBA (8.0 mL). The solution was stirred at room temperature for 5.0 min of reaction. The obtained AuNP-MBA-rBSA-FA, AuNR-MBA-rBSA-FA or AuNS-MBA-rBSA-FA dispersion was centrifuged (8000 rpm, 10 min), washed twice and kept in the fridge for further use.

8


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Table 1. Preparation and characterization of AuNP-MBA and AuNP-MBA-rBSA-FA. CAuNPs C4-MBA CrBSA-FA SERS Nomenclature (mg/mL) (µM) (µg/mL) Intensity a

a

AuNP-MBA1

0.02

60

-

2040±121

AuNP-MBA2

0.02

10

-

1080±87

AuNP-MBA3

0.02

1

-

510±39

AuNP-MBA4

0.02

0.1

-

30±11

AuNP-MBA1-rBSA-FA1

0.02

60

0.6

2910±173

AuNP-MBA1-rBSA-FA2

0.02

60

1.0

2700±167

AuNP-MBA1-rBSA-FA3

0.02

60

2.5

2940±65

0.02 60 6.0 2160±75 AuNP-MBA1-rBSA-FA4 -1 The SERS signal intensity of the nanoparticles measured at 1078 cm (Mean ± SE, n=3).

Table 2. Preparation and characterization of AuNR-MBA and AuNR-MBA-rBSA-FA.

a

Nomenclature

CAuNRs (mg/mL)

C4-MBA (µM)

CrBSA-FA (µg/mL)

SERS Intensity a

AuNR-MBA1

0.02

60

-

680±165

AuNR-MBA2

0.02

10

-

440±25

AuNP-MBA3

0.02

1

-

170±11

AuNR-MBA4

0.02

0.1

-

10±3

AuNR-MBA1-rBSA-FA1

0.02

60

3

1980±133

AuNR-MBA1-rBSA-FA2

0.02

60

12

4070±316

AuNR-MBA1-rBSA-FA3

0.02

60

25

3420±236

AuNR-MBA1-rBSA-FA4

0.02

60

60

1980±118

-1

The SERS signal intensity of the nanoparticles measured at 1078 cm (Mean ± SE, n=3).

Table 3. Preparation and characterization of AuNS-MBA and AuNS-MBA-rBSA-FA.

a

Nomenclature

CAuNS (mg/mL)

C4-MBA (µM)

CrBSA-FA (µg/mL)

SERS Intensity a

AuNS-MBA1

0.02

60

-

890±73

AuNS-MBA2

0.02

10

-

680±39

AuNS-MBA3

0.02

1

-

330±20

AuNS-MBA4

0.02

0.1

-

150±14

AuNS-MBA1-rBSA-FA1

0.02

60

0.6

-

AuNS-MBA1-rBSA-FA2

0.02

60

1.0

5940±121

AuNS-MBA1-rBSA-FA3

0.02

60

2.5

2900±303

AuNS-MBA1-rBSA-FA4

0.02

60

6.0

1950±245

-1

The SERS signal intensity of the nanoparticles measured at 1078 cm (Mean ± SE, n=3). 9



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2.6 Characterization of the nanoparticles The nanoparticles were characterized by transmission electron microscope (TEM, JEOL-2100, JEOL, Japan), UV-vis spectroscopy (T10CS, Beijing Purkinje General Instrument Co., Ltd, China), X-ray photoelectron spectroscopy (XPS, AXIS ULTRA, Kratos, America). The Raman spectra were observed on a confocal microprobe Raman system (Renishaw inVia Reflex, UK). The laser wavelength was fixed at 785 nm. The diameter of the laser beam was tuned to be around 1.0 mm according to previous protocols.30 The range of the scattering spectra was set from 400 to 1500 cm-1 and the time of data acquisition was set to 1.0 s. The laser power was 140 mW for the optimization of 4-MBA and 280 mW for other SERS spectra. In addition, the Au concentration of the solution of AuNPs,

AuNP-MBA-rBSA,

AuNP-MBA-rBSA-FA,

AuNRs,

AuNR-MBA-rBSA,

AuNR-MBA-rBSA-FA, AuNS, AuNS-MBA-rBSA and AuNS-MBA-rBSA-FA were all determined utilizing inductively coupled plasma optical emission spectrometry (ICP-OES) (Optima 2100DV instrument, Perkin Elmer, USA). 100 µL of the nanoparticles were first treated with 5.0 mL of HNO3, 0.5 mL of HClO4 and 0.3mL of H2O2 at 150 °C for 30 min. Then, the samples were kept at room temperature for natural cooling. After that, the samples were transferred into reaction kettles and the reaction was continued for 4.0 h at 180 oC. The samples were cooled down to room temperature at natural conditions, diluted using MilliQ water (25 mL), and then observed by means of ICP-OES. Au standard solutions with different concentrations (prepared using 5% of HNO3) were also measured for construction of a calibration curve, which was used for measuring the Au concentration of our samples. The nanoparticle concentrations shown in this study are all the corresponding Au concentration.

2.7 Cell culture HepG2 cells (human hepatocellular carcinoma cell line) and HeLa cells (human cervical cancer cell line) were cultured in DMEM medium containing fetal bovine serum (FBS, 10 wt %), penicillin (100 units/mL) and streptomycin (100 mg/mL). The incubation temperature was 37oC, and the CO2 10


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concentration in the humidified atmosphere was 5 %.36

2.8 Detection of cancer cells in blood samples Animal experiments in this study were all carried out according to the protocols approved by IACUC. The blood samples were drawn from the rabbit heart and then injected into vacutainer tubes, which had contained lithium heparin. The tubes were shaken vigorously and used for the detection of cancer cells. For study of the sensitivity of the SERS nanoparticles, 4~400 HeLa cells in 4.0 mL of PBS were first mixed with the rabbit blood (4.0 mL). The mixed blood samples (8.0 mL) were mixed with 2.0 mL of peripheral blood lymphocyte separation medium, and then centrifuged at 400 × g for 25 min. The monocyte cells including white blood cells and HeLa cells were collected into clean centrifuge tubes for PBS washing (twice). Then, the washed and centrifuged cells were dispersed in PBS (3.0 mL). Next, the aqueous solution of AuNP-MBA-rBSA-FA, AuNR-MBA-rBSA-FA or AuNS-MBA-rBSA-FA (100 µL, 0.5 mg/mL) was added into the cell dispersion (3.0 mL, in PBS), and cultured in 37 oC of incubator. After 30 min of incubation, the samples were centrifuged at 400 × g for 5.0 min, washed three times using PBS. The final centrifuged samples were dissolved in 0.2 mL of PBS, and then tested by the above-mentioned Raman instrument. For study of the specificity of the SERS nanoparticles, the similar protocol was used except one different point, which is that the rabbit blood (4.0 mL) with or without HeLa (or HepG2) cells was mixed with the SERS nanoparticles (100 µL, 0.5 mg/mL in MilliQ water). The cell number in the 4.0 mL of blood is 10 for AuNP-MBA-rBSA-FA or AuNR-MBA-rBSA-FA, but 4 for AuNS-MBA-rBSA-FA.

3. RESULTS AND DISCUSSION 3.1 Synthesis and optimization of the SERS-active nanoparticles The SERS-active nanoparticles are constructed based on the AuNP, AuNR and AuNS as shown in 11



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Scheme 1. 4-MBA (i.e. Raman reporter molecule) is first linked to the AuNP, AuNR or AuNS via Au-S bond to obtain the SERS signal. The rBSA is then used to modify the AuNP-MBA, AuNR-MBA or AuNS-MBA for stabilization purpose. In addition, the thin rBSA layer can reduce the non-specific catching or uptake by healthy cells in blood. The targeting molecule FA is used to increase the specificity and sensitivity toward cancer cells among much more normal blood cells. The Raman signal of the nanoparticles is the spectrum of Raman reporter 4-MBA, which is related to the morphology of gold nanoparticles and 4-MBA amount. Therefore, the nanoparticle shape and the higher amount of 4-MBA binding (i.e. SERS enhancing molecule) are responsible for the better detection. In addition, it’s well known that the SERS signal intensity should increase with increase of the gold nanoparticle asymmetry (at same mass of Au and Raman reporter molecule).16,33,34 Therefore, at the same conditions, the SERS intensity of AuNS-MBA should stronger than that of AuNR-MBA, and that of AuNP-MBA should be lowest. However, it’s unclear that which shape of the SERS-active nanoparticles is best for detection of CTCs because the best dosage of 4-MBA, rBSA and FA may be different due to the different stability of the nanoparticles. The aggregated nanoparticles cannot be used for detection of CTCs using our presented method because they may precipitate with the normal blood cells together under centrifugation resulting a “false positive result”. In this study, the modification method and the dosage of 4-MBA, rBSA and FA are optimized in accordance with the nanoparticle stability and SERS signal intensity. The three optimized SERS-active nanoparticles with different shape and without aggregation are utilized for cancer cell detection and their LODs are compared. The SERS-active nanoparticles are first prepared by a three-step method, i.e. the AuNP, AuNR or AuNS is modified with 4-MBA, rBSA and FA step by step. Regarding the optimizing of 4-MBA dosage, Table S1 represents the preparation conditions and SERS intensity of AuNP-MBA1’-4’, AuNR-MBA1’-4’ and AuNS-MBA1’-4’, and Figure S1 displays the corresponding SERS signal. We can see that the higher 4-MBA concentration in the range of 25-250 µM, the stronger SERS 12


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signal intensity (Table S1 & Figure S1). Because the 4-MBA concentration higher than 250 µM leads to aggregation of the nanoparticles, 250 µM is selected as the optimized condition for the subsequent investigation. Regarding optimization of rBSA dosage, the preparative conditions and SERS intensity of the AuNP-MBA1’-rBSA1-5, AuNR-MBA1’-rBSA1-5 and AuNS-MBA1’-rBSA1-5 nanoparticles are shown in Table S2, and the corresponding SERS signals are presented in Figure S2. Although SERS intensities of these nanoparticles are not weak, the stability of them is not good because they precipitate when shelf-time is longer than three days. Because the higher concentration of 4-MBA leads to worse nanoparticle stability, it was decreased to 60 µM for further optimization of rBSA dosage. Table S3 presents the preparation conditions and SERS signal intensities of AuNP-MBA1-rBSA1-5, AuNR-MBA1-rBSA1-5 and AuNS-MBA1-rBSA1-5, and Figure S3 presents corresponding SERS signals. These nanoparticles are stable. The rBSA concentration cannot be too high because over package of the nanoparticles by rBSA reduces the SERS intensity, and cannot be too low because less rBSA package results in less FA conjugation amount on the nanoparticle surfaces.30 In view of the SERS intensity and FA conjugation, the rBSA concentration was respectively fixed at 0.625, 1.25 and 0.625 µg/mL as optimal values for AuNP-MBA1, AuNR-MBA1 and AuNS-MBA1 in the following experiments. Regarding the optimizing of FA dosage, AuNP-MBA1-rBSA3, AuNR-MBA1-rBSA4 and AuNS-MBA1-rBSA3 with optimized dosages of 4-MBA and rBSA were used for FA conjugation. However, the obtained nanoparticles are all not stable. That’s because the –COOH on the surface of AuNP-MBA1-rBSA3, AuNR-MBA1-rBSA4 or AuNS-MBA1-rBSA3 may also be activated during activation of FA by EDC. In that case, the reaction between the –COOH and –NH2 on the surface of AuNP-MBA1-rBSA3, AuNR-MBA1-rBSA4 or AuNS-MBA1-rBSA3 may result in aggregation of the nanoparticles. In order to solve this problem, the SERS-active nanoparticles are subsequently prepared by a two-step method, i.e. the FA is conjugated to rBSA and the obtained rBSA-FA is purified and directly used to modify the AuNP-MBA, AuNR-MBA or AuNS-MBA. 13



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Figure 1. SERS spectra of the AuNP-MBA1-4 (a) and AuNP-MBA1-rBSA-FA1-4 (b).

Figure 2. SERS spectra of the AuNR-MBA1-4 (a) and AuNR-MBA1-rBSA-FA1-4 (b). 14


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Regarding the optimizing of 4-MBA and rBSA-FA dosages, Table 1-3 show the nanoparticles’ preparation conditions and SERS signal intensities of the nanoparticles, and Figure 1-3 show corresponding SERS spectra. The 4-MBA concentration is optimized to be 60 µM due to the strong SERS intensity. Because more packages of the nanoparticles by rBSA-FA reduces the SERS intensity, the rBSA-FA concentration was respectively fixed at 2.5, 25 and 2.5 µg/mL as optimal values in the following experiments.

Therefore,

the

SERS

nanoparticles

of

AuNP-MBA1-rBSA-FA3,

AuNR-MBA1-rBSA-FA3 and AuNS-MBA1-rBSA-FA2 are further characterized by TEM and UV-vis spectroscopy, and utilized for cancer cell detection in blood samples.

Figure 3. SERS spectra of the AuNS-MBA1-4 (a) and AuNS-MBA1-rBSA-FA1-4 (b).

Figure S4 presents the relationship between SERS signal intensities and 4-MBA concentrations in the range of 1.0-60 µM. Although there is no linear correlation, the SERS signal intensities 15



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increase with the increase of 4-MBA concentrations.

3.2 Characterization of the optimized nanoparticles The XPS analysis for AuNP-MBA1 is shown in Figure S5. We can find the peaks of S, C, O and Au, which demonstrates that the nanoparticles contain elements of S, C, O and Au. Therefore, we can conclude that 4-MBA is successfully conjugated onto the surface of AuNPs via Au-S bond. Figure 4a and Figure 4b show TEM (transmission electron microscope) images of the AuNPs and AuNP-MBA1-rBSA-FA3. It is found that the dispersity of them is good and their sizes are very close, indicating a very thin modification layer of rBSA-FA. Figure 4c shows TEM image of AuNP-MBA1-rBSA-FA3 with a negative stain using the 0.2% uranyl acetate (pH 4.5). It is found that the shell of rBSA-FA on the surface of nanoparticles is obvious and very thin. This is greatly beneficial to the SERS intensity because thick protection layer on the particle surface reduces the SERS signal.30,31 Figure 4d represents UV-vis spectra of the AuNPs and AuNP-MBA1-rBSA-FA3. The absorption peak of AuNPs locates at 550 nm. However, it shifts to 538 nm for AuNP-MBA1-rBSA-FA3, which results from the functionalization of 4-MBA and rBSA-FA.

Figure 4. Characterization of the AuNPs and AuNP-MBA1-rBSA-FA3. (a, b, c): TEM images of the AuNPs (a), AuNP-MBA1-rBSA-FA3 (b) and AuNP-MBA1-rBSA-FA3 with a negative stain using the 0.2% uranyl acetate (pH 4.5) (c); (d): UV-vis spectra of the AuNPs and AuNP-MBA1-rBSA-FA3.

16


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Figure 5. Characterization of the AuNRs and AuNR-MBA1-rBSA-FA3. (a, b, c): TEM images of the AuNRs (a), AuNR-MBA1-rBSA-FA3 (b) and AuNR-MBA1-rBSA-FA3 with a negative stain using the 0.2% uranyl acetate (pH 4.5) (c); (d): UV-vis spectra of the AuNRs and AuNR-MBA1-rBSA-FA3.

Figure 6. Characterization of the AuNS and AuNS-MBA1-rBSA-FA2. (a, b, c): TEM images of the AuNSs (a), AuNS-MBA1-rBSA-FA2 (b) and AuNS-MBA1-rBSA-FA2 with a negative stain using the 0.2% uranyl acetate (pH 4.5) (c); (d): UV-vis spectra of the AuNS and AuNS-MBA1-rBSA-FA2.

17



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Figure 5 represents the TEM images and UV-Vis spectra of AuNRs and AuNR-MBA1-rBSA-FA3. The TEM images indicate that modification of 4-MBA and rBSA-FA also cannot affect the particle size and the rBSA-FA protection layer on the surface of AuNRs is also very thin. In addition, the absorption peak has a slight shift from 880 to 918 nm. That’s because the refractive index around the AuNRs changes with introducing of the organic molecules. The TEM images and UV-Vis spectra of AuNSs and AuNS-MBA1-rBSA-FA2 are shown in Figure 6. It can be seen that the particle size of AuNSs and AuNS-MBA1-rBSA-FA2 are also very close, and the thickness of rBSA-FA shells on the surface of AuNSs is also very thin. And the absorption peak shifts from 616 nm of AuNSs to 580 nm of AuNS-MBA1-rBSA-FA2. Compared with that of AuNSs, the absorption peak of AuNS-MBA1-rBSA-FA2 becomes very wide. That may be ascribed to the change of local geometry due to the modification of 4-MBA and rBSA-FA. In addition,

the

sizes

of

AuNP-MBA1-rBSA-FA3,

AuNR-MBA1-rBSA-FA3

and

AuNS-MBA1-rBSA-FA2 are all around 60 nm, which is an important condition for comparison of the sensitivities of them for detection of CTCs. When the concentrations of element Au in AuNP-MBA-rBSA-FA, AuNR-MBA-rBSA-FA and AuNS-MBA-rBSA-FA are same (i.e. 20 µg/mL), the average SERS enhancement factors (EF) of three nanoparticles were respectively 103, 103 and 104 according to the formula of EF = (ISERS/IRaman) × (NRaman/NSERS), where ISERS or IRaman is respectively the peak intensity of SERS or normal Raman spectrum of 4-MBA, while NSERS or NRaman is respectively the 4-MBA number of moles for the nanoparticles-attached or the free molecule samples in the aqueous solution.41 The average SERS enhancements most come from electromagnetic enhancement which also could be calculated by finite-difference time-domain (FDTD) simulations.41-43

3.3 Cancer cell detection without enrichment process CTCs shed from solid tumors are ultra-rare in peripheral blood with as few as one single CTC per 106~107 leukocytes.44-46 Here, the three optimized SERS-active nanoparticles with similar particle 18


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size,

similar

modifications

and

different

shape

(i.e.

AuNP-MBA1-rBSA-FA3,

AuNR-MBA1-rBSA-FA3 and AuNS-MBA1-rBSA-FA2) are utilized to detect cancer cells in peripheral blood of rabbit. Evaluations for the specificity and sensitivity of our three SERS-active nanoparticles are operated on HepG2 and HeLa cells because they are respectively folate receptor alpha (FRα) negative cells and FRα positive cells and the WBCs have a low FRα expression.35,36 The limit of detection (LOD) is determined at the lowest cell concentration in the fitting line of SERS intensity versus cell concentration (i.e. the inset plot of Figure 7b, 8b, 9b). Figure 7 presents the detection sensitivity and specificity of the AuNP-MBA1-rBSA-FA3 for HeLa cells in peripheral blood of rabbit. Figure 7a shows the SERS signals of rabbit blood (4.0 mL, ~2.0 × 107 WBCs) with HeLa cells (1-100 cells/mL) after incubation with AuNP-MBA1-rBSA-FA3. We can see that the higher concentrations of HeLa cells result in stronger SERS intensities. Figure 7b shows a relationship between the SERS intensity and the HeLa cell concentration in blood samples. It is a fitting line for the SERS intensity as a function of HeLa cell concentration ranging from 3 to 100 cells/mL. We can see that the linear relationship is not so bad with a R2 larger than 0.98, which demonstrates the potential application of our AuNP-MBA1-rBSA-FA3 for cancer cells quantitative analysis in blood samples. The LOD of 3 cells/mL is lower than our previously reported value (i.e. 5 cells/mL).30

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Figure 7. Detection sensitivity (a,b) and specificity (c) of the AuNP-MBA1-rBSA-FA3 for HeLa cells in peripheral blood of rabbit. (a): SERS signals of rabbit blood (4.0 mL, ~2.0 × 107 WBCs) with 1-100 HeLa cells/mL after incubation with AuNP-MBA1-rBSA-FA3. (b): Plot of the SERS signal intensity versus HeLa cell concentration in peripheral blood of rabbit. (c): SERS signals of rabbit blood (4.0 mL, ~2.0 × 107 WBCs) without or with 10 HeLa (or 10 HepG2) cells incubated with AuNP-MBA1-rBSA-FA3 or AuNP-MBA1-rBSA.

20


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The detection specificity of the AuNP-MBA1-rBSA-FA3 is further evaluated for 3 cancer cells in 1.0 mL of blood. Figure 7c shows the SERS signals of rabbit blood (4.0 mL, ~2.0 × 107 WBCs) without or with 10 HeLa (or 10 HepG2) cells after incubation with AuNP-MBA1-rBSA-FA3 or AuNP-MBA1-rBSA. The SERS signal is considered to be significant when the signal to noise ratio (S/N) is larger than 10. It’s found that the SERS signal at 1078 cm-1 is obvious for the rabbit blood samples with HeLa cells incubated with AuNP-MBA1-rBSA-FA3 due to S/N > 10. However, regarding the rabbit blood samples incubated with AuNP-MBA1-rBSA-FA3, or the rabbit blood samples with HepG2 cells incubated with AuNP-MBA1-rBSA-FA3, or the rabbit blood sample with HeLa cells incubated with AuNP-MBA1-rBSA, no corresponding SERS signal is found due to S/N < 10. The healthy blood cells cannot disturb the detection because the HepG2 cells were mixed with the rabbit blood samples before detection. Consequently, it can be concluded that our AuNP-MBA1-rBSA-FA3 has a high specificity for HeLa cell detection in peripheral blood of rabbit. The detection sensitivity and specificity of the AuNR-MBA1-rBSA-FA3 in the rabbit blood samples are shown in Figure 8. Figure 8a presents the SERS signals of rabbit blood (4.0 mL) with 1-100 HeLa cells/mL after incubation with AuNR-MBA1-rBSA-FA3. A similar result is found that the higher HeLa cell concentration, the stronger SERS intensity. Figure 8b shows a plot of the SERS intensity versus HeLa cell concentration in peripheral blood of rabbit. From the inset plot, it is found that the relationship between the SERS intensity and the HeLa cell concentration ranging from 3 to 100 cells/mL is linear (R2 = 0.975593), and the LOD is 3 cells/mL. The reliability of the AuNR-MBA1-rBSA-FA3 is a little bit worse than that of the AuNP-MBA1-rBSA-FA3 because of the low value of R2.

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Figure 8. Detection sensitivity (a,b) and specificity (c) of the AuNR-MBA1-rBSA-FA3 for HeLa cells in peripheral blood of rabbit. (a): SERS signals of rabbit blood (4.0 mL, ~2.0 × 107 WBCs) with 1-100 HeLa cells/mL after incubation with AuNP-MBA1-rBSA-FA3. (b): Plot of the SERS signal intensity versus HeLa cell concentration in peripheral blood of rabbit. (c): SERS signals of rabbit blood (4.0 mL, ~2.0 × 107 WBCs) without or with 10 HeLa (or 10 HepG2) cells incubated with AuNR-MBA1-rBSA-FA3 or AuNR-MBA1-rBSA. 22


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The detection specificity of AuNR-MBA1-rBSA-FA3 is also verified for cancer cells in peripheral blood of rabbit at a concentration of 3 cells/mL. Figure 8c presents the SERS signals of rabbit blood (4.0 mL) without or with 10 HeLa (or 10 HepG2) cells after incubation with AuNR-MBA1-rBSA-FA3 or AuNR-MBA1-rBSA. The SERS peak at 1078 cm-1 is evident for rabbit blood samples with HeLa cells incubated with AuNR-MBA1-rBSA-FA3 (S/N > 10), but not for the rabbit blood samples incubated with AuNR-MBA1-rBSA-FA3, or the rabbit blood with HepG2 cells incubated with AuNR-MBA1-rBSA-FA3, or the rabbit blood with HeLa cells incubated with AuNR-MBA1-rBSA (S/N < 10). Therefore, our AuNR-MBA1-rBSA-FA3 also has a high specificity for HeLa cell detection in the rabbit blood samples. In addition, the specificity of the AuNR-MBA1-rBSA-FA3 is higher than that of the AuNP-MBA1-rBSA-FA3 due to the stronger SERS intensity. We also applied the AuNS-MBA1-rBSA-FA2 for cancer cell detection in peripheral blood of rabbit without enrichment process. Figure 9a shows the SERS signals of rabbit blood (4.0 mL) with 1-100 HeLa cells/mL incubated with AuNS-MBA1-rBSA-FA2 nanoparticles. It is obvious that the higher HeLa cell concentration, the stronger SERS intensity. Figure 9b shows a plot of the SERS signal intensity versus the HeLa cell concentration in the rabbit blood samples. The inset plot shows the SERS intensity as a function of HeLa cell concentration ranging from 1 to 100 cells/mL. The linear relationship (R2 = 0.98012) demonstrates that the AuNS-MBA1-rBSA-FA2 nanoparticles could be used to measure cancer cells quantitatively. The LOD of 1 cell/mL is lower than that of the above two SERS nanoparticles (3 cells/mL) and the reported lowest value (5 cells/mL).30

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Figure 9. Detection sensitivity (a,b) and specificity (c) of the AuNS-MBA1-rBSA-FA2 for HeLa cells in peripheral blood of rabbit. (a): SERS signals of rabbit blood (4.0 mL, ~2.0 × 107 WBCs) with 1-100 HeLa cells/mL after incubation with AuNS-MBA1-rBSA-FA2. (b): Plot of the SERS signal intensity versus HeLa cell concentration in peripheral blood of rabbit. (c): SERS signals of rabbit blood (4.0 mL, ~2.0 × 107 WBCs) without or with 4 HeLa (or 4 HepG2) cells incubated with AuNS-MBA1-rBSA-FA2 or AuNS-MBA1-rBSA.

The specificity of our AuNS-MBA1-rBSA-FA2 nanoparticles is also investigated for cancer cell 24


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detection in peripheral blood samples at its LOD value (i.e. 1 cell/mL). Figure 9c presents the SERS signals of rabbit blood (4.0 mL) without or with 4 HeLa (or 4 HepG2) cells incubated with AuNS-MBA1-rBSA-FA2 or AuNS-MBA1-rBSA. The SERS peak at 1078 cm-1 is obvious for rabbit blood with HeLa cells incubated with AuNS-MBA1-rBSA-FA2 nanoparticles (S/N > 10). However, similar SERS signal is not found for other groups (S/N < 10). Because the concentration of the positive cells is only 1 cell/mL, we can conclude that our AuNS-MBA1-rBSA-FA2 nanoparticles are very specific for cancer cell detection in peripheral blood of rabbit. Influence of the laser power on the SERS signal intensity is evaluated using the AuNS-MBA1-rBSA-FA2 nanoparticles (Figure S6). We can see that the higher laser power we use, the stronger SERS signal intensity we can get. In order to get best detection limits, we use 100 % of laser power (i.e. 280 mW) for cancer cell detection. The interaction between HeLa cells and AuNS-MBA1-rBSA or AuNS-MBA1-rBSA-FA2 is also observed via TEM (Figure S7). Figure S7a shows the TEM image of HeLa cells incubated with AuNS-MBA1-rBSA for 30 min, and Figure S7b,c show those of HeLa cells incubated with AuNS-MBA1-rBSA-FA2 for 30 min. It can be seen that almost no AuNS-MBA1-rBSA nanoparticles are found inside cells or clustered on the surface of cells because no FA ligand is conjugated onto the surface of nanoparticles. However, a few AuNS-MBA1-rBSA-FA2 nanoparticles are found inside the cells and lots of AuNS-MBA1-rBSA-FA2 nanoparticles are clustered on the cell surfaces because the ligand FA of nanoparticles can recognize the FRα of the cells. Therefore, our observed SERS signals are sum of the signals from the two families of nanoparticles (i.e. inside cells and clustered on the cell surfaces). The above results demonstrate that our SERS nanoparticles based on AuNPs, AuNRs and AuNSs (i.e. AuNP-MBA-rBSA-FA, AuNR-MBA-rBSA-FA, and AuNS-MBA-rBSA-FA) can all be utilized for CTC detection without enrichment process in the blood with high specificity, and the AuNS-MBA-rBSA-FA is the best one due to its supersensitivity. In this study, the addition of cancer cells to blood samples is used as a model for application of 25



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Page 26 of 34

the SERS-active nanoparticles modified with FA. Because most of CTCs overexpress epithelial cell adhesion molecule (EpCAM)2-4, we will replace the FA with anti-EpCAM antibody for CTC detection in the blood samples of cancer patients.

4. CONCLUSIONS In summary, we developed three kinds of new SERS-active nanoparticles based on AuNPs, AuNRs and AuNSs with similar particle size, similar modifications and different shape for CTC detection in blood samples without enrichment process. The modification method and the dosage of 4-MBA, rBSA and FA are optimized in accordance with the stability and SERS signal intensity of the composite nanoparticles. The SERS-active nanoparticles are prepared by a three-step method (i.e. the 4-MBA, rBSA and FA are used to modify the AuNP, AuNR or AuNS step by step) or by a two-step method (i.e. the FA is first conjugated to rBSA and the obtained rBSA-FA is directly used to modify the AuNP-MBA, AuNR-MBA or AuNS-MBA), and the two-step method is confirmed to be a better one. The dosage of 4-MBA is optimized to be 60 µM for 0.02 mg/mL of AuNPs, AuNRs and AuNSs. The dosage of rBSA-FA is respectively optimized to be 2.5, 25 and 2.5 µg/mL for AuNP-MBA, AuNR-MBA and AuNS-MBA. TEM images demonstrate that the rBSA modification layer of the SERS nanoparticles (i.e. AuNP-MBA-rBSA-FA, AuNR-MBA-rBSA-FA and AuNS-MBA-rBSA-FA) is very thin, which is greatly beneficial to the SERS intensity. Under the optimal

conditions,

the

detection

results

reinforce

that

our

AuNP-MBA-rBSA-FA,

AuNR-MBA-rBSA-FA, and AuNS-MBA-rBSA-FA can all be utilized for CTC detection in the blood without enrichment process with high specificity, and the AuNS-MBA-rBSA-FA is the best one due to its supersensitivity, whose LOD (1 cell/mL) is much lower than the reported lowest value (5 cells/mL).

■ ASSOCIATED CONTENT Supporting Information 26


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The Supporting Information is available free of charge on the ACS Publications website. Table S1-3: Preparation conditions and results for optimization of 4-MBA or rBSA concentration; Figure S1-3: SERS spectra of the AuNP-MBA, AuNR-MBA, AuNS-MBA, AuNP-MBA-rBSA, AuNR-MBA-rBSA and AuNS-MBA-rBSA; Figure S4: Plot of the SERS signal intensity versus the 4-MBA concentration; Figure S5: XPS spectra of the lyophilized AuNP-MBA1; Figure S6: Plot of the SERS signal intensity versus the laser power using the AuNS-MBA1-rBSA-FA2 nanoparticles; Figure S7: Characterization of the interaction between HeLa cells and AuNS-MBA1-rBSA or AuNS-MBA1-rBSA-FA2 (PDF).

■ AUTHOR INFORMATION Corresponding authors *Email: [email protected]. Phone: +86 574 87617278. *Email: [email protected]. Phone: +86 574 86685039. Notes The authors declare no competing financial interest.

■ ACKNOWLEDGMENTS

This work is financially supported by National Natural Science Foundation of China (Grant Nos. 51411140243, 61571278, 21305148 and 31128007), Youth Innovation Promotion Association of Chinese Academy of Sciences (2016269), NSFC-Guangdong Province Joint Project on National Supercomputer Centre in Guangzhou (NSCC-GZ) (Aiguo WU), Key Breakthrough Program of Chinese Academy of Sciences (KGZD-EW-T06) from CAS and Bureau of Science and Technology of Ningbo Municipality City (Grant Nos. 2014B82010, and 2015B11002) and Natural Science Foundation of Ningbo (2015A610080 and 2014A610159).

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Graphic Abstract Three kinds of new SERS (surface-enhanced Raman scattering) active nanoparticles based on spherical gold nanoparticles (AuNPs), gold nanorods (AuNRs) and gold nanostars (AuNSs) with similar particle size, similar modifications and different shape are developed for direct detection of CTCs in the blood with supersensitivity and high specificity.

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Improved SERS-Active Nanoparticles with Various Shapes for CTC

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