Design of a Biocompatible and Ratiometric Fluorescent probe for the

Oct 22, 2018 - This strategy realized the highly sensitive detection of CTCs in whole blood ... dynamics evaluation.3 Quantification of CTCs in patien...
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Design of A Biocompatible and Ratiometric Fluorescent probe for the Capture, Detection, Release and Reculture of rare number CTCs Yanyan Yu, Yuan Yang, Jinhua Ding, Si Meng, Chenglin Li, and Xiaoxing Yin Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b02625 • Publication Date (Web): 22 Oct 2018 Downloaded from http://pubs.acs.org on October 22, 2018

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Analytical Chemistry

Design of A Biocompatible and Ratiometric Fluorescent probe for the Capture, Detection, Release and Reculture of rare number CTCs Yanyan Yu,†,‡,ǁ Yuan Yang,†, ǁ Jinhua Ding,† Si Meng,† Chenglin Li,*,† Xiaoxing Yin*,† †

Jiangsu Key Laboratory of New Drug Research and Clinical Pharmacy, Xuzhou Medical University, 209 Tongshan Road, Xuzhou 221004, Jiangsu, P.R.China ‡

Department of Pharmaceutical Analysis, School of Pharmacy, Xuzhou Medical University, 209 Tongshan Road, Xuzhou 221004, Jiangsu, P.R.China ABSTRACT: Circulating tumor cells (CTCs) served as an important biomarker for tumor recurrence and prediction of prognosis. However, selective capture and quantification of CTCs from whole blood was still full of challenge due to the extremely scare number of CTCs. Moreover, how to keep a high cell viability after capture remained to be solved. Here, we described a ratiometric fluorescent probe for the efficient capture and accurate determination of CTCs by conjugating graphitic carbon nitride quantum dots (g-CNQDs) with gold nanoclusters (AuNCs) and further linking with anti-EpCAM antibody to acquire the CTC-specific immune probe. In this probe, AuNCs protected by albumin V bovine played the role as the fluorophore reference and anti-EpCAM-attached g-CNQDs acted as both the response signal and specific recognition element for sensing CTCs. In the presence of CTCs, the quenched fluorescence of the immune probe at 500 nm was recovered due to the detachment of anti-EpCAM from the probe, while the intensity at 650 nm was essentially unchanged. This strategy realized the highly sensitive detection of CTCs in whole blood down to one CTC. Furthermore, it was demonstrated that the designed probe allowed capturing living CTCs with minimal cell damage. The subsequent reculture of captured cells for proliferation revealed that after a 7d proliferation, almost 28 MCF-7 cells were obtained from one target cell. The immune probe was successfully applied into capture and detection of CTCs from clinical cancer patients. Our data suggested the good potential of fluorescent probe for the clinical diagnosis of cancers.

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Cancer is the second major cause of death in the industrialized world. Treatment of primary tumors has progressed steadily, whereas metastases still occur frequently and are responsible for about 90% of cancer deaths.1 Circulating tumor cells (CTCs) are tumor cells disseminated from primary or metastasis sites that have then travelled through the bloodstream to other tissues of the body,2 and have considered a reliable biomarker for early cancer detection, diagnosis, prognostic, predictive, stratification and pharmacodynamics evaluation.3 Quantification of CTCs in patient blood provides new and valuable information about managing cancer. However, compared to billions of red blood cells and millions of white blood cells in 1 mL of blood, the number of CTCs is rather scarce with only 1~10 CTCs per milliliter on average for an active tumor.4 Consequently, development of an effective enrichment process will be a critical step for detecting and characterizing CTCs down to a frequency of 1 ~ 10 cells per 1 mL of blood. Epithelial cell adhesion molecule (EpCAM)-based immunomagnetic separation is the most commonly adapted strategy in CTCs enrichment, in which desirable capture specificity and efficiency are usually available, as most cancer cells overexpress EpCAM.5,6 Significant progresses have been achieved when the commercial CellSearch system was approved by Food and Drug Administration (FDA) as the only authorized CTC diagnostic system for enumeration of CTCs in patients with breast, prostate, and metastatic colorectal cancers. Guided by this strategy, our group also developed a functional and

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biocompatible immunomagnetic nanosphere based probe for the capture of multiple types of CTCs.7 However, although CTC enumeration by this system provides prognostic value in cancer patients, the captured CTCs are non-viable and cannot be released for downstream analysis or ex vivo cell culture. Consequently, increasing efforts have been devoted on how to maintain cell integrity and functions for follow-up experiments to interrogate and characterize the captured cells on the condition that a high capture efficiency can be acquired. Notably, apart from the efficient capturing of rare CTCs from the complex cell microenvironment, precise quantification of captured CTCs is also crucial for assessing the status of cancer patients and their drug response.8 Driven by this need, over the past decade, several types of techniques have been developed and applied such as cytologic testing, fluorescence imaging, magnetic resonance imaging, computerized tomography, Xray radiography, flow cytometry, surface-enhanced Raman scattering (SERS) and ultrasound.9-13 However, these emerging technologies are usually time-consuming and laborintensive, and the sensitivity is often limited by the enrichment degree of CTCs. Therefore, the development of an efficient and simple method, which not only allows for imaging of the captured CTCs, but also enables quantitative analysis, would dramatically increase the use of CTCs in diagnostics and prognostics. Nanostructured materials provide a new set of tools to overcome current limitations with CTCs capture and cells often

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show enhanced topological interactions with nanostructured interfaces.14-17 So far, two-dimensional graphitic carbon nitride quantum dots (g-CNQDs) have been considered a useful platform for the development of fluorescence biosensor, due to its high fluorescence quantum yield and excellent biocompatibility, which holds great promise for real life applications, including live cell analysis.18-20 In comparison with organic fluorophores, g-CNQDs have advantageous optical and electronic properties, such as size- and composition-tunable fluorescent emission from visible to infrared wavelengths, large absorption coefficients across a wide spectral range and high levels of brightness and photostability.21-23 In recent years, gCNQDs – based fluorescent probes have been efficiently designed for selectivity and sensitivity determination of H2O2/glucose,20 biothiol,24 ascorbic acid,25 zeatin,26 glucose,27 etc. Gold nanoclusters (AuNCs) are a new type of luminescent nanomaterial for fluorescence analysis because of their good biocompatibility, high electrocatalytic activity, and unique optical property.28 The synthesis of AuNCs often involves a kind of ligand (proteins or amino acids) to prevent them from growing into larger Nano crystals. For example, AuNCs protected by BSA can exhibit enhanced properties with a large Stokes shift, good biocompatibility, and low cytotoxicity due to its small size and BSA shell.29-32 Compared with single-signal fluorescence intensity-based assays, the dual-output ratiometric fluorescence technique that normalizes the variation from environmental changes provides more accurate measurement in analytical applications, especially for cells and other complex biological samples.33,34 In this regard, we combined two classical fluorescent nanomaterials, g-CNQDs and AuNCs into fabricating one ratiometric fluorescent nanoprobe (Au@g-CNQDs) and applied into efficient capture and quantitative detection of CTCs from cancer patients for the first time (Scheme 1). To achieve this goal, BSA chelated AuNCs were firstly synthesized, which were then covalently bonded to g-CNQDs. The fluorescence of AuNCs kept unchanged and thus could be served as reference signal for providing built-in correction to avoid environmental effects. Meanwhile, g-CNQDs acting as the responsive fluorescence signal were conjugated with the specific receptor of CTCs, anti-EpCAM, to form an immune fluorescent probe. The fluorescence intensity (FI) of g-CNQDs was quenched after attachment of anti-EpCAM due to aggregation of gCNQDs particles. Following the addition of a certain amount of CTCs into the immune probe, specific recognition between CTCs and anti-EpCAM occurred and anti-EpCAM was detached from the probe, as a result of which, the FI was recovered. Consequently, variations of fluorescence intensity ratios between g-CNQDs and AuNCs (Fg-CNQDs / FAuNCs) could be utilized as the quantitative index for the ratiometric determination of CTCs. Our results demonstrated that the designed immune probe was not only capable of specifically capturing CTCs from complex whole blood of cancer patients but also could accurately quantify the number of captured CTCs. We also realized the identification of CTCs by three-color immunocytochemistry (ICC), cell viability evaluation after capturing and reculture of the captured CTCs. To our best knowledge, report on the accurate quantification of CTCs by nanostructured fluorescent probe, in combination with identification, cell reculture was still seldom. This work represented an important step toward the application of ratiometric fluo-

rescence detection of CTCs, which provided a promising potential to advance individualized antitumor therapies.

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Scheme 1. Schematic diagram showing the fluorescent probe for the capture, detection, release and reculture of CTCs. EXPERIMENTAL SECTION

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Materials and Reagents. Gold chloride trihydrate (HAuCl4 •3H2O, ≥99.9%) and urea (≥ 99.5%) were purchased from Aladdin-Reagent Company (Shanghai, China). Sodium citrate was bought from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). Albumin V bovine (BSA) and Triton X100 were obtained from VICMED Company (Xuzhou, China). Monoclonal anti-EpCAM produced in mouse, carbodiimide (EDC) and N-hydroxysuccinimide (NHS) were from SigmaAldrich (USA). FITC labeled goat anti-mouse IgG, Alexa Fluor®594 labeled anti-Cytokeratin 19 monoclonal antibody (CK 19) and Alexa Fluor®488 labeled anti-CD45 (CD 45) monoclonal antibody were obtained from Abcam (Shanghai, China). MTT cell proliferation and cytotoxicity test kit and Annexin V-FITC Apoptosis detection kit were bought from KeyGEN BioTECH Corp., Ltd (Nanjing, China). Calcein-AM / PI double stain kit was obtained from Tiandz, Inc (Beijing, China). The clinical whole blood in EDTA-K2 anti-coagulated tubes were provided by Xuzhou Medical University Affiliated Hospital. All starting materials were analytical grade from commercial sources and used without further purification. Water used throughout the whole experiment was purified with Millipore system. Instruments and cell culture. These sections have been put into Supporting Information. Preparation of anti-EpCAM conjugated Au@g-CNQDs immune probe. Firstly, the AuNCs were prepared by a classical method according to previous reports.35,36 Aqueous HAuCl4 solution (5 mL, 10 mM, 37°C) was added to BSA solution (5 mL, 50 mg/mL, 37°C) under vigorous stirring. Two minutes later, NaOH solution (0.5 mL, 1 M) was introduced, and the mixture was kept stirring at 37°C for 12 h. The color of the solution changed from light yellow to light brown, and then to deep brown. Finally, the resultant was stored at 4 °C for further use. The synthesis of g-CNQDs were as followed:37 1.68 mmol urea and 0.14 mmol sodium citrate were mixed in an agate mortar and grounded to a uniform powder that was placed in an autoclave and heated to 170°C for 1 h in a drying oven. The

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Analytical Chemistry resultant yellowish mixture was washed by ethanol and centrifugation (12,000 rpm, 10 min) for three times, and then dried for 24 h in the drying oven to obtain g-CNQDs. Finally, to link AuNCs with g-CNQDs efficiently, 2 mL g-CNQDs (60 µg / mL) were firstly activated in 10 mg / mL EDC/NHS mixture dissolved in 400 µL PBS at room temperature with gentle shaking for 30 min. After that, 500 µL AuNCs were added into the activated g-CNQDs solutions for about 4 h with continuous shaking at room temperature. Finally, the resultant composite was stored at 4 °C for further use. To conjugate anti-EpCAM onto the above prepared Au@gCNQDs surface, 200 µL Au@g-CNQDs were reacted with 20 µL anti-EpCAM (0.5 mg / mL) for 4 h with continuous shaking at room temperature to obtain the required immune probe. When not in use, the probe should be stored at 4 °C. Ratiometric determination of CTCs by the immune probe. The capture and determination of CTCs was performed by fluorescent method. 500 µL of the immune probe were incubated with a certain amount cells suspended in 1 mL PBS for 30 min at room temperature. Subsequently, the fluorescence intensity of the incubated solution was measured at 500 and 650 nm with excitation at 400 nm. For blood samples, a defined number of MCF-7 was spiked into 1 mL of healthy human blood to obtain mimic clinical samples and then monitored the fluorescence in the same way as described above. ICC identification of CTCs in mixed cells and cell viability assessment after capture. These information could be found in Supporting Information. Released cell reculture. After incubation with the immune probe for 30 min at room temperature and collected by centrifugation, the released “cell-anti-EpCAM” entirety from the immune probe, were resuspended in a new cell culture dish (diameter 22 mm), with a total volume of 2 mL of 1640 culture medium to investigate the proliferative performance in one week in cell incubator (37°C and 5% CO2). The medium was changed every 2~3 days. The proliferated cells were observed under optical microscope every 24 h in one week. Clinical application of the immune probe. 15 cancer patients’ and 7 healthy human’ blood samples were collected and treated with the immune probe as described above. Typically in each assay, 500 µL of the immune probe was added into 1 mL blood and incubated for 30 min and detected the florescence intensity. According to the obtained linear curve equation, the number of CTCs in the blood sample could be quantitatively calculated. To obtain a high accuracy of the quantification, 3 mL blood was collected for each patient and three determinations were carried out on every patient. The calculated CTCs numbers were expressed as mean ± s.d. This result was further verified by three - color ICC method and the captured cells were observed under confocal fluorescence microscope. Cells that with phenotypes of CK19 positive and DAPI positive but CD45 negative were enumerated as CTCs.

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Figure 1. Typical TEM images of AuNCs (a), g-CNQDs (b), Au@g-CNQDs (c) and the immune probe (d). 85

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RESULTS AND DISCUSSION

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Structural characterizations of AuNCs, g-CNQDs, Au@g-CNQDs and the immune probe. In this work, AuNCs and g-CNQDs were combined together by covalent bonding to form a ratiometric fluorescent nanoprobe. Due to its high surface area, g-CNQDs provided enough binding sites for antiEpCAM immobilization. And the excellent biocompability

was beneficial for cell adhesion. The high resolution TEM images of AuNCs, g-CNQDs, Au@g-CNQDs, immune nanoprobe were displayed in Figure 1a-d. As could be seen, both AuNCs and g-CNQDs were uniformly mono-dispersed with average size of 2 ~ 3 nm respectively (Figure 1a, b). From the inset in Figure 1a, we could observe that the crystal lattice fringes of AuNCs were 0.227 nm, which corresponded to the spacing of crystal plane of Au.38 Moreover, the crystal lattice fringes of g-CNQDs (inset in Figure 1b) showed the representative image of an individual nanoparticle, indicating the high crystallinity with a lattice parameter of 0.303 nm.37 It was apparent that AuNCs were surrounded by g-CNQDs after AuNCs were conjugated with g-CNQDs (Figure 1c), indicating the successful preparation of Au@g-CNQDs. When antiEpCAM was further modified, the separated Au@g-CNQDs particles aggregated (Figure 1d) and the round shape of these particles became irregular. Because of the abundant functional groups on the surface of g-CNQDs and the rather small sizes, anti-EpCAM could be easily adsorbed onto g-CNQDs and interacted with g-CNQDs, which brought the particles close to each other and resulted in aggregations.19,39

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To better analyze the chemical composition and states of AuNCs, g-CNQDs, Au@g-CNQDs and the immune probe, XPS spectra of C 1s, N 1s and Au 4f were studied. As presented in Figure S1a, the XPS survey spectra showed a predominant graphitic C 1s peak at 284 eV, an N 1s peak at 400 eV and an O 1s peak at 530 eV. The spectrum of C 1s (Figure S1b) exhibited two peaks located at 284.7 and 288.0 eV on the four samples, respectively, which could be attributed to the standard reference carbon and sp2 - bonded carbon (N-C=N), respectively.40,41 Moreover, compared with AuNCs and gCNQDs, the intensity of the two peaks obtained on Au@gCNQDs and immune probe surfaces became higher and the positions of the peak gradually shifted to a lower energy, which was due to the coupling process. Moreover, after antiEpCAM was modified on Au@g-CNQDs, a new and weak peak at 286.1 eV was observed, corresponding to the C-N bonding, indicating that anti-EpCAM was successfully immobilized onto the surface of Au@g-CNQDs layers.42 The N 1s spectrum showed a clear peak at 399.9 eV for AuNCs, gCNQDs and Au@g-CNQDs samples, belonging to C-N-C

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(Figure S1c). Moreover, after anti-EpCAM modification, the original peak blue-shifted to 399.5 eV, indicating the formation of covalent -N-C bond in the immune probe between the carboxylic acid groups in Au@g-CNQDs and the amine groups in anti-EpCAM.42 The atomic percent of N 1s peak was found to be 2.54, 12.28, 10.03, 15.62, respectively. The AuNCs displayed a dominant component of Au 4f7/2 and Au 4f5/2 located at 83.5 and 87.3 eV, respectively as shown in Figure S1d, which was consistent with Au0 and Au+.35 These two peaks were missing in g-CNQDs but visible in Au@gCNQDs and immune probe. The FT-IR and Raman spectra of AuNCs, g-CNQDs, Au@g-CNQDs and the immune probe were presented in Figure S2. Optical property and fluorescent sensing of the immune probe toward CTCs. To further explore the optical properties of Au@g-CNQDs fluorescent probe, UV-vis absorption and fluorescent spectra were investigated (Figure 2a). In the presented UV-vis spectrum, a typical absorption peak at 400 nm was observed, which was almost the same as that of the maximum excitation peak of Au@g-CNQDs. The most intense fluorescence from Au@g-CNQDs appeared under 400 nm excitation and had dual maximum emission peaks located at 500 and 650 nm, belonging to g-CNQDs and AuNCs, respectively. The spectra of them were found to be consistent with reported literatures.35-37 To capture CTCs selectively from whole blood, antiEpCAM was modified onto Au@g-CNQDs surface to obtain the required immune probe. From the result in Figure 2b, the strong FI of Au@g-CNQDs at 500 nm was significantly weakened when anti-EpCAM was conjugated with Au@gCNQDs, while the peak of AuNCs at 650 nm kept constant (red curve). The decline in the FI of g-CNQDs was possibly due to that the modification of anti-EpCAM brought originally isolated g-CNQDs particles into close proximity with each other and therefore aggregation occurred.43-45 This result was consistent with TEM characterizations that the g-CNQDs particles became aggregated in the presence of anti-EpCAM (Figure 1d). However, upon additions of a certain number of CTCs into the immune probe, the FI at 500 nm was found to be recovered (blue curve). This phenomenon could be explained that anti-EpCAM had stronger interactions with CTCs than to the functional groups on g-CNQDs, and thus they were released from g-CNQDs in the presence of CTCs, consequently inducing disaggregation of g-CNQDs and FI recover.39 Thereby, a sensitive quantitative method based on the fluorescence changes of Au@g-CNQDs was established for the ratiometric detection of rare CTCs based on the fluorescence ratio between g-CNQDs and AuNCs (Fg-CNQDs / FAuNCs). Before applying the probe into capture and determination of CTCs, we investigated the anti-fouling ability from whole blood and stability of the immune probe. A certain amount of the immune probe was respectively added to human whole blood into a centrifuge tube and incubation for some time, and then the fluorescence intensity were measured. The recovery was calculated according to the following equation: recovery = Fc / F0 × 100%, where Fc and F0 was denoted as the FI of the immune probe at 500 nm after incubating in blood and PBS, respectively. This experiment was performed three times and the recovery was estimated to be (88.97 ± 0.39) %, revealing an acceptable anti-fouling ability of the immune probe in the complicated blood environment. The immune probe was found

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to be stable over a long period of storage time, which was reflected from the fact that the calculated Fg-CNQDs / FAuNCs ratios were almost the same with rather minor floating when the immune probe was stored at 4°C for one, two, three, four weeks (Figure S3a). Besides, we also monitored the bioactivity of immune probe toward CTCs by comparing the fluorescence enhancement factors [(F – F0) / F0] (F and F0 were the fluorescence intensity of the immune probe at 500 nm in the presence and absence of CTCs, respectively) upon additions of CTCs into the immune probe after the probe was stored for different time. As was seen from Figure S3b, the obtained [(F – F0) / F0] values changed little with increasing storage time. These evidences confirmed that the immune probe could resist the potential interferences from the complicated whole blood environment and kept enough stability and bioactivity for the following experiments.

Figure 2. (a) UV-vis, excitation and emission spectra of Au@g-CNQDs. (b) Fluorescence spectra of Au@g-CNQDs, immune probe in the absence and presence of MCF-7. (c-e) Confocal microscopic images of the released MCF-7 cells on the 24-well plates after incubating with FITC-conjugated goat anti-mouse IgG under bright field (c), fluorescente field (d) and merge of bright and fluorescent fields (e). Scale bar represented 50 µm. Illustration of detection principle. As depicted above, the feasibility of the Au@g-CNQDs - based fluorescence assay has been demonstrated by observing the FI changes upon addition of tumor cells, in which the quenched fluorescence could be basically recovered in the presence of CTCs due to the detachment of anti-EpCAM from the immune probe caused by its specific interaction with CTCs. To further illustrate the detection principle, we used optical microscopy to image the captured cells. MCF-7 cells were initially incubated in 24-well plates for 24 h, then anti-EpCAM modified Au@g-CNQDs were added and incubated with cells for 30 min at 37°C, after that, the supernatant was removed and the remained cells on the plates were washed with PBS for three times. Then, FITCconjugated goat anti-mouse IgG, which was used to mark the appearance of anti-EpCAM, was added and reacted for 2 h. After that, the supernatant was removed away and cells on the plates were washed again. The fluorescence microscopic images were taken for the cells and shown in Figure 2c-e. Obviously, the green fluorescence from FITC-conjugated goat antimouse IgG was observed under confocal microscope, indicating the appearance of anti-EpCAM on target cell surfaces, which meant that when cells were incubated with the immune

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Analytical Chemistry probe, due to its much stronger interaction with cells than to the functional groups on g-CNQDs, anti-EpCAM and cells as a whole, were detached from the immune probe. The separation from nanomaterials made it advantageous for the following cell analysis, including reculture and viability assessment. This result was highly consistent with our fluorescence quantitative assay, which confirmed the rationality of our strategy for the accurate sensing of CTCs.

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Figure 3. (a) The calculated [(F – F0) / F0] values in the presence of MCF-7, HCT116, A549, HepG2, THP-1, Hela, Hek293 and MCF-10A cells; (b) Fluorescence spectra of the immune probe upon additions of various number of MCF-7 cells into PBS and healthy human blood (c). (d) Regression analysis of Fg-CNQDs / FAuNCs versus the number of MCF-7 cells spiked in PBS and whole blood. (Error bar represented the standard deviations, n = 3). Specificity and sensitivity toward CTCs determination. To assess the targeting specificity of the prepared immune probe toward CTCs determination, we examined the fluorescence responses of the immune probe upon binding with other EpCAM-positive (A549, HepG2, HCT116) cells and negative cells (THP-1, Hela, Hek293 and MCF-10A). As presented in Figure 3a, similar to MCF-7, with the same cell concentration (350 / mL), additions of the other three type EpCAM-positive cells all induced recovered FI of the probe, while the [(F – F0) / F0] value was almost the same as that of blank probe after the immune probe was incubated with the four negative cells. Meanwhile, as another control experiment, we used the unmodified Au@g-CNQDs instead of the immune probe to incubate with the above mentioned tumor cells and extreme low [(F – F0) / F0] values were obtained compared with those of immune probe. These results demonstrated that our immune probe was only specific and effective for tumor cells but negative to white blood cells. Under the optimal conditions (the optimization was presented in Surpporting Information, Figure S4), the immune probe was applied for the quantitative detection of CTCs in PBS and whole blood mediums. As shown in Figure 3b and 3c, both in the two mediums, with the increase of MCF-7 numbers, the peak FI of the probe at 500 nm gradually increased, while the peak intensity at 650 nm remained constant. The calculated FgCNQDs / FAuNCs ratios were found to be increasing linearly with cell numbers over a wide range from 2 to 400 cells / mL. The regression equations were y = 0.0099 x + 3.15 and y = 0.0031

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x + 2.99, with the two correlation coefficients (R2) of 0.996 and 0.997 in PBS and whole blood, respectively, where y and x were denoted as Fg-CNQDs / FAuNCs ratios and MCF-7 cell numbers. We noticed that in the absence of CTCs, the FI of the immune probe at 500 and 650 nm in blood were both stronger than those in PBS, especially the peak at 650 nm. As a result, all of the calculated Fg-CNQDs / FAuNCs values were lower than those obtained in PBS. We speculated that the complicated environment of the whole blood, which contained multiple proteins, billions of red blood cells and millions of white blood cells, etc, destroyed the interactions between antiEpCAM and Au@g-CNQDs to some extent due to steric effects. Thus, the degree of Au@g-CNQDs aggregations in blood was lesser and therefore the FI was stronger. Noticeably, the slope of the linear plot obtained in whole blood was found lower than that in PBS medium (0.0091 and 0.0031 in PBS and whole blood, respectively) (Figure 3d), which was possibly caused by the blockage from red blood cells and the reduced interaction chance between the immune probe and the target CTCs.46,47 Therefore, the captured CTC numbers by the probe decreased. In this study, the limit of detection was calculated to be one cell at 3σ, which held prominent superiority over other detection assays (Table S1). The enhanced detection ability of this immune fluorescence assay could be ascribed to the application of g-CNQDs that provided enlarged surface area for immobilization of anti-EpCAM to specifically recognize CTCs and inherent strong fluorescence emission. Re-culture of the released cells and viability assessment. As there was an extremely low number of CTCs in the bloodstream, the proliferation of rare captured CTCs would be a significant attempt for further molecular characterization and functional analysis. As the detection principle illustrated above, after incubating MCF-7 cells with the immune probe under the optimal conditions and then centrifuged, the released cells could be collected in the supernatant. In order to demonstrate the dynamic proliferation behavior of captured cells, the collected cells were re-cultured for seven days successively at 37°C under 5% CO2 in RPMI-1640 medium. As shown in Figure 4, not only quantities of MCF-7 cells but single MCF-7 cell kept excellent proliferative performance in one week. After a 7d proliferation, almost 28 MCF-7 cells were obtained from one target cell. We then determined the viability of the captured cells by counting the numbers of dead and live cells after isolation stained with Calcein AM and PI, respectively. Calcein AM was a kind of dye which can traverse the live cell membrane and be hydrolysed by esterase to form calcein with green fluorescence,4,48 while propidium iodide (PI) was a fluorescence stain that cannot pass through intact cell membranes but readily passes through damaged membranes and binds with DNA,48 producing bright red fluorescence. In this way, we could make a distinction between live and dead cells by the color observation of their fluorescence. Images were taken under the fluorescence microscope shown in Figure S5a. The viability rate was calculated to be 94.5 ± 2.6%, indicating that most of the MCF-7 cells remained viable after release. The high cell viability was primarily due to the excellent biocompatibility of the immune probe. To verify this statement, we carried out MTT assay and cell apoptosis experiments. As demonstrated in Figure S5b, no obvious decline in cell viability was detected in any of the MCF-7 cell samples, which were treated with increasing amounts of the immune probe. The

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lowest cell viability was found to be 94.13%. The results of cell apoptosis (Figure S5c and 5d) showed that compared with the apoptosis rate of control group (Q2 + Q3) (4.20%), the value in the experimental group was 4.35% (Q2 + Q3), displaying slightly elevated. These results were quite consistent with MTT assay that our designed probe possessed good biocompatibility and had rather low toxicity for cells.

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Figure 4. The re-culture process of the released cells in one week. The scale bars represented 20 µm. ICC identification of CTCs in mixed cells. The captured CTCs were further identified by immunocytochemical (ICC) analysis. Initially, MCF-7 cells were spiked with THP-1 cells, which were negative to anti-EpCAM. Then, an immunostaining approach was adopted to distinguish CTCs from the mixture cells. Cells were stained DAPI to identify nucleated cells, anti-CD45 to differentiate WBCs from cancer cells, and anticytokeratin (CK), as CK was a gold-standard marker for the identification of epithelial cells in the blood.49 As shown in Figure S6, cells that exhibited strong CK19 expression and negligible CD45 signals were identified as tumor cells, while cells that presented high CD45 and low CK19 expression levels were counted as WBCs. Clinical application of the immune probe. On the basis of the excellent analytical performance of the immune probe toward CTCs, to further explore the clinical application of the immune assay, 500 µL immune probe was incubated with 1 mL blood samples from healthy human and cancer patients for 30 min and then detected the recovered fluorescence as depicted above. 15 cancer patients, including breast, lung, colon, gastric and liver cancer and 7 healthy human were involved. To obtain a high accuracy, three determinations were carried out on each patient. The fluorescence spectra of the immune probe toward blood samples from cancer patients were presented in Figure S7. After calculation, CTCs in the bloods of 15 cancer patients were all detected, while no CTC was found in any healthy human samples (Table 1 and Table S2). To demonstrate the accuracy of the method, CTCs were further

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identified and counted by the three-color ICC as depicted above, which were DAPI + / CK19 + / CD45 – for CTCs and WBCs were DAPI + / CK19 - / CD45 + (Figure 5). As could be confirmed, the counted CTCs numbers under fluorescence microscope were mostly consistent with the fluorescent results (Table 1). The images of 7 CTCs, including a CTC cluster and 5 single cells, from 1 mL of blood of patient # 12 were shown in Figure S8. These results suggested that our constructed immune probe could be reliably applied to the detection and identification of CTCs from clinical cancer patient blood. Table 1. Quantitative and counted CTC numbers in 1 mL blood sample from patients with epithelial cancers. NO

Cancer Type

Gender

Age

Treatment

Detected CTCs (mean ± s.d)

Counted CTCs

1

Breast

Female

49

Treated

7.3 ± 0.17

7

2

Breast

Female

62

Treated

9.9 ± 0.35

10

3

Breast

Female

56

Treated

15.7 ± 0.75

16

4

Breast

Female

46

Treated

5.1 ± 0.51

5

5

Breast

Female

36

Treated

11.0 ± 0.35

11

6

Lung

Female

69

Treated

18.3 ± 0.35

19

7

Lung

Female

75

Treated

15.1 ± 0.06

15

8

Lung

Male

64

Treated

11.1 ± 0.50

11

9

Liver

Male

71

Untreated

22.2 ± 0.81

22

10

Liver

Male

66

Untreated

4.8 ± 0.35

5

11

Gastric

Male

61

Untreated

29.7 ± 1.00

30

12

Gastric

Male

50

Untreated

7.1 ± 1.30

7

13

Gastric

Male

61

Untreated

3.3 ± 0.51

3

14

Gastric

Female

64

Untreated

17.3 ± 0.46

17

15

Colon

Male

49

Treated

2.2 ± 0.51

2

Figure 5. (a-d) Microscopic images of CTC from cancer patients’ blood and identified with the three-color ICC method. Merged: merge of nucleus (DAPI), CK19 (Alexa Fluor® 594) and CD45 (Alexa Fluor® 488). Scale bar represented 20 µm.

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Analytical Chemistry CONCLUSIONS

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In this contribution, a fluorescent stategy based on the“ offto-on” mechanism of Au@g-CNQDs was developed for capturing, detecting, releasing and reculturing rare number CTCs using a facile approach, exihibiting excellent capture specificity, sensitivity and good celluar biocompability. Our data demonstrated that the employment of the two classical nanomaterials provided not only more surface area for antiEpCAM immobilization, but also a biocompatible plateform for keeping a high cell viability after capture, which realized the proliferation of rare target cells. Further application of this immue probe into capturing and detection of CTCs from clinical caner patients indicated the good potential of the developed technology as a reliable biomarker to predict disease progression and diagnosis of cancer patients.

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ASSOCIATED CONTENTS Supporting Information

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The Supporting Information is available free of charge on the ACS Publications website. It included instrument details; cell culture and collection; XPS, FT-IR and Raman characterizations; stability investigation; optimization process; cell viability evaluation and CTC identification; fluorescence spectra of clinical samples.

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AUTHOR INFORMATION 25

Corresponding Author

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*Phone/Fax: +86 516 8326-2009, e-mail: [email protected] *Phone/Fax: +86 516 8326-2009, e-mail: [email protected] 90

Author Contributions ǁ

30

These authors contributed equally.

Notes The authors declare no competing financial interest.

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ACKNOWLEDGMENT

35

This work was financially supported by the National Natural Science Foundation of China (no. 21675137), Natural Science Foundation of Jiangsu Province (no. BK20161170) and China Postdoctoral Science Special Foundation (no. 2016T90504).

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