A Supersensitive CTC Analysis System Based on Triangular Silver

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A Supersensitive CTC Analysis System Based on Triangular Silver Nanoprisms and SPION with Function of Capture, Enrichment, Detection and Release Huimin Ruan, Xiaoxia Wu, Chengcheng Yang, Zihou Li, Yuanzhi Xia, Ting Xue, Zheyu Shen, and Aiguo Wu ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.7b00825 • Publication Date (Web): 05 Feb 2018 Downloaded from http://pubs.acs.org on February 14, 2018

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ACS Biomaterials Science & Engineering

A Supersensitive CTC Analysis System Based on Triangular Silver Nanoprisms and SPION with Function of Capture, Enrichment, Detection and Release

Huimin Ruan,†,‡ Xiaoxia Wu,† Chengcheng Yang,ǁ Zihou Li,† Yuanzhi Xia,† Ting Xue,† Zheyu Shen,*,†,§ Aiguo Wu*,†

†

CAS 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, 1219 Zhong-guan West Road, Ning-bo, Zhe-jiang 315201, P. R. China. ‡

University of Chinese Academy of Sciences, 19 A Yu-quan Road, Shi-jing-shan District, Beijing

100049, P. R. China ǁ

State Key Laboratory Oncogenes and Related Genes, Shanghai Cancer Institute, School of

Biomedical Engineering, Shanghai Jiao Tong University, 800 Dong-chuan Road, Min-hang District, Shanghai 200030, China. §

Laboratory of Molecular Imaging and Nanomedicine, National Institute of Biomedical Imaging

and Bioengineering, National Institutes of Health, 9000 Rockville Pike, Bethesda, Maryland 20892, United States.

*Corresponding Authors Tel.: +86 574 87617278 / 574 86685039; Email: [email protected], or [email protected] ORCID Zheyu Shen: 0000-0002-0350-375X Aiguo Wu: 0000-0001-7200-8923

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ABSTRACT Detection of circulating tumor cells (CTCs) may be applied for diagnosis of early tumors like a liquid biopsy. However, the sensitivity remains a challenge because CTCs are extremely rare in peripheral blood. In this study, we developed a supersensitive CTC analysis system based on triangular silver nanoprisms (AgNPR) and superparamagnetic iron oxide nanoparticles (SPION) with function of capture, enrichment, detection and release. The AgNPR was encoded with MBA (i.e. 4-mercaptobenzoic acid ) and modified with rBSA (i.e. reductive bovine serum albumin) and FA (i.e. folic acid) generating organic/inorganic composite nanoparticle MBA-AgNPR-rBSA-FA, which has the function of surface-enhanced Raman scattering (SERS). The optimized SERS nanoparticles (i.e. MBA3-AgNPR-rBSA4-FA2) can be utilized for CTC detection in blood samples with high sensitivity and specificity, and the LOD (i.e. limit of detection) reaches to 5 cells per mL. In addition, the SPION was also modified with rBSA and FA generating magnetic nanoparticle SPION-rBSA-FA.

Our

supersensitive

CTC

analysis

system

composes

of

MBA3-AgNPR-rBSA4-FA2 and SPION-rBSA-FA nanoparticles, which were applied for capture (via interaction between FA and FRα), enrichment (via magnet) and detection (via SERS) of cancer cells from blood samples. The results demonstrate that our supersensitive CTC analysis system has a better sensitivity and specificity than the SERS nanoparticles alone, and the LOD is up to 1 cell/mL. The flow cytometry and LSCM (i.e. laser scanning confocal microscope) results indicate the CTCs captured, enriched, and isolated by our supersensitive CTC analysis system can also be further released (via adding excessive free FA) for further cell expansion, and phenotype identification.

KEYWORDS: circulating tumor cells, triangular silver nanoprisms, superparamagnetic iron oxide nanoparticles, detection, phenotype identification.

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INTRODUCTION Malignant tumors are one of the most common diseases, and most of tumor deaths are caused by tumor metastasis. Although diagnosis of early tumor is the key to save patients’ lives, early tumors are not easy to be found because the patients with early tumors have no obvious symptoms.1-3 It is believed that CTCs (i.e. circulating tumor cells) circulate in blood vessels and CTC detection may be applied for diagnosis of early tumors like a liquid biopsy.4-6 Nevertheless, CTCs are extremely rare in peripheral blood usually (1 mL blood includes several CTCs, ~ 107 leukocytes and ~ 5×109 erythrocytes).7-8 Therefore, development of a supersensitive method for CTC detection is very attractive in the past 10 years. SERS (i.e. surface-enhanced Raman scattering) is a supersensitive detection method, which could be used for the characterization at molecular levels.9-10 Compared with other spectroscopic techniques, SERS spectrum has the advantages of good selectivity, high sensitivity, no light bleaching, anti-interference, fast detection, and so on. Thus, the SERS spectrum has a wide range of applications, e.g. environmental analysis and biomedical diagnosis.11-12 For the Raman reporter molecules adsorbed on noble metal nanostructures, the Raman signal increases 106 times or more (i.e. SERS effect).13-14 Therefore, SERS technique is a powerful tool to realize supersensitive detection of CTCs. Wen et al. reported a kind of new magnetic nanospheres that can capture CTCs for further highly sensitive detection. They drew real blood from patients with tumors for evaluation of CTC capture efficacy.15 It is also reported that silicon-nanopillar arrays could be used for sensitive detection of CTCs in blood samples via enhancing topographic interactions.16 There are many other strategies that can be used for efficient CTC capture including immunomagnetic particles,17 aptamers,18 graphene oxide (rGO) films19 and microfluidic devices.20-21 Previously, we reported composite nanoparticles with SERS function constructed from AuNPs (i.e. spherical gold nanoparticles), which can be used for detection of CTCs without enrichment. The LOD (limit of detection) reaches to 5 cells per mL.22 After that, in order to increase the sensitivity, we further proposed 3 types of SERS-active gold particles, whose particle size and modifications were similar, 3



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but shapes are different (i.e. spheres, stars, and rods). These nanoparticles were utilized for detection of CTCs in blood samples. It was found that the SERS-active nanostars were most sensitive with 1 cell/mL of LOD, which is the reported lowest value.23 However, the solution stability of the SERS-active nanostars is not very high. Some precipitation forms if the time of room temperature storage is long up to a few days. In addition, the SERS-active nanostars cannot be used to enrich CTCs before detection, and the CTCs after detection cannot be used for further cell expansion, phenotype identification and molecular analysis. In order to overcome these problems, in this study, we developed a supersensitive CTC analysis system based on triangular silver nanoprisms (AgNPR) and superparamagnetic iron oxide nanoparticles (SPION) with function of capture, enrichment, detection and release. As shown in Figure 1 a, 4-mercaptobenzoic acid (MBA) was associated onto AgNPR surface via Ag-S bond. The MBA-labeled AgNPR (MBA-AgNPR) was further modified with rBSA (i.e. reductive bovine serum albumin) to increase the stability and reduce the nonspecific recognition by normal cells in the blood. After that, FA (i.e. folic acid) was grafted to the AgNPR surface by chemical reaction between –COOH of FA and –NH2 of rBSA to obtain MBA-AgNPR-rBSA-FA nanoparticles. In addition, SPION was also modified with rBSA and FA to obtain SPION-rBSA-FA (Figure 1 b). The supersensitive

CTC

analysis

system

was

developed

based

on

the

mixture

of

MBA-AgNPR-rBSA-FA and SPION-rBSA-FA, which can both specifically recognize cancer cells including ovarian cancer, kidney cancer, breast cancer, lung cancer, etc. The optimized CTC analysis system was applied for capture (via interaction between FA and folate receptor alpha (FRα)), enrichment (via magnet) and detection (via SERS) of cancer cells that are dispersed in the blood (Figure 1 c). This method is very sensitive with 1 cell per mL of LOD that is equal to the reported lowest value. The relationship between the cancer cell concentration (1-100 cells per mL) and the SERS intensity is linear (R2 = 0.993) that enables the supersensitive and quantitative analysis of CTCs. Furthermore, the CTCs captured, enriched, and isolated by our CTC analysis system can also be released (via adding excessive free FA) for further cell expansion, phenotype 4


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identification and molecular analysis (Figure 1 c). In addition, the electromagnetic enhancement, which is much higher than the chemical enhancement, could be very high up to 1010−1011. The SERS mainly results from the electromagnetic enhancement. The asymmetric shape of the nanoparticles can enhance the electromagnetic enhancement.23 Therefore, we used nanoprisms instead of nanospheres because the nanoprisms have stronger electromagnetic enhancement compared to nanospheres.

MATERIALS AND METHODS Materials. Hydrogen peroxide (H2O2), silver nitrate(AgNO3), trisodium citrate dehydrate (C6H5Na3O7·2H2O) were purchased from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China). Poly(vinyl pyrrolidone) (PVP, MW ∼58 000 g/mol), sodium borohydride (NaBH4), FeCl3·6H2O (99%), FeCl2·4H2O (99%), NaOH (99.99%), diethyleneglycol (DEG, 99%), N-hydroxysuccinimide (NHS), folic acid (FA), 4,6-diamidino2-phenylindole (DAPI), bovine serum albumin (BSA), sodium borohydride (NaBH4), 4-mercaptobenzoic acid (MBA), 1-ethyl-3-[(dimet-hyllamino)propyl] carbodiimide hydrochloride (EDC·HCl), and Ammonia solution (NH3·H2O, GR) were purchased from Aladdin Reagent Co. Ltd. (Shanghai, China). N-Hydroxysuccinimide (NHS) was ordered from Sigma-Aldrich. Dulbecco’s modified Eagle medium (DMEM) were ordered from Gibco Life Technologies. FITC-labeled anti-CK8 monoclonal antibody was purchased from Abcam. A peripheral blood lymphocyte separation medium was purchased from Beijing Slolarbio Science and Technology Co. Ltd. (China). The glasswares were washed using aqua-regia (HCl : HNO3 = 3:1 (v/v)) and Milli-Q water before using.

Synthesis of Triangular Silver Nanoprisms (AgNPR). AgNPR was synthesized at room temperature in water phase on the basis of reported methods with minor modifications.24-26Typically, a mixture of AgNO3 (0.5 mL, 20 mM), C6H5Na3O7·2H2O (6.0 mL, 30 mM), PVP (6.0 mL, 0.7 mM), and H2O2 (240 µL, 30 wt %) were added into pure water (99.5 mL). Under vigorous stirring at room 5



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temperature, fresh NaBH4 (100 mM, 1.0 mL) was added rapidly into the above-mentioned mixtures. After the solution color changed to pale yellow, it was stored in a dark room. After 2 h with a color change to blue, the sample was stored in refrigerator for further use.

Preparation of the CTCs Specific SERS Nanoparticles. MBA molecule, a Raman reporter, was conjugated to AgNPR based on reported methods.13, 22 Briefly, various concentrations (25 ~ 500 µM) of MBA solutions (40µL) in tetrahydrofuran were charged to the AgNPR solution (8.0 mL), respectively. The reaction was kept at room temperature with magnetic stirring. After 2.0 min of reaction, SERS intensities of the solutions were characterized. After that, rBSA was used to stabilize the AgNPR with conjugation of MBA (i.e. MBA-AgNPR). In that case, the nonspecific recognition of MBA-AgNPR by normal cells in blood can also be reduced. Typically, a rBSA solution (80 µL, 2.5 ~ 20 µg/mL) was mixed with 4.0 mL of MBA-AgNPR solution. After 5.0 min of reaction, the obtained samples of rBSA-stabilized MBA-AgNPR (MBA-AgNPR-rBSA) were characterized by a Raman instrument. Subsequently, the MBA-AgNPR-rBSA was modified with FA (a targeting ligand) via an amidation reaction.27,28 Carboxyl groups of the FA were first activated using EDC and NHS. Typically, FA (40.0 mg), EDC (32.0 mg) and NHS (19.2 mg) were added into phosphate-buffered saline (PBS, pH 7.4, 10 mM, 50 mL). After 8.0 h of reaction, the solution of activated FA (i.e. FA-NHS, 0.2 ~ 2.0 mL) was mixed with the obtained MBA-AgNPR-rBSA solution (12.0 mL), and additional PBS was charged to make 14 mL of total volume. The reaction was then continued for 16 h under magnetic stirring. The resulted MBA-AgNPR-rBSA-FA samples (i.e. FA-conjugated MBA-AgNPR-rBSA) were added into filter tubes (Amicon Ultra-15, Millipore, MWCO 3.0 kDa) and centrifuged at 4,500 rpm. The supernatants were transferred to new tubes for UV-vis analysis. The purified MBA-AgNPR-rBSA-FA was dissolved in Milli-Q water (2.0 mL) for next usage. The FA conjugation contents on the MBA-AgNPR-rBSA-FA nanoparticles were determined via the UV-vis and calculated in accordance with a previously reported method.28 6


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Synthesis of the CTCs Specific Magnetic Nanoparticles. SPION was synthesized by a reported polyol method with minor modifications.29,30 Typically, a solution (2.0 mL) with 2.0 M of FeCl3·6H2O and 1.0 M of FeCl2·4H2O was charged to DEG (100 mL) drop by drop. In order to remove O2, the mixture was bubbled using N2 for more than 50 min, and refluxed at 170 °C for 15 min. After that, 50 mL of ammonia solution was injected in 1.0 hour (i.e. 20 mL for the first injection, and then 10 mL for each injection every 20 min). After addition of ammonia solution, the reaction was continued 1.0 h at 170 °C. After that, the reaction was terminated by cooling at room temperature. The resulted SPION was rinsed using 1.0 M of HNO3 and pure water, respectively. Finally, the SPION was concentrated using a magnet (neodymium). The SPION was further functionalized with rBSA to decrease the nonspecific recognition by normal cells in the blood. 0.5 mL of rBSA solution (10 mg/mL) was added into the SPION solution (12.0 mL, CFe = 3.67 mM). After 2.0 h of reaction, 32.0 mg EDC, 19.2 mg NHS, and 4.0 mL of FA solution (0.8 mg/mL) was added into the obtained SPION-rBSA dispersion. The solutions were then stirred at room temperature. After 16.0 h, the resulted SPION-rBSA with FA conjugation (i.e. SPION-rBSA-FA) was concentrated using a neodymium magnet and rinsed using Milli-Q water. The FA conjugation contents on SPION-rBSA-FA nanoparticles were determined via the UV-vis and calculated in accordance with a previously reported method.28

Nanoparticle Characterization. The nanoparticle morphology and size were observed via a transmission electron microscopy (TEM, JEOL. 2100, Tokyo, Japan). The UV-vis absorption was measured by a UV-vis spectroscopy (T10CS, Beijing Purkinje General Instrument Co., Ltd., China). The Raman spectra were obtained from a Raman instrument (i.e. confocal microprobe, Renishaw inVia Reflex, Wolton-under-Edge, U.K.). 785 nm of laser wavelength was used. The Raman scattering spectra were observed in the range of 400 ~ 1500 cm−1. The data acquisition time was fixed at 1.0 s, and the power of laser was set to 280 mW. The SERS spectra were observed from 7



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liquid samples with homogeneous SERS hotspot. The laser beam diameter was adjusted to ~ 1.0 mm to capture lots of SERS hotspot at the same time. The particle size distributions were determined by a DLS (i.e. dynamic light scattering) instrument (Nano-ZS, Malvern, England). The FT-IR (i.e. Fourier transform infrared) spectra were observed on a FT-IR spectrometer (Cary660+620, Agilent, USA). Weight loss analysis of SPION and SPION-rBSA are measured using thermogravimetric and derivative thermogravimetric analysis (TG-DTG, Pyris Diamond, Perkin-Elmer, USA). Surface elemental analysis of SPION and SPION-rBSA were determined by XPS (X-ray photoelectron spectroscopy, AXIS ULTRA DLD, Shimadzu, Japan). A physical property measurement system (PPMS, Model-9, Quantum Design, USA) was used to measure the M-H curves of nanoparticles in the range of -30 ~ 30 kOe at 300 K.

CTC Detection in Mixed Cells by the SERS Nanoparticles. In order to evaluate the sensitivity and specificity, the as-prepared SERS nanoparticles (i.e. MBA3-AgNPR-rBSA4-FA2) were used for CTC detection by mixing the cells with overexpression of FRα (i.e. FRα-positive) to those without FRα expression (i.e. FRα-negative). HepG2 (FRα-negative cells), HeLa (FRα-positive cells), and MCF-7 ( FRα-positive cells) were chosen in this study because they have different expression levels of FRα. Our SERS nanoparticles should be more sensitive to HeLa cells than MCF-7 cells due to the higher FRα expression.22 For the sensitivity study, MBA3-AgNPR-rBSA4-FA2 solution (0.2 mg/mL, 1.0 mL) was added into DMEM (3.0 mL) including 2.5 × 106 HepG2 cells and 30 ~ 3000 MCF-7 (or HeLa cells). The samples were cultured in a 37 oC incubator for 30 min, and then centrifuged (500 × g, 5.0 min) and rinsed thrice by PBS. The obtained samples were dispersed in 200 µL PBS and detected using the aforementioned Raman equipment. The specificity of our SERS nanoparticles was also investigated by the above-mentioned method. The only difference is that 1.0 mL of MBA3-AgNPR-rBSA4-FA2 or MBA3-AgNPR-rBSA4 (0.2 mg/mL) was added into DMEM (3.0 mL) including 2.5 × 106 HepG2 cells or 2.5 × 106 HepG2 cells 8


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plus 30 HeLa (or MCF-7) cells.

CTC Detection in the Rabbit Blood by the SERS Nanoparticles. Animal experiments were all performed according to the approved protocols of the animal care and use committee of Ningbo University. Rabbit blood was drawn from the rabbit hearts, and injected into vacutainer tubes including lithium heparin followed with vigorous shaking. For the sensitivity study, 20~20,000 HeLa (or MCF-7) cells in PBS (2.0 mL) were added into rabbit blood (2.0 mL) and dispersed via shaking. The solutions were then mixed with 2.0 mL of peripheral blood lymphocyte separation medium. After centrifugation at 400 ×g for 25 min, the WBCs (white blood cells, ∼1.0 × 107) and MCF-7 (or HeLa) cells in the low-density cell layer were taken out, washed and centrifuged 2 times using PBS. The final purified cells were dispersed in PBS (3.0 mL). 0.2 mg/mL of MBA3-AgNPR-rBSA4-FA2 (1.0 mL) was then added into the 3.0 mL of cells. After 30 min of incubation at 37 °C, the cells were centrifuged at 400 × g for 5.0 min, rinsed 3 times using PBS, and dispersed in PBS (200 µL). The resultant cell samples were finally detected by the aforementioned Raman instrument. For the specificity study, 20 HeLa (or MCF-7) cells in PBS (2.0 mL) were added into rabbit blood (2.0 mL) followed with shaking. After that, the samples were mixed with peripheral blood lymphocyte separation medium (2.0 mL). The separated WBCs (∼1.0 × 107) and MCF-7 (or HeLa) cells were incubated with 1.0 mL of MBA3-AgNPR-rBSA4-FA2 or MBA3-AgNPR-rBSA4 (0.2 mg/mL). Other steps were same with the above mentioned protocol.

Capture, Enrichment, and Detection of CTCs in the Rabbit Blood. For a study of the sensitivity, 2.0 mL of HeLa cells in PBS (2 ~ 200 cells per mL) were added into centrifuge tubes containing rabbit blood (2.0 mL) followed with shaking. The solutions were then mixed with peripheral blood lymphocyte separation medium (2.0 mL). After centrifugation at 400 × g for 25 min, the WBCs (∼1.0 × 107) and HeLa cells in the low-density cell layer were taken out, washed 9



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and centrifuged 2 times using PBS. The final purified cells were dispersed in PBS (3.0 mL). 1.0 mL of MBA3-AgNPR-rBSA4-FA2 (0.2 mg/mL) and 300 µL of SPION-rBSA-FA (3.67 mM) were then added into 3.0 mL of the cells. After 30 min of incubation at 37 °C, the target cells were then collected with a magnet to remove the nontarget cells, and washed twice with PBS, and dispersed in PBS (200 µL). The resultant cell samples were finally detected by the aforementioned Raman instrument. For the specificity study, 4 HeLa cells in PBS (2.0 mL) were added into rabbit blood (2.0 mL) followed with shaking. After that, the samples were mixed with peripheral blood lymphocyte separation medium (2.0 mL). The separated WBCs (∼1.0 × 107) and HeLa cells were incubated with 1.0 mL of MBA3-AgNPR-rBSA4-FA2 (or MBA3-AgNPR-rBSA4, 0.2 mg/mL) plus 300 µL of SPION-rBSA-FA (or SPION-rBSA, 3.67 mM). Other steps were same with the above mentioned protocol.

Release of the Isolated CTCs for Further Phenotype Identification. The CTCs captured, enriched, and isolated by our CTC analysis system were released via adding excessive free FA for further cell expansion, phenotype identification and molecular analysis. Typically, 2000 HeLa cells in PBS (2.0 mL) was mixed with the rabbit blood (2.0 mL) in centrifuge tubes, added into 2.0 mL of peripheral blood lymphocyte separation medium. After centrifugation at 400 × g for 25 min, the WBCs (∼1.0 × 107) and HeLa cells in the low-density cell layer were taken out, washed and centrifuged 2 times using PBS. The final purified cells were dispersed in PBS (3.0 mL). 1.0 mL of MBA3-AgNPR-rBSA4-FA2 (0.2 mg/mL) and 300 µL of SPION-rBSA-FA (3.67 mM) were then added into 3.0 mL of the cells. After 30 min of incubation at 37 °C, the target cells were collected with a magnet to remove the nontarget cells, and rinsed 2 times using PBS. The final obtained cells were dispersed in complete DMEM containing excess FA (i.e. 50 µg/mL) to release the obtained cells from our nanomaterials. After cell expansion via culture at 37 oC in Falcon® Culture Slide (8 Well, Corning) for 7 days, the cells were rinsed 2 times using PBS, and then treated with 10


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paraformaldehyde (4 %) for 30 min, Triton X-100 (0.1 %) for 5.0 min, and BSA (1.0 %) for 30 min. Finally, the cells were stained with a solution of DAPI (10 µg/mL) and FITC-labeled anti-cytokeratin 8 monoclonal antibody (FITC-CK8, 1.25 µg/mL) for 2 h in refrigerator. After that, the samples were observed on a laser scanning confocal microscope (LSCM) imaging system (TCS SP5 II, Leica, Germany). The isolated CTCs were also analyzed by flow cytometry. Typically, 10,000 HeLa cells (FITC-CK8 stained) in PBS (2.0 mL) were added into the rabbit blood (2.0 mL, ∼1.0 × 107 WBCs) in centrifuge tubes. The FITC-CK8 stained HeLa cells were isolated from the mixtures using our CTC analysis system and the above-mentioned protocol. The final obtained cells were dispersed in 0.5 mL of PBS and measured by a flow cytometer (BD FACSCalibur, America). In addition, WBCs from rabbit blood as a negative control and FITC-CK8 stained HeLa cells as a positive control were also measured by the flow cytometer. Data analysis was performed using the flow cytometry analysis software (FlowJo 7.6).

RESULTS AND DISCUSSION Synthesis and Characterization of the SERS Nanoparticles. Our supersensitive CTC analysis system includes AgNPR-based SERS nanoparticles and SPION-based magnetic nanoparticles, whose preparation is shown in Figure 1. AgNPR prepared by a thermal method with minor modifications24, 31 was modified with MBA molecule (i.e. a Raman reporter) via Ag-S bonds. In order to reduce the nonspecific recognition by the normal cells in the blood, the MBA-AgNPR was further stabilized with rBSA, whose plenty of thiol groups can be associated with the AgNPR surface via Ag-S bonds forming a very thin layer of hydrophilic macromolecules. In addition, the targeting ligand FA was conjugated via an amidation reaction in the presence of EDC and NHS to enhance the specificity of MBA-AgNPR-rBSA to a few CTCs in plenty of normal healthy cells in blood. Synthesis conditions of the SERS active nanoparticles were optimized as shown in Table S1. It is 11



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found that the higher is the CMBA ranging from 25 to 500 µM, the stronger is the intensity of SERS signal (Table S1 & Figure S1). The optimal CMBA was chosen to be 100 µM for subsequent studies because higher CMBA (> 100 µM) can reduce the nanoparticle stability. The relative SERS intensity of MBA3-AgNPR-rBSA1 is very low at 20.0 µg/mL of CrBSA (Table S1 & Figure S1). However, MBA3-AgNPR-rBSA2-4 nanoparticles with 2.5 ~10 µg/mL of CrBSA have much stronger SERS intensities. The optimal rBSA concentration is considered as 2.5 µg/mL due to the strong SERS intensity. In addition, the FA conjugation content (FCC) and SERS intensity both increase with increasing the feeding FA concentration. Because higher FA concentrations (> 85.7) result in aggregation of the SERS active nanoparticles, the CFA was optimized to be 85.7 µg/mL. The morphologies of AgNPR and MBA-AgNPR-rBSA-FA were observed by TEM (Figure 2 a, b). Both nanoparticles are well dispersed, and MBA-AgNPR-rBSA-FA still keeps almost identical size and shape with AgNPR, which could be further confirmed by the corresponding DLS data of AgNPR and MBA3-AgNPR-rBSA4-FA2 (Figure 2 c). The DLS and TEM data demonstrate that it is really very thin for the protection layer rBSA, which benefits to decreasing the weakening effect of rBSA on SERS intensity. The UV-vis spectra of AgNPR and MBA3-AgNPR-rBSA4-FA2 (Figure 2 d) show that the absorption peak is 680 nm for AgNPR, but shifts to 710 nm for MBA3-AgNPR-rBSA4-FA1. The molecules of MBA, rBSA and FA may induce slight change of the refractive index surrounding AgNPR, which contributes to the red shift.32 In addition, the larger particle size (Figure 2 c) also contributes to the red shift.

Preparation and Characterization of the Magnetic Nanoparticles. The SPION was synthesized using a polyol method.33 TEM images in Figure 3 a, b confirm that our SPION and SPION-rBSA-FA are uniform and their water dispersity is good. The hydrodynamic diameters are measured to be 15.5 and 21.4 nm for SPION and SPION-rBSA-FA by DLS (Figure 3 c). The M-H curves (Figure 3 d) demonstrate that our SPION and SPION-rBSA-FA are both superparamagnetic. The Ms values of our SPION and SPION-rBSA-FA are respectively 83 and 75 emu g−1, which are 12


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comparable to other reported SPIONs.34 The Ms value of SPION-rBSA-FA is very similar with that of the SPION, which indicates that rBSA modification and FA conjugation cannot induce nanoparticle aggregation because aggregation of SPION can result in a much higher Ms value.35 Figure S2 a shows the XPS spectrum, which suggests that the component of our SPION is Fe3O4. The high resolution TEM (HRTEM) image in Figure S2 b shows the (311) and (220) planes of our SPION. The interplanar distance is respectively 2.53 and 2.97 Å. In the FT-IR spectra (Figure S3 a), the peak of 1250 cm-1 for SPION-rBSA corresponds to the C-H stretching in –CH3 from rBSA, and that of 1550 cm-1 corresponds to the C=O stretching in carboxylate (i.e. –COO–) from rBSA. The C-O stretching at 1250~1300 cm-1 in –COOH disappeared, which demonstrates the loss of hydrogen for –COOH. Figure S3 b, c show thermogravimetry (TG) and differential thermogravimetry (DTG) curves of SPION and SPION-rBSA. The weight loss of both SPION and SPION-rBSA increases with increasing temperature. However, the weight loss of SPION-rBSA is much higher than that of SPION at high temperatures (> 400 oC). In addition, the weight loss rate of SPION-rBSA is also much faster than that of SPION. These results demonstrate that the rBSA is successfully grafted onto the surface of SPION. The DLS data show the good stability of mixture of SPION-rBSA-FA and MBA3-AgNPR-rBSA4-FA2 that are used for detection and isolation of CTCs (Figure S4).

CTC Detection in Mixed Cells by the SERS Nanoparticles. The sensitivity and specificity of our SERS active nanoparticles (i.e. MBA3-AgNPR-rBSA4-FA2) were tested in the mixture of HepG2 and HeLa (or MCF-7) cells because they are respectively negative or positive cells with or without overexpression of FRα.36,37 The sensitivity and specificity of MBA3-AgNPR-rBSA4-FA2 for detection of MCF-7 (or HeLa) cells among 2.5 × 106 HepG2 cells are show in Figure 4. Figure 4 a, b show the SERS signals of 10 ~ 1000 cells/mL of MCF-7 (or HeLa) cells in 2.5 × 106 HepG2 cells. It is found that the stronger SERS intensity results from the higher cell density of HeLa or MCF-7 cells ranging from 10 to 13



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1000 cells/mL. The detection specificity of the MBA3-AgNPR-rBSA4-FA2 for 30 MCF-7 (or HeLa) cells among 2.5 × 106 HepG2 cells is further verified as shown in Figure 4 c, d. A SERS peak at 1588 cm-1 can be clearly found for 30 HeLa cells among 2.5 × 106 HepG2 cells with incubation of MBA3-AgNPR-rBSA4-FA2, but the corresponding SERS peak cannot be seen for 2.5×106 HepG2 cells with incubation of MBA3-AgNPR-rBSA4-FA2, or 30 HeLa cells among 2.5×106 HepG2 cells incubated with MBA3-AgNPR-rBSA4 (Figure 4 c). Figure 4 d shows the similar results for the detection of 30 MCF-7 cells among 2.5×106 HepG2 cells. In addition, from Figure 4 a, b, we can see that our MBA3-AgNPR-rBSA4-FA2 has a higher sensitivity for HeLa cells than MCF-7 cells due to the higher FRα expression of HeLa cells than MCF-7 cells.10

CTC Detection in the Rabbit Blood by the SERS Nanoparticles. Our SERS active nanoparticles (i.e. MBA3-AgNPR-rBSA4-FA2) are also used for detection of MCF-7 or HeLa cells in rabbit blood. Figure 5 a shows the SERS signals of rabbit blood (∼ 1.0 × 107 WBCs) with 1 ~ 5000 HeLa cells/mL after incubation with MBA3-AgNPR-rBSA4-FA2 nanoparticles. The stronger SERS intensity results from the higher HeLa cell density ranging from 1 to 5000 cells/mL. Figure 5 b shows the intensity of SERS signal versus the HeLa cell density in rabbit blood. A linear relationship (R2 = 0.9819) is found between the SERS intensity and HeLa cell density ranging from 5 to 100 cells/mL (inset plot), which demonstrates that our SERS nanoparticles could be utilized for detection of HeLa cells, and the LOD reaches to 5 cells/mL. The SERS signals of rabbit blood (∼ 1.0 × 107 WBCs) with or without HeLa cells (5 cells/mL) after incubation with MBA3-AgNPR-rBSA4-FA2 or MBA3-AgNPR-rBSA4 nanoparticles are shown in Figure 5 c. The SERS peak at 1588 cm−1 can be clearly found for the rabbit blood containing HeLa cells after incubation with MBA3-AgNPR-rBSA4-FA2. However, the corresponding SERS peak cannot be found for the rabbit blood containing HeLa cells after incubation with MBA3-AgNPR-rBSA4, or the rabbit blood without HeLa cells after incubation with MBA3-AgNPR-rBSA4-FA2. Therefore, these results demonstrate that our MBA3-AgNPR-rBSA4-FA2 is very specific to HeLa cells in the 14


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rabbit blood. The detection of MCF-7 cells among rabbit blood shows a similar result (Figure 6). The above results reinforce that our MBA3-AgNPR-rBSA4-FA2 nanoparticles can be utilized for CTC detection in blood samples with high sensitivity and specificity.

Capture, Enrichment, and Detection of CTCs in Rabbit Blood by our Supersensitive CTC Analysis System. Our supersensitive CTC analysis system includes SERS active nanoparticles (i.e. MBA3-AgNPR-rBSA4-FA2) and magnetic nanoparticles (SPION-rBSA-FA), which can be applied for capture (via interaction between FA and FRα), enrichment (via magnet) and detection (via SERS) of cancer cells from blood samples (Figure 1 c). We used HeLa cells for the following study due to the higher FRα expression. Figure 7 a shows the SERS signals of rabbit blood containing 1 ~ 100 HeLa cells per mL after incubation with MBA3-AgNPR-rBSA4-FA2 and SPION-rBSA-FA, isolation by magnet, and detection by a Raman instrument. The higher is the HeLa cell density, the stronger is the SERS intensity. Figure 7 b shows the relationship between the SERS intensity and HeLa cell density in the rabbit blood. There is a linear relationship between the SERS intensity and the HeLa cell density in the range of 1 ~ 100 cells per mL (R2 = 0.99345), and the LOD is 1 cells/mL (inset plot). Figure 7 c shows the SERS signals of rabbit blood with or without HeLa cells (1 cell/mL) incubated with MBA3-AgNPR-rBSA4-FA2 (or MBA3-AgNPR-rBSA4) plus SPION-rBSA-FA (or SPION-rBSA). We can see a SERS peak at 1588 cm−1 for the sample of rabbit blood with HeLa cells (1 cell/mL) after

incubation

with

MBA3-AgNPR-rBSA4-FA2

and

SPION-rBSA-FA.

However,

the

corresponding SERS peak cannot be seen for the rabbit blood with HeLa cells (1 cell/mL) incubated with

MBA3-AgNPR-rBSA4

plus

SPION-rBSA-FA,

or


plus

SPION-rBSA. In addition, we also cannot find the corresponding SERS peak for the rabbit blood without HeLa cells after incubation with MBA3-AgNPR-rBSA4-FA2 and SPION-rBSA-FA. The results demonstrate that our supersensitive CTC analysis system can be utilized for the capture, 15



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enrichment, and detection of CTCs in rabbit blood with a better sensitivity and specificity than the SERS nanoparticles alone (i.e. MBA3-AgNPR-rBSA4-FA2).

Release of the Isolated CTCs for Further Phenotype Identification. The CTCs captured, enriched, and isolated by our supersensitive CTC analysis system were further released by co-culture with excessive free FA. The released CTCs were then cultured at 37 oC for cell expansion, phenotype identification and molecular analysis. Figure 8 shows the LSCM images of HeLa cells expanded for 7 days and stained with DAPI (EX at 405 nm, EM at 447 nm) and FITC-CK8 (EX at 488 nm, EM at 525 nm). The FITC-CK8 is FITC-labeled anti-cytokeratin 8 monoclonal antibody that can be used as a marker for epithelial cell differentiation as well as a tool for tumor identification and classification. The FITC-CK8 does not react with non-epithelial tissues and cells including WBCs. The green signals in Figure 8 demonstrate that the cells captured, enriched, and isolated by our supersensitive CTC analysis system are our target cells (i.e. HeLa cells). In addition, the cells captured, enriched, and isolated using our supersensitive CTC analysis system were also analyzed by flow cytometry. Figure 9 shows flow cytometry analysis of WBCs (from rabbit blood, negative control), FITC-CK8 stained HeLa cells (positive control), and isolated cells from rabbit blood (∼1.0 × 107 WBCs plus 10,000 FITC-CK8 stained HeLa cells). It is found that the isolated cells have two peaks that respectively correspond to WBCs (negative control) and FITC-CK8 stained HeLa cells (positive control). This result indicates that the isolated cells include majority of HeLa cells and a few of WBCs. As shown in Table S2, the total content of silver and iron before the releasing process are 200 µg and 255 µg, respectively. After the releasing process, the remained contents of silver and iron are respectively 19.4 ± 0.6 % and 27.5 ± 0.9 %. Figure S5 shows the microscopic images of HeLa cells stained with calcein AM (green, for living cells) and PI (red, for dead cells) after the releasing process. The HeLa cells without any treatments were used as a control. Almost no difference was found between the HeLa cells after the releasing process and the control, which indicates that the 16


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remained nanoparticles cannot influence the cell growth.

CONCLUSIONS In this study, we developed a supersensitive CTC analysis system based on triangular silver nanoprisms (AgNPR) and superparamagnetic iron oxide nanoparticles (SPION) with function of capture, enrichment, detection and release. The AgNPR was encoded with MBA and modified with rBSA and FA to generate composite nanoparticle MBA-AgNPR-rBSA-FA (i.e. SERS active nanoparticles). The SERS active nanoparticles were optimized to be MBA3-AgNPR-rBSA4-FA2 nanoparticles via SERS signal intensity and nanoparticle stability, and then were used for CTC detection in the mixed cells (i.e. negative HepG2 plus positive HeLa (or MCF-7) cells). After that, the MBA3-AgNPR-rBSA4-FA2 nanoparticles were also used for CTC detection in rabbit blood with

addition

of

HeLa

(or

MCF-7)

cells.

The

results

demonstrate

that

our

MBA3-AgNPR-rBSA4-FA2 nanoparticles could be applied for CTC detection with high sensitivity and specificity. The LOD is 5 cells per mL. In addition, the SPION was also modified with rBSA and FA to generate composite nanoparticle SPION-rBSA-FA (i.e. magnetic nanoparticles). Our supersensitive

CTC

analysis

system

composes

of


and

SPION-rBSA-FA nanoparticles, which were applied for capture (via interaction between FA and FR α), enrichment (via magnet) and detection (via SERS) of cancer cells from blood samples. The results demonstrate that our supersensitive CTC analysis system has a better sensitivity and specificity than the SERS nanoparticles alone (i.e. MBA3-AgNPR-rBSA4-FA2). The LOD is only 1 cell per mL. The LSCM and flow cytometry results demonstrate that the CTCs captured, enriched, and isolated by our supersensitive CTC analysis system can also be further released (via adding excessive free FA) for further cell expansion, phenotype identification and molecular analysis.

ASSOCIATED CONTENT 17



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Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Table S1. Synthesis conditions and characterization results. Figure S1: SERS spectra. Figure S2: XPS spectrum and HR-TEM image. Figure S3: FTIR spectra, thermogravimetry and differential thermogravimetry curves.

AUTHOR INFORMATION Corresponding Authors *E-mail: [email protected] *E-mail: [email protected] ORCID Zheyu Shen: 0000-0002-0350-375X Aiguo Wu: 0000-0001-7200-8923 Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS This work is supported in part by Youth Innovation Promotion Association of Chinese Academy of Sciences (2016269) (Z.S.), National Natural Science Foundation of China (Grant No. 51761145021, U1501501, and U1432114), the Public Welfare Technology Application Research Project of Zhejiang Province (Grant No. 2017C33129), the National Key Research & Development Program (Grant No. 2016YFC1400600), Production-study-research cooperation project of Ningbo online technology market (Grant No. 2017B310003), Funding from Zhejiang Zuoyun Biological Technology Co., Ltd (Grant No. Y00428SA18), Hundred Talents Program of Chinese Academy of Sciences (2010-735).

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Figure 1. Schematic illustration for preparation of our supersensitive CTC analysis system based on AgNPR and SPION (a, b), and its application for capture, enrichment, detection and release of CTCs (c).

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Figure 2. Characterization of the AgNPR (control) and SERS active MBA3-AgNPR-rBSA4-FA2 nanoparticles. TEM images of AgNPR (a) and MBA3-AgNPR-rBSA4-FA2 (b). (c): Size distributions of AgNPR and MBA3-AgNPR-rBSA4-FA2 in Milli-Q water at room temperature. (d): UV-vis spectra of AgNPR and MBA3-AgNPR-rBSA4-FA2.

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Figure 3. Characterization of SPION and SPION-rBSA-FA. (a, b): TEM images of the SPION and SPION-rBSA-FA showing well dispersed nanoparticles. (c): Size distributions of SPION and SPION-rBSA-FA in Milli-Q water at room temperature. (d): M-H curves of SPION and SPION-rBSA-FA at 300 K.

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Figure 4. Detection sensitivity and specificity of our MBA3-AgNPR-rBSA4-FA2 nanoparticles for HeLa (or MCF-7) cells among HepG2 cells. (a, b): SERS signals of 30-3000 HeLa or MCF-7 cells among 2.5 × 106 HepG2 cells (3.0 mL) incubated with MBA3-AgNPR-rBSA4-FA2 nanoparticles. (c, d) SERS signals of the 2.5 × 106 HepG2 cells or 2.5 × 106 HepG2 cells plus 30 HeLa (or MCF-7) cells (3.0 mL) incubated with MBA3-AgNPR-rBSA4-FA2 or MBA3-AgNPR-rBSA4 nanoparticles.

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Figure 5. Detection sensitivity (a, b) and specificity (c) of the MBA3-AgNPR-rBSA4-FA2 for HeLa cells in rabbit blood. (a): SERS signals of rabbit blood with 5 ~ 5000 HeLa cells/mL after incubation with MBA3-AgNPR-rBSA4-FA2; (b) plot of the SERS signal intensity as a function of HeLa cell concentrations in rabbit blood; (c) SERS signals of rabbit blood without or with HeLa cells (5 cell/mL) incubated with MBA3-AgNPR-rBSA4-FA2 or MBA3-AgNPR-rBSA4.

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Figure 6. Detection sensitivity (a, b) and specificity (c) of the MBA3-AgNPR-rBSA4-FA2 for MCF-7 cells in rabbit blood. (a): SERS signals of rabbit blood with 5 ~ 5000 MCF-7 cells/mL after incubation with MBA3-AgNPR-rBSA4-FA2; (b) plot of the SERS signal intensity as a function of MCF-7 cell concentrations in rabbit blood; (c) SERS signals of rabbit blood without or with MCF-7 cells (5 cell/mL) incubated with MBA3-AgNPR-rBSA4-FA2 or MBA3-AgNPR-rBSA4.

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Figure 7. Detection sensitivity (a, b) and specificity (c) of the MBA3-AgNPR-rBSA4-FA2 and SPION-rBSA-FA for HeLa cells in rabbit blood. (a) SERS signals of rabbit blood with 1-100 HeLa cells/mL after incubation with MBA3-AgNPR-rBSA4-FA2 and SPION-rBSA-FA, isolation by magnet and detection by a Raman instrument; (b) plot of the SERS signal intensity as a function of HeLa cell concentrations in rabbit blood; (c) SERS signals of rabbit blood without or with HeLa cells (1 cell/mL) incubated with MBA3-AgNPR-rBSA4-FA2 (or MBA3-AgNPR-rBSA4) plus SPION-rBSA-FA (or SPION-rBSA).

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Figure 8. LSCM images of HeLa cells after isolation from rabbit blood using our supersensitive CTC analysis system, and expansion via culture at 37 oC for 7 days. (a): Bright field. (b): Nucleus stained with DAPI (excitation 405 nm, emission 447 nm). (c): FITC-CK8 (excitation 488 nm, emission 525 nm). (d): Merge of nucleus (DAPI) and CK (FITC). Scale bar: 30 µm.

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Figure 9. Flow cytometry analysis of WBCs (from rabbit blood, negative control), FITC-CK8 stained HeLa cells (positive control), and isolated cells from rabbit blood (∼1.0 × 107 WBCs plus 10,000 FITC-CK8 stained HeLa cells) using our supersensitive CTC analysis system based on AgNPR and SPION. Excitation wavelength: 543 nm. Emission channel: 600-660 nm. Voltage: 412 V.

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Title: A Supersensitive CTC Analysis System Based on Triangular Silver Nanoprisms and SPION with Function of Capture, Enrichment, Detection and Release

Huimin Ruan, Xiaoxia Wu, Chengcheng Yang, Zihou Li, Yuanzhi Xia, Ting Xue, Zheyu Shen,* Aiguo Wu*

Table of Contents A supersensitive CTC analysis system can be used for capture (via interaction between FA and folate receptor alpha), enrichment (via magnet) and detection (via SERS) of cancer cells from blood samples, and the isolated CTCs can also be released (via adding excessive free FA) for further cell expansion, phenotype identification and molecular analysis.


A Supersensitive CTC Analysis System Based on Triangular Silver

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