Aptamer-Functionalized and Gold Nanoparticle Array-Decorated

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Aptamer-Functionalized and Gold Nanoparticle Array-Decorated Magnetic Graphene Nanosheets Enable Multiplexed and Sensitive Electrochemical Detection of Rare Circulating Tumor Cells in Whole Blood Baoting Dou, Lin Xu, Bingying Jiang, Ruo Yuan, and Yun Xiang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.9b02403 • Publication Date (Web): 16 Jul 2019 Downloaded from pubs.acs.org on July 18, 2019

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Analytical Chemistry

Aptamer-Functionalized and Gold Nanoparticle Array-Decorated Magnetic Graphene Nanosheets Enable Multiplexed and Sensitive Electrochemical Detection of Rare Circulating Tumor Cells in Whole Blood Baoting Dou,† Lin Xu,‡ Bingying Jiang,*,‡ Ruo Yuan† and Yun Xiang*,† †

Key Laboratory of Luminescent and Real-Time Analytical Chemistry, Ministry of Education, School of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715, PR China ‡

School of Chemistry and Chemical Engineering, Chongqing University of Technology, Chongqing 400054, PR China

*Corresponding authors. E-mails: [email protected] (B. Jiang); [email protected] (Y. Xiang). ABSTRACT The identification and monitoring of circulating tumor cells (CTCs) in human blood has a pivotal role for the convenient diagnosis of different cancers. However, it remains a major challenge to monitor these CTCs because of their extremely low abundances in human blood. Here, we describe the synthesis of a new aptamer-functionalized and gold nanoparticle (AuNP) array-decorated magnetic graphene nanosheet recognition probe to capture and isolate the rare CTCs from human whole blood. In addition, by employing the aptamer/electroactive species-loaded AuNP signal amplification probes, multiplexed electrochemical detection of these low levels of CTCs can be realized. The incubation of the probes with the sample solutions containing the target CTCs can lead to efficient separation of the CTCs and result in the generation of two distinct voltammetric peaks on a screen printed carbon electrode, whose potentials and current intensities, respectively, reflect the identity and number of CTCs for multiplexed ACS Paragon Plus Environment 1

Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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detection of the Ramos and CCRF-CEM cells with detection limits down to 4 and 3 cells mL-1. With the successful demonstration of the concept, further extension of the developed sensing strategy for the determination of various CTCs in human whole blood for the screening of different cancers can be envisioned in the near future. INTRODUCTION According to a cancer statistic by the National Cancer Center in China, over 90% of cancer mortality is caused by cancer metastases.1 Many evidences indicate that cancer cells first escape from primary tumors and enter peripheral bloodstream, later rapidly proliferate in other parts of the body as a critical route for cancer metastasis and ultimately deplete the organs of the body.2,3 These cancer cells that disseminate from primary tumors or metastatic sites to the bloodstream and circulate in the blood vessels are named as circulating tumor cells (CTCs).4 Importantly, CTCs can be easily accessed by minimally invasive blood into all disease sites and at multiple time points during the disease, offering CTCs as “liquid biopsy” means of primary tumors or metastatic lesions.5,6 In comparison with conventional tumor biopsies, measuring and analyzing CTCs from whole blood can supply insightful information for estimating the disease status with less invasive procedures and small probability of false positives. However, the capture and determination of CTCs in whole blood is of extreme challenge, owing to their low abundance in blood, which is usually in the range of 1 to 100 cells per milliliter with the co-existence of millions of hematologic cells.7,8 Over the past decade, there have been crucial research endeavors devoted to the detection of CTCs, and these reported methods can be roughly classified into two categories: the strategies using physical separation and the assays based on biological affinities. Techniques based on the differences of physical characteristics (e.g., size, density, and dielectric properties) between CTCs and other cells, such as the microfilter approach,9,10

deterministic lateral

displacement method,11

and

density gradient

centrifugation,12 have proven to be promising approaches in CTC assays for metastatic cancers. However, these techniques encounter the obstacles of complicated experimental setups, the loss of ACS Paragon Plus Environment 2

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CTCs and low sample purity. This further encourages the development of assays based on biological affinities for CTC detection. Methods based on biological affinities13,14 typically utilize the capture molecules including cytokeratins and antibodies against the epithelial cell adhesion molecules (anti-EpCAM) or aptamers loaded on special carriers for capturing and detecting CTCs. One of the good examples is the commercially available product from CellSearch (Veridex, Raritan, NJ) approved by the U.S. food and drug administration (FDA) for monitoring CTCs, which utilizes anti-EpCAM antibody-coated immunomagnetic beads for identification of the target CTCs.15 However, EpCAM are expressed on 70% of tumor cells, which can potentially cause significant false positive signals and the compromise of accuracy and precision.16 In addition, CTCs may undergo epithelial to mesenchymal transition (EMT) during metastasis, which could further result in the loss of efficacy for antibody-antigen recognition.17,18 Thus, further efforts should be devoted to techniques based on biological affinities for more efficient and convenient detection of CTCs. In this regard, based on the aptamer-functionalized and gold nanoparticle array-decorated magnetic graphene nanosheet (AuNPs-Fe3O4-GS) capture probes and the electroactive species-loaded AuNP amplification signal probes, we report herein an electrochemical sensing strategy for ultrasensitive and multiplexed monitoring of CTCs in human whole blood. Aptamers are a class of oligonucleotide sequences with specific recognition functions, which can essentially fold into specific three-dimensional structures and specifically bind to different biological targets with high affinity.19-21 In addition to the excellent binding ability that rivals antibodies, nucleic acid aptamers are simple to synthesis, easy to modify, stable and lack of immunogenicity.22-25 These intrinsic advantages make them desirable alternative recognition candidates for biological detections. A series of nucleic acid aptamers against CTCs in blood has been screened through the cell-based systematic evolution of ligands by exponential enrichment (cell-SELEX) technology.26-28 The cell aptamers can not only discriminate whole cells from control cells, but also can distinguish high homologies from immunophenotypes, which are conducive to accurate classification of CTCs in blood cells and early diagnosis of hematological tumors. For example, the Sgc8 and Td05 aptamers exhibit excellent selectivity and high affinity toward the target ACS Paragon Plus Environment 3


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leukemia cells and the Ramos cells, respectively.[26,28-30] In our sensor design, aptamers are linked to the AuNPs-Fe3O4-GS for the preparation of the capture probes with two major advantages. First, owing to the many dispersed AuNPs, the loading amount of the aptamers can be significantly enhanced for efficient capture of the target CTCs. Second, because of the magnetic property of the AuNPs-Fe3O4-GS, convenient isolation of the captured CTCs from whole blood can be realized. Besides, when coupled with electroactive species-loaded AuNP amplification signal probes with well-resolved potentials, sensitive and multiplexed detection of the target CTCs can be achieved. EXPERIMENTAL SECTION Materials and reagents: Ramos (Burkitt’s lymphoma circulating cancer cells), CCRF-CEM (leukemia lymphoma circulating cancer cells), SK-BR-3 and MCF-7 (human breast cancer cells), and A549 (human lung cancer cells) were purchased from the cell bank of the type culture collection of the Chinese Academy of Sciences (Shanghai, China), and the cell culture reagents including Roswell Park Memorial Institute (RPMI) 1640 medium and Dulbecco’s Modified Eagle Medium (DMEM) were supplied by Dingguo Biological Technology Co., Ltd. (Chongqing, China). Bovine serum albumin (BSA), iron trichloride hexahydrate (FeCl36H2O), 6-ferrocenyl-1-hexanethiol (Fc-SH), ethanediamine, thionine (Thi), ethylene glycol (EG), L-lysine, gold (III) chloride trihydrate (HAuCl43H2O), sodium acetate anhydrous (NaOAc) and glucose were supplied by Sigma-Aldrich Shanghai Trading Co., Ltd. (Shanghai, China). Disposable screen-printed carbon electrodes (SPCEs) each containing a silver pseudo-reference electrode, a carbon auxiliary electrode and a carbon working electrode (3 mm in diameter) were supplied by Zensor R&D Co., Ltd. (Taichung, Taiwan). Graphene oxide (500 nm in size) was supplied by Nanjing XFNANO Materials Tech Co. Ltd (Nanjing, China). The HPLC-purified aptamer sequences (Td05 and Sgc8) adopted from previous literatures26,28 were synthesized by Sangon Biotech Co., Ltd in Shanghai. Sgc8: 5’-ATC TAA CTG CTG CGC CGC CGG GAA AAT ACT GTA CGG TTA GAT TTT TT -(CH2)6-SH-3’; Td05: 5’-AAC ACC GGG AGG ATA GTT CGG TGG CTG TTC AGG GTC TCC TCC CGG TGT TTT TT -(CH2)6-SH-3’. ACS Paragon Plus Environment 4

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Synthesis of the AuNPs-Fe3O4-GS: The Fe3O4-modified graphene (Fe3O4-GS) was first synthesized as follows. FeCl36H2O (0.05 g) was homogeneously dissolved in EG (10 mL), and NaOAc (0.15 g), ethanediamine (5 mL) and graphene oxide (0.05 g) were successively added to the solution under vigorous agitating for 0.5 h. Next, the mixture was heated to 200 °C and maintained for 8 h in a teflon-lined stainless steel autoclave, followed by cooling down to 25 °C. Finally, the obtained product was rinsed thrice with ethanol and water alternately, and dried at 50 °C under high vacuum to obtain the Fe3O4-GS. The Fe3O4-GS (10 mg) was further dispersed in ultrapure water (1 mL), followed by the addition of L-lysine (183 mg) and sonication for 20 min. The mixture was then centrifuged (8000 rpm, 2 min) and re-dispersed in a fresh L-lysine solution (1 mL, 1 mM), and the above procedure was repeated twice, before which the nanocomposite was centrifuged and re-dispersed in water (1 mL). Next, the Au seeds with the size range of 3 ~ 7 nm (0.5 mL, 0.25 mM), which were synthesized according to one of the previous literatures,31 were dropped into the resulted intermediate products and agitated for 0.5 h. Afterward, the mixture was separated by a magnet and the precipitates were dispersed in ultrapure water (3.5 mL), and HAuCl4 solution (500 μL, 10 mM) was added to the above mixture, and then adjusted pH to 10.9. Finally, glucose solution (1.5 mL, 100 mM) was injected drop by drop and the mixture was agitated for 9.5 h, resulting in the growth of AuNPs on Fe3O4-GS to form the AuNPs-Fe3O4-GS. The final products were separated by a magnet and rinsed thrice with phosphate buffered saline (PBS, 100 mM, pH 7.4), as well as finally re-dispersed in PBS. Immobilization of aptamers on the AuNPs-Fe3O4-GS: The Td05 or Sgc8 aptamer sequences (200 μL, 10 μM) were first incubated with TCEP (1 mM) for 1 h to reduce the disulfide bonds, and then were mixed with the as-prepared AuNPs-Fe3O4-GS nanocomposite (500 μL, 1 mg mL-1) and slowly agitated for 5 h at 25 °C. Subsequently, the nanocomposite was collected with an external magnet and rinsed thrice with PBS, and then re-dispersed in 5% BSA for 0.5 h to block the active sites. After being washed with PBS again, the resulted Td05/AuNPs-Fe3O4-GS or Sgc8/AuNPs-Fe3O4-GS bioconjugates were dispersed in PBS (50 μL) for further use. ACS Paragon Plus Environment 5


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Preparation of the electroactive species-loaded AuNP amplification signal probes: The AuNPs with the diameter of 20 nm were first synthesized through reduction of HAuCl4 with trisodium citrate,32 and the functionalization of the AuNPs with the electroactive species (Fc-SH or Thi) and the aptamers was carried out by following the reported protocols with slight modifications.33,34 In brief, the TCEP-treated Td05 aptamer (200 μL, 10 μM) and Fc-SH in hexane (100 μL, 5 mM) were mixed with AuNPs (500 μL, 0.25 mM) in PBS and agitated for 24 h. The Td05 and Fc-SH could be assembled onto the AuNPs through the formation of the Au-S bonds. The resulting (Td05+Fc-SH)/AuNPs was centrifuged (12000 rpm, 10 min) and washed with hexane and re-suspended in PBS buffer (50 μL) and stored at 4 °C for further use. (Sgc8+Thi)/AuNPs was prepared with similar procedures. Briefly, the TCEP-treated Sgc8 aptamer (200 μL, 10 μM) and Thi (100 μL, 5 mM) were mixed with AuNPs (500 μL, 0.25 mM) in PBS buffer and stirred for 24 h, followed by centrifugation and rinsing with PBS buffer, then re-suspended in PBS buffer (50 μL). Cell culture: A549 and MCF-7 cells were cultured in DMEM medium containing 10% FBS and 1% penicillin/streptomycin. CCRF-CEM, Ramos and SK-BR-3 cells were cultured in RPMI 1640 medium containing 100 IU mL-1 penicillin-streptomycin and 10% fetal bovine serum (FBS). The culture of the cells was conducted at 37 °C in a 5% CO2 incubator, and cell experiments were carried out until the cells reached 80% confluency. Multiplexed and amplified monitoring of CTCs in buffer and whole blood: Ramos and CCRF-CEM cells were centrifuged for 5 min at 1000 rpm separately to detach them from the culture medium. After discarding the supernatant, sterile PBS was subsequently added to obtain fresh cell suspensions at different concentrations. Then, the capture probes (5 μL of Td05/AuNPs-Fe3O4-GS and 5 μL of Sgc8/AuNPs-Fe3O4-GS) and signal probes (50 μL of (Td05+Fc-SH)/AuNPs and 50 µL of (Sgc8+Thi)/AuNPs) were incubated with the cell suspensions (1 mL) containing various concentrations of CCRF-CEM and Ramos cells at 37 ºC with gentle agitation for 20 min. After being separated by a magnet and rinsed twice using PBS, the resulted cell complexes were re-dispersed in PBS (50 μL) and ACS Paragon Plus Environment 6

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transferred onto the SPCE for immediate electrochemical measurements with a magnet sitting beneath the working electrode. Whole blood specimens from healthy volunteers and leukemia patients from the Daping Hospital of Chongqing (Chongqing, China) were collected in ethylenediaminetetraacetic acid (EDTA) tubes, and the subsequent detection of CTCs in whole blood was performed with identical procedures except the capture and signal probes were incubated with whole blood samples containing the spiked target CTCs (1 mL) at different concentrations of Ramos and CCRF-CEM cells. Apparatus and electrochemical measurements: The microscopic image was obtained using an Olympus IX70 optical microscopy with an objective lens (200×). Transmission electron microscopy (TEM) photographs were acquired on a JEM 1200EX microscope operated at 120 kV. All electrochemical measurements were conducted on a computer-controlled CHI 852C electrochemical workstation (CH Instruments, Shanghai, China). Square wave anodic stripping voltammetry (SWASV) analysis was performed in an electrochemical cell comprising the Ag/AgCl reference electrode, glassy carbon working electrode (3 mm in diameter) and platinum wire auxiliary electrode. Deposition was first carried out with accumulation time of 150 s at -1.4 V with gently stirring, and then stripping was conducted after a 15 s rest period from 0.0 to +1.0 V with frequency of 15 Hz, a step potential of 4 mV, and amplitude of 25 mV. Square wave voltammetry (SWV) was carried out from -0.4 V to +0.5 V with a frequency of 25 Hz, an amplitude of 25 mV and a step potential of 4 mV. RESULTS AND DISCUSSION

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Scheme 1. Illustration of the synthesis of (A) the aptamer-functionalized AuNPs-Fe3O4-GS capture probes and (B) the aptamer/electroactive species-loaded AuNP amplification signal probes. (C) The capture, isolation and amplified and multiplexed detection of the target CTCs in whole blood. The principle for CTC detection in blood. Scheme 1 outlines the designed strategy for the capture and amplified and multiplexed monitoring of CTCs in whole blood. As shown in Scheme 1A, Fe3O4-GS was first obtained through an one-step solvothermal method of the mixture of FeCl3∙6H2O, NaOAc, ethanediamine and graphene oxide. L-lysine is further used as the connecter between the Au seeds and Fe3O4-GS by taking the advantage of the two functional groups (NH2 and COOH) of L-lysine. Nucleation and growth of AuNPs on the Au seeds is realized in the presence of HAuCl4 with the assistance of the glucose reductant to obtain the AuNPs-Fe3O4-GS nanocomposites. The Td05 and Sgc8 aptamers are immobilized onto the surface of AuNPs-Fe3O4-GS by Au-S bond to prepare the aptamer-functionalized AuNPs-Fe3O4-GS capture probes. In addition, Thi and Fc-SH with well-resolved potentials are selected as redox probes in this work for simultaneous electrochemical detection of different CTCs. The aptamers and Fc-SH are assembled onto the surface of the AuNPs through the formation of the Au-S bond while Thi is immobilized via the combination of Au-S bond and electrostatic interaction between Thi and the AuNPs to obtain the aptamer/electroactive species-loaded AuNP amplification signal probes (Scheme 1B). Scheme 1C displays the diagram for the capture and simultaneous monitoring of two types of CTCs in whole blood. When the blood sample is incubated ACS Paragon Plus Environment 8

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with the aptamer-functionalized AuNPs-Fe3O4-GS capture probes and the aptamer/electroactive species-loaded AuNP amplification signal probes, the target CCRF-CEM and Ramos cells can be effectively recognized and captured by the corresponding probes, and the application of the external magnet leads to the separation of the target cells from other cells in large excess (e.g., leukocyte and erythrocyte). After washing and transferring the cell/probe conjugates onto the sensor electrode, SWV is performed to obtain the voltammogram, in which the peak current intensities and distinct potentials of the electroactive species can thus respectively be related to the concentration and identity of the target cells.

Figure 1. TEM images of (A) Fe3O4-GS, (B) Au seeds-attached Fe3O4-GS, (C) AuNPs-Fe3O4-GS; (D) TEM-EDS of AuNPs-Fe3O4-GS; Photographs of AuNPs-Fe3O4-GS dispersed in PBS solution (E) without and (F) with the application of an external magnet. Characterization of the AuNPs-Fe3O4-GS nanocomposite. The morphologies and elemental composition of the synthesized nanomaterials were first characterized by TEM. As shown in Figure 1A, ACS Paragon Plus Environment 9


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the Fe3O4/GS obtained by the facile one-step solvothermal method exhibits that the Fe3O4 particles are uniformly anchored on the graphene sheets. The addition of the Au seeds solution to the Fe3O4/GS leads to the adsorption of a large number of Au seeds on the surface of Fe3O4-GS (Figure 1B). It can also be observed that the Au seeds are not only located on the exposed graphene nanosheets but also deposited around Fe3O4 through the L-lysine connecter. Importantly, the growth of AuNPs on the surface-adsorbed Au seeds with the addition of HAuCl4 and glucose is evidenced by the increase of the size of the Au seeds (Figure 1C), and the AuNPs are uniformly distributed on the graphene nanosheets. To further confirm the element composition, TEM energy-dispersive X-ray spectroscopy (TEM-EDS) characterization of AuNPs-Fe3O4-GS was performed. As shown in Figure 1D, typical peak components corresponding to the Fe, Au, O, and C elements respectively can be observed, clearly suggesting the successful synthesis of the AuNPs-Fe3O4-GS. Besides, the AuNPs-Fe3O4-GS can be well-dispersed in PBS buffer (Figure 1E), as well as concentrated and separated on the side of a centrifuge tube within 20 s with the application of an external magnet (Figure 1F), demonstrating the magnetic feature of the AuNPs-Fe3O4-GS.

Figure 2. (A) Microscopic image of CCRF-CEM cells (150 cells mL-1) incubated with Sgc8/AuNPs-Fe3O4-GS for 30 min. (B) SWASV responses of (a) SK-BR-3 (150 cells mL-1) and (b) CCRF-CEM cells (150 cells mL-1) incubated with (Sgc8+Thi)/AuNPs for 30 min. Binding of the probes to the target CTCs. Bright-field microscopic imaging was used to verify the association of the aptamer-functionalized AuNPs-Fe3O4-GS capture probe with the CTCs by using the CCRF-CEM cells as the model cells. According to Figure 2A, after the incubation of the Sgc8/AuNPs-Fe3O4-GS capture probes with the target CCRF-CEM cells and the separation of the cell ACS Paragon Plus Environment 10

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composites from the solution with a magnet, it is clear that the Sgc8/AuNPs-Fe3O4-GS are attached to the cells because of the highly specific interaction between the aptamers linked to the Sgc8/AuNPs-Fe3O4-GS and the target CCRF-CEM cells. For confirming the attachment of (Sgc8+Thi)/AuNPs to the CCRF-CEM, SWASV analysis of AuNPs was performed after incubating the signal probes with the CCRF-CEM, followed by separation of the cell complexes with centrifugation of the solution at 1000 rpm for 5 min. The acidic bromine-bromide solution (100 μL, 1.0 M HBr-0.1 mM Br2) was used for dissolving the AuNPs attached to the CCRF-CEM cells for 20 min, and 3-phenoxypropionic acid (10 μL, 2 mM in 0.1 M HBr) was then dropped and incubated for 5 min to eliminate the excess Br2, followed by SWASV measurement of the Au3+ ions. As displayed in Figure 2B, there is no obvious current response after incubating the control SK-BR-3 cells with the (Sgc8+Thi)/AuNPs signal probes (curve a). On the contrary, a remarkable current response can be observed when (Sgc8+Thi)/AuNPs are treated with the CCRF-CEM cells, demonstrating the successful binding of (Sgc8+Thi)/AuNPs to the CCRF-CEM cells.

Figure 3. SWV responses of the mixture of the capture probes and signal probes incubated with different target CTCs: (A) without the target CTCs, (B) CCRF-CEM cells (150 cells mL-1), (C) Ramos cells (150 cells mL-1) and (D) both CCRF-CEM cells (150 cells mL-1) and Ramos cells (150 cells mL-1).



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Simultaneous monitoring of Ramos and CCRF-CEM cells in buffer. After the successful verification of the association of the probes with the CTCs, the concept for simultaneous electrochemical detection of different captured CTCs in buffer with our method was examined. Figure 3 exhibits the SWV responses of the mixture of the capture probes and signal probes incubated with different solutions. As displayed in Figure 3A, negligible current response can be obtained in the absence of the target CTCs. However, the incubation of the probes with the CCRF-CEM cells (150 cells mL-1) contributes to a large current response at -0.22 V (Figure 3B), which is derived from the electrochemical oxidation of the Thi species loaded on AuNPs. Similarly, the presence of Ramos cells (150 cells mL-1) results in a significant current peak at +0.30 V (Figure 3C), corresponding to the oxidation of the many Fc-SH tags on AuNPs bound to the target CTCs, while the current signal at -0.22 V is not affected. The above comparisons clearly indicate that this sensing method can be employed to monitor either Ramos or CCRF-CEM cells. Importantly, when the mixture of the Ramos and CCRF-CEM cells are treated with the probes, obvious increase of current response at the corresponding two distinct and well-resolved potentials can be observed (Figure 3D), which confirms the capability of the proposed strategy for multiplexed detection of CTCs.

Figure 4. Effects of (A) the volume ratio of Sgc8 to Thi, (B) the volume ratio of signal probes to capture probes and (C) incubation time on the current responses of the sensors. Error bars: SD, n=3. Experimental optimizations. The electrochemical performance of this sensor may be influenced by the parameters such as the volume ratio of the aptamer to the electrochemical species, the volume ratio of the signal probes to the capture probes and incubation time. Therefore, by using the detection of the CCRF-CEM cells as the model, the effect of the volume ratio of Sgc8 to Thi used for the synthesis of ACS Paragon Plus Environment 12

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the signal probes was studied firstly. As shown in Figure 4A, when CCRF-CEM cells (150 cells mL-1) are incubated with the signal probes prepared by using different volume ratios, the peak current increases with increasing volume ratio of Sgc8 to Thi until 2:1, followed by a sharp decrease, which can be attributed to the competition between the Sgc8 aptamer and Thi on the surface of AuNPs. When the AuNPs bind to a large number of Sgc8 aptamers, the binding ability of the signal probes to the target CTCs can be enhanced. However, the excessive aptamers will hinder the anchoring of the Thi tags on the surface of the AuNPs, resulting in a reduced current signal. These obtained results indicate that the best performance of signal probes can be achieved with the volume ratio of Sgc8 to Thi at 2:1. Furthermore, Figure 4B displays that the current response first increases with increasing volume ratio of signal probes to capture probes from 1 to 10 and decrease thereafter. This is due to the fact that excessive signal probes can potentially block the binding sites on the target CTCs surface, leading to low capture efficiency to the target CTCs. As a result, the volume ratio at 10:1 of signal probes to capture probes was used in the following experiments. As displayed in Figure 4C, when the incubation time of the probes and the target cells were varied from 5 to 40 min, the obtained peak current response is elevated with the extension of the incubation time and levels off after 20 min, which suggests the saturation of the binding of the probes to the target CTCs at 20 min. Consequently, the incubation time of 20 min was chosen for subsequent experiments.

Figure 5. (A) SWV responses of the sensors to various concentrations of Ramos and CCRF-CEM cells. From a to j: 0, 5, 50, 80, 100, 150, 200, 300, 400, 500 cells mL-1. The resulting calibration plots of peak current vs. the concentrations of (B) the CCRF-CEM cells and (C) Ramos cells. Error bars: SD, n=3. Analytical performance of the method for the detection of CTCs. After optimizing the experimental parameters, we proceeded to evaluate the dependence of peak current on the ACS Paragon Plus Environment 13


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concentrations of the two target CTCs for validating multiplexed detection. It can be observed in Figure 5A that both current peaks from electrochemical oxidation of Thi and Fc gradually increase with elevated concentrations of the Ramos and CCRF-CEM cells. According to Figure 5B and 5C, both calibration plots exhibit good linear relationships between the SWV peak currents and the concentrations of CTCs, and the linear ranges are from 5 to 500 cells mL-1 for Ramos (i = 0.02771c + 0.07985) and CCRF-CEM (i = 0.04419c + 0.24425) cells with the correlation coefficients (R)-square of the linear regression of 0.9936 and 0.9957, respectively. Furthermore, according to the 3σ rule, the detection limits for Ramos and CCRF-CEM cells are calculated to be 4 and 3 cells mL-1, respectively. Such detection limits for simultaneous monitoring of CTCs are comparable with or even more sensitive than some other reported approaches (Table 1). Additionally, the relative standard deviations (RSD) of 4.9% and 5.3% were obtained based on six repetitive measurements for the monitoring of CCRF-CEM at 200 cells mL-1 and Ramos at 200 cells mL-1, respectively, validating the desirable reproducibility of the proposed sensor for multiplexed monitoring of CTCs. Table 1 Comparison of the various strategies for monitoring of CTCs Linear range Detection limit Technique Amplification strategy Reference -1 (cells mL ) (cells mL-1) Electrochemical

Array of nanochannel-ion channel hybrid

100 to 1×106

100

35

Electrochemical

Fe3O4@nanocage core-satellite hybrids

50 to 1×107

34

36

Electrochemical

Direct plasmon-enhanced electrochemistry

5 to 1×105

5

37

Portable pressure meter

CuO/Co3O4 heterojunction nanofibers

50 to 1×104

50

38

UV-vis spectrometry

Cyclic enzymatic signal amplification

100 to 104

40

39

Electrochemical

Aptamer/electroactive species-loaded AuNP probes

5 to 500

4 and 3

This work


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Figure 6. Selectivity of this sensing strategy for the target CCRF-CEM and Ramos cells against other control cells. Error bars: SD, n=3. With the aim of investigating the specificity of the established electrochemical strategy, various cancer cells including SK-BR-3 (1500 cells mL-1), MCF-7 (1500 cells mL-1) and A549 (1500 cells mL-1) were incubated with the designed probes, and the measured peak currents were displayed in Figure 6. No remarkable increase of peak current can be obtained despite the presence of excess other cancer cells when compared with the blank experiment (without Ramos and CCRF-CEM cells), whereas significant current increases can be obtained in the existence of the target Ramos (150 cells mL-1) and CCRF-CEM cells (150 cells mL-1), which is ascribed to the selective recognition of the designed probes. Moreover, the mixture of the target CTCs and the other cancer cells leads to similar current responses with the presence of two target cells, confirming the excellent anti-interference capability of this methodology.

Figure 7. (A) Peak current responses of the sensors to various concentrations of spiked Ramos and CCRF-CEM cells in whole blood. From a to e: 0, 5, 50, 100, 200 cells mL-1. (B) Detection of CTCs in sample blood from healthy volunteers (1-3) and leukemia patients (4-6). Error bars: SD, n=3.



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Amplified detection of CTC in whole blood. The capture and multiplexed monitoring of CTCs in human whole blood was further studied to investigate the potential clinical application of this sensor. Different concentrations of Ramos and CCRF-CEM cells were first added into the whole blood sample from the healthy volunteer, which were incubated with the probes and the corresponding current responses obtained from the captured cells on the sensor were recorded for analysis. The insignificant peak current responses of the whole blood sample in the absence of the target CTCs shown in Figure 7A (column a) indicate the high specificity of the probes for the target CTCs even under the complex biological environments. Moreover, the current intensity enhances gradually as the number of the Ramos and CCRF-CEM cells increases. Although, the current responses in whole blood are slightly lower than that in buffer, the detection of a low number of the target CTCs can still be clearly achieved. The detection of CTCs in real sample blood from healthy volunteers and leukemia patients has also been performed. As shown in Figure 7B, very small current responses from the samples of the healthy volunteers (sample 1-3) are observed while the current intensity corresponding to the CCRF-CEM cells from the leukemia patients (sample 4-6) show significant increases, suggesting that our developed sensor has great potentials to be applied for the detection of CTCs in whole blood for cancer diagnosis. CONCLUSIONS In conclusion, a multiplexed and ultrasensitive electrochemical sensor for the monitoring of CTCs in whole blood has been demonstrated. Such a sensor utilizes the highly specific aptamer-functionalized AuNPs-Fe3O4-GS capture probes and the aptamer/electroactive species-loaded AuNP amplification signal probes for the capture and amplified monitoring of rare CTCs. The specific aptamers immobilized on the AuNP array-decorated magnetic graphene sheets enable efficient recognition and capture of the target CTC cells and the electroactive species-loaded AuNPs lead to substantial signal amplification, the combination of the two effects results in the successful monitoring of two rare CTCs in blood samples. With the selection of more aptamers that can target different CTCs in the future, the


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proposed strategy can thus offer a new platform for the detection of different CTCs in human blood for convenient cancer diagnosis. AUTHOR INFORMATION Corresponding Author E-mails: [email protected] (B. Jiang); [email protected] (Y. Xiang). Notes The authors declare no competing financial interest. ACKNOWLEDGMENT This work was supported by National Natural Science Foundation of China (No. 21675128) and Fundamental Research Funds for the Central Universities (XDJK2017A001). REFERENCES (1) Chen, W. Q.; Zheng, R. S.; Baade, P. D.; Zhang, S. W.; Zeng, H. M.; Bray, F.; Jemal, A.; Yu, X. Q.; He, J. Cancer Statistics in China. Ca-Cancer J. Clin. 2015, 66, 115-132. (2) Mehlen, P.; Puisieux, A. Metastasis: a Question of Life or Death. Nat. Rev. Cancer 2006, 6, 449-458. (3) Yu, M.; Bardia, A.; Aceto1, N.; Bersani, F.; Madden, M. W.; Donaldson, M. C.; Desai, R.; Zhu, H. L.; Comaills, V.; Zheng, Z. L.; Wittner, B. S.; Stojanov, P.; Brachtel, E.; Sgroi, D.; Kapur, R.; Shioda, T.; Ting, D. T.; Ramaswamy, S.; Getz, G.; Iafrate, A. J.; Benes, C.; Toner, M.; Maheswaran, S.; Haber, D. A. Ex Vivo Culture of Circulating Breast Tumor Cells for Individualized Testing of Drug Susceptibility. Science 2014, 345, 216-220. (4) Ashworth, T. R. A Case of Cancer in which Cells Similar to Those in the Tumors were Seen in the Blood after Death. Aust. Med. J. 1869, 14, 146-147. (5) Adams, A. A.; Okagbare, P. I.; Feng, J.; Hupert, M. L.; Patterson, D.; Gottert, J.; McCarley, R. L.; Nikitopoulos, D.; Murphy, M. C.; Soper, S. A. Highly Efficient Circulating Tumor Cell Isolation from Whole Blood and Label-Free Enumeration Using Polymer-Based Microfluidics with an Integrated Conductivity Sensor. J. Am. Chem. Soc. 2008, 130, 8633-8641. (6) Lin, M.; Chen, J. F.; Lu, Y. T.; Zhang, Y.; Song, J. Z.; Hou, S.; Ke, Z. F.; Tseng, H. R. Nanostructure Embedded Microchips for Detection, Isolation, and Characterization of Circulating Tumor Cells. Acc. Chem. Res. 2014, 47, 2941-2950. (7) Cristofanilli, M.; Budd, G. T.; Ellis, M. J.; Stopeck, A.; Matera, J.; Miller, M. C.; Reuben, J. M.; Doyle, G. V.; Allard, W. J.; Terstappen, L.; Hayes, D. F. Circulating Tumor Cells, Disease Progression, and Survival in Metastatic Breast Cancer. New Engl. J. Med. 2004, 351, 781-791. (8) Li, P.; Stratton, Z. S.; Dao, M.; Ritz, J.; Huang, T. J. Probing Circulating Tumor Cells in Microfluidics. Lab Chip 2013, 13, 602-609. ACS Paragon Plus Environment 17


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For TOC only:


Aptamer-Functionalized and Gold Nanoparticle Array-Decorated

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