An aptamer-based dual-functional probe for rapid and specific

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An aptamer-based dual-functional probe for rapid and specific counting and imaging of MCF-7 cells Bin Yang, Beibei Chen, Man He, Xiao Yin, Chi Xu, and Bin Hu Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b04927 • Publication Date (Web): 08 Jan 2018 Downloaded from http://pubs.acs.org on January 8, 2018

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Analytical Chemistry

An aptamer-based dual-functional probe for rapid and specific counting and imaging of MCF-7 cells Bin Yang, Beibei Chen, Man He, Xiao Yin, Chi Xu, Bin Hu* Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), Department of Chemistry, Wuhan University, Wuhan 430072, China

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Corresponding [email protected]

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+86-27-68754067;

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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

Abstract Development of multimodal detection technologies for accurate diagnosis of cancer at early stages is in great demand. In this work, we report a novel approach using an aptamer-based dual-functional probe for rapid, sensitive and specific counting and visualization of MCF-7 cells by inductively coupled plasma-mass spectrometry (ICP-MS) and fluorescence imaging. The probe consists of a recognition unit of aptamer to catch cancer cells specifically, a fluorescent dye (FAM) moiety for fluorescence resonance energy transfer (FRET)-based “off-on” fluorescence imaging as well as gold nanoparticles (Au NPs) tag for both ICP-MS quantification and fluorescence quenching. Due to the signal amplification effect and low spectral interference of Au NPs in ICP-MS, an excellent linearity and sensitivity were achieved. Accordingly, a limit of detection of 81 MCF-7 cells and a relative standard deviation of 5.6% (800 cells, n=7) were obtained. The dynamic linear range was 2×102-1.2×104 cells, and the recoveries in human whole blood were in the range of 98-110%. Overall, the established method provides quantitative and visualized information of MCF-7 cells with a simple and rapid process, and paves the way for a promising strategy for biomedical research and clinical diagnostics.

Keywords: MCF-7 cell; aptamer; FRET; inductively coupled plasma mass spectrometry; fluorescence imaging

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Introduction Cancer is one of the most serious threats to public health worldwide, it still causes high mortality rate even with the developed level of clinic medical treatment nowadays.1 According to the latest cancer statistics released by the National Central Cancer Registry of China, 4.29 million new cancer cases and 2.81 million cancer deaths occurred in China in 2015.2 Specifically, breast cancer is the most commonly cancer among women, which accounts for 15% of all new cancers in women. The main cause of cancer-related death is cancer metastasis, which may largely attribute to the tumor cells dislodged from tumors into the blood also known as circulating tumor cells (CTCs).3 Early and accurate detection of CTCs buried in the ocean of numerous normal and/or benign cells is therefore significant as a general strategy to monitor and prevent the development of cancer, and also as a guide to the effective therapeutic treatments.4 To address this great challenge, novel approaches for rapid, sensitive, noninvasive, reliable, and accurate detection of extremely rare CTCs in clinical blood samples are in great demand. Up to now, many efforts have been made regarding the detection of CTCs to improve cancer diagnosis and prognosis, including flow cytometry,5 electrochemical method,6 quantitative real-time reverse transcriptase-polymerase chain reaction assay,7 inductively coupled plasma-mass spectrometry (ICP-MS)-based method,8 etc. Among all these methods, ICP-MS combined with varieties of element-labeling strategies has emerged and become a promising approach for quantitative bioanalysis in recent years. By applying suitable elemental tags as endogenous elemental tags to label target objects, ICP-MS-based methods have been extended to the determination of various biomolecules including peptides,9 proteins,10,11 DNA,12,13 and RNA,14 as well as cells15,16 since the pioneering work reported by Zhang et al. in 2001.17 Owing to the superior performance of ICP-MS for element-specific analysis, ICP-MS-based methods usually exhibit extraordinary advantages such as high sensitivity and accuracy, wide dynamic linear range, robust resistance to the matrix, and multiplex detection capability for elements and/or isotopes. Nevertheless, the limitations of ICP-MS-based methods should still be noted especially for cell analysis. As a destructive detection technique, ICP-MS has to face the challenge of providing in situ noninvasive image which would offer unrivaled spatiotemporal resolution and help to exclude false-negative/positive results. Therefore, a combination of ICP-MS-based quantitative bioanalysis

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with fluorescence imaging is urgently needed to offer more comprehensive and valuable information over each single assay for more reliable diagnosis of disease and better patient treatment. To address these issues, we have previously proposed two kinds of strategies to bridge the barrier between optical imaging and ICP-MS quantification by introducing quantum dots18 and upconversion nanoparticles19 as multifunctional tags, respectively. Moreover, Zhang and co-workers reported an integrin-targeted trifunctional probe which comprised a guiding unit, an europium chelate tag, and a fluorescent moiety for seeing and counting cancer cells.20 However, these reported methods more or less suffer from time-consuming operations and complicated fabrication. Besides, the antibody-involved method also has several drawbacks such as high cost and limited analysis targets. Therefore, more effort should be taken for the development of simpler, less time consuming, less expensive, and more straightforward approaches which combine fluorescence imaging and ICP-MS quantification. With the advances in nanotechnology, innumerable nanomaterials have been synthesized and widely utilized in various fields. Among them, gold nanoparticles (Au NPs) possess distinct chemical, physical, and biological properties, and have been demonstrated a good application potential in many fields especially in biomedical analysis.21,22 Au NPs are stable, biocompatible, slightly toxic, and have a large molar extinction coefficient and broad energy bandwidth. Moreover, Au NPs are also a kind of attractive elemental tags with low background in biological sample and satisfactory signal amplification effect.23,24 Compared to those metal chelates tags containing only one ICP-MS detectable atom in each chelate, a 15 nm diameter Au NP contains ∼106 Au atoms, which could potentially amplify the signal by up to six orders of magnitude, and would significantly improve the sensitivity of the assay.25 Aptamers are single-stranded oligonucleotide or peptide sequences which can recognize and bind to specific targets through folding into unique secondary or tertiary structures. They are generated by an in vitro screening process amplification technique known as SELEX (systematic evolution of ligands by exponential enrichment) from a large random oligonucleotide or peptide sequence pool.26 Aptamers have the ability to bind with a variety of targets such as small organic molecules, proteins, metal ions, and even entire cells with high affinity and selectivity.27

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Compared to antibodies, these chemically synthesized molecules offer distinct advantages such as smaller sizes, lower cost, better stability over a wide range of temperature, solvents, and pH, and a lack of immunogenicity, all of which make aptamers an ideal alternative to protein antibodies.28 Accordingly, several aptamer-based colorimetric biosensors,29 electrochemical biosensor,30 and fluorescent biosensor31 have been reported for the detection of cancer cells. Liu and co-workers demonstrated an approach for fluorescent imaging and mass quantification of mIgM in live cells by confocal fluorescence microscope and ICP-MS via a single probe composed of an aptamer and a silver cluster.32 Unfortunately, the practical application potential of this method was not explored by real biological samples analysis. Inspired by this idea, we attempt to develop an aptamer dual-functional probe for rapid and specific counting and imaging of cancer cells by fluorescence microscope and ICP-MS. To demonstrate the feasibility, human breast cancer cell MCF-7 was chosen as a model cell line in this work. A 25-base oligonucleotide (sequence from 5´ to 3´: GCA GTT GAT CCT TTG GAT ACC CTG G) selected by Ferreira et al. has been widely confirmed as possessing a specific binding property for mucin 1 protein (MUC1), which is overexpressed on the surface of MCF-7 cell.33 In addition, rapid separation of target cells from complex biological matrix under mild conditions was achieved with the introduction of magnetic beads (MBs) in the probes, which not only greatly shortened the analysis time but also extended the application to real clinical samples.

Experiment Apparatus An Agilent 7500a ICP-MS (Agilent Technologies, Japan) equipped with a Babington nebulizer was used to determine Au by monitoring

197

Au. The operating parameters for ICP-MS detection

are summarized in Table S1. An H-7000FA transmission electron microscope (TEM, Hitachi, Japan) was used to obtain the TEM images with an acceleration voltage of 100 kV. Fluorescence spectra were obtained by an LS 55 spectrophotometer (PerkinElmer, U.S.A.) with the synchronous scan method. The fixed wavelength difference (∆λ) of synchronous scanning fluorescence spectroscopy was set at 22 nm, and slit widths of excitation and emission were both set at 10 nm.

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An AxioObserver Z1 inverted fluorescent microscope (Zeiss, Germany) were utilized for observation and fluorescence imaging. Materials and Reagents Streptavidin-modified magnetic beads (SA-MBs) (1 µm, 10 mg mL-1) were purchased from Beaverbio Co., Ltd. (Suzhou, China). Streptavidin-modified gold nanoparticles (SA-Au NPs) (15 nm, 0.4 mg mL-1) were obtained from Biosynthesis Biotech Co., Ltd. (Beijing, China). All oligonucleotides used in this study were HPLC-purified and synthesized by Sangon Biotechnology Co., Ltd. (Shanghai, China). Their sequences are listed as follows: FAM-modified aptamer (Apt-FAM): 5´-biotion-TTT TTG CAG TTG ATC CTT TGG ATA CCC TGG-FAM-3´ Complementary to Apt-FAM (c-Apt): 5´-biotion-TTT TTC CAG GGT ATC CA-3´ The buffers involved in this work are as follows: binding buffer solution contains 10 mM Tris-HCl, 1 mM EDTA, 1 M NaCl, and 0.05% Tween-20 (v/v) (pH 7.4); hybridization buffer solution contains 10 mM Tris-HCl, 1 mM EDTA, and 150 mM NaCl (pH 7.4); washing buffer solution is a mixture of 10 mM Tris-HCl, 1 mM EDTA, and 0.1% Tween-20 (v/v) (pH 7.4); phosphate-buffered saline (PBS) is a mixture of 137 mM NaCl, 2.7 mM KCl, 1.9 mM KH2PO4, and 8.1 mM Na2HPO4 (pH 7.4). All reagents were of analytical grade at least and used without further purification. Milli-Q ultrapure water (18.2 MΩ·cm, Millipore, France) was used throughout this work. Cell Culture The target MCF-7 cells (human breast carcinoma cell line), the control HepG2 cells (human hepatocellular carcinoma cell line) and SCC-7 cells (squamous cell carcinoma cell line) used in this study were purchased from American Type Culture Collection (ATCC, U.S.A.). MCF-7, HepG2 and SCC-7 cells were maintained in Dulbecco's modified Eagle's medium (DMEM, Gibco, U.S.A.) supplemented with 10% (v/v) fetal bovine serum (FBS, PAN, Germany), 100 U mL-1 penicillin, and 100 U mL-1 streptomycin. Cells were cultured at 37 °C in an atmosphere of humidified 5% CO2. Cells were detached by trypsinization with 0.25% trypsin and 0.02% EDTA in PBS buffer. Preparation of MB-Apt-FAM-Au NP conjugate

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The design of the dual-functional probe is shown in Figure 1. Firstly, Apt-FAM was immobilized on the surface of SA-MBs via a specific binding between streptavidin and biotin. Briefly, 100 µL of 10 mg mL-1 SA-MBs were washed three times with 200 µL of washing buffer solution, and then re-dispersed in 300 µL of binding buffer solution. Subsequently, 200 µL of 1 µM biotinylated Apt-FAM was added into the SA-MBs dispersion, and then the mixture was incubated at 37 °C for 60 min with gentle shaking to immobilize the Apt-FAM on the surface of SA-MBs. After washing with 200 µL of washing buffer solution for three times to remove excess aptamers, the Apt-FAM-modified MBs were re-suspended in 500 µL of hybridization buffer solution for the following use. In another vial, 100 µL of 5 µM biotinylated c-Apt was mixed with 25 µL of 0.4 mg mL-1 SA-Au NPs in 400 µL binding buffer solution, and then the mixture was incubated at 37 °C for 60 min with gentle shaking to immobilize the c-Apt on the surface of SA-Au NPs via biotin-streptavidin specific interaction. The as-prepared c-Apt-modified Au NPs were separated by centrifugation at 12000 g for 15 min and washed with 200 µL of washing buffer solution for three times, followed by re-dispersing in 500 µL of hybridization buffer solution. Accordingly, 500 µL of the as-prepared Apt-FAM-modified MBs were added to the c-Apt-modified Au NPs re-dispersing solution, and the mixture was incubated at 37 °C for 60 min to form a MB-Apt-FAM-Au NP conjugate through complementary base pairing. The product was separated and washed with 200 µL of washing buffer solution for five times under an external magnetic field. Finally, the product was re-dispersed in 200 µL of PBS for further use. Fluorescence imaging and ICP-MS determination of MCF-7 cells For fluorescence imaging, 7 µL of above prepared MB-Apt-FAM-Au NP conjugates were added to MCF-7 cell suspension in 200 µL of PBS. Then, the mixture was incubated at 37 °C for 180 min with gentle shaking. During the incubation, the target MCF-7 cells would bind with the aptamer modified on MBs specifically, and release Au NPs at same time. The MB-bound MCF-7 cells can be collected with a magnet, and the fluorescence image of MCF-7 cells was observed under blue light excitation. For ICP-MS determination, 100 µL of supernatant was collected after magnetic separation, and followed by introducing into ICP-MS for monitoring the signal of 197Au. As a result, the number of target cells can be obtained according to the signal intensity of indirectly. All of the experiments were performed in triplicate unless otherwise stated.

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197

Au


Blood sample analysis Fresh human whole blood of healthy people was obtained from the Zhongnan Hospital of Wuhan University (Wuhan, China) according to the rules of the local ethical committee. Spiking experiments were conducted to evaluate the application potential of the proposed method to real biological samples. A certain number of target MCF-7 cells in different levels were spiked into lysed human blood, and then subjected to the proposed method followed by ICP-MS determination.

Results and Discussion Design of the dual-functional probe The detailed principle of this experiment is illustrated in Figure 1. The dual-functional probe is composed of a MB moiety for separation and collection of target cells, an aptamer unit to recognize target cells via the overexpression of MUC1 on the surface of MCF-7 cells, a fluorescent dye moiety for fluorescence imaging as well as an Au NP tag. The ingenious design of this probe is the utilization of Au NPs as a quencher and reporter, which provides both an “off-on” fluorescence signal via fluorescence resonance energy transfer (FRET) and an elemental tag for ICP-MS determination. The excellent performance of Au NPs serve as elemental tags in ICP-MS-based quantitative bioanalysis has been widely confirmed, which may attribute to their sensitive response, low spectral interference in ICP-MS as well as low background in biological samples. Furthermore, Au NPs can also act as fluorescent quenchers in this probe to form an “off-on” system for fluorescence imaging, since Au NPs have a wide absorption band and strong fluorescence quenching ability. In the absence of the target cells, the Apt-FAM-modified MBs can bind with the c-Apt-modified Au NPs to form stable MB-Apt-FAM-Au NP conjugates via complementary base pairing. In this case, the fluorescence of FAM is quenched due to FRET from the FAM to the Au NPs quencher. Whereas, when the target cells are added and come into contact with the probe, the target cells would compete with c-Apt-modified Au NPs for binding to the Apt-FAM immobilized on the MBs. It turns out that the c-Apt-modified Au NPs are released from the conjugates due to a stronger binding force between the target cells and the aptamers. After magnetic separation, the

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released Au NPs can be easily separated from the MB-bound target cells and excess unreacted MB-Apt-FAM-Au NP conjugates. The amount of released Au NPs in the supernatant is in direct proportion to the amount of target cells, and can be accurately determined using Au signal in ICP-MS. Meanwhile, since the Au NPs quencher is released, the fluorescence image of target cells can be observed with the fluorescence recovery of FAM. In this way, it provides a dual-modal sensing platform for rapid, sensitive and specific counting and imaging of MCF-7 cells. Characterization of the as-prepared dual-functional probe TEM was used to characterize the morphologies of MB-Apt-FAM-Au NP conjugates. As the TEM images shown in Figure 2a, MB-Apt-FAM-Au NP conjugates had a uniform size around 1 µm with good dispersity. Furthermore, no Au NPs were observed on the surface of the bare MBs (Figure 2b) after they reacted with c-Apt-modified Au NPs; whereas, several Au NPs (judging from the size difference, the diameter of Au NPs was about 15 nm) were clearly observed on the surface of the Apt-FAM-modified MBs (Figure 2c). In addition, the precise amount of Au NPs anchored on MBs was further determined with ICP-MS to confirm the formation of MB-Apt-FAM-Au NP conjugates by means of digestion with HNO3. The results of ICP-MS analysis indicated that 14.9 ± 0.5 ng of Au NPs was anchored on 1 µg of Apt-FAM-modified MBs. However, as for the original MBs without Apt-FAM modification, approximately 30-fold less amount of Au NPs was detected, indicating that few c-Apt-modified Au NPs could attach to the surface of unmodified MBs through nonspecific adsorption. All the above experimental results clearly indicate the successful formation of MB-Apt-FAM-Au NP conjugates via complementary base pairing. Optimization of experimental conditions To achieve the optimal analytical performance, several experimental factors involved in this procedure were examined and optimized, including the density of Apt-FAM immobilized on MBs, amount of the dual-functional probe, and incubation time for the dual-functional probe and target MCF-7 cells. In the proposed method, target MCF-7 cells were recognized by the aptamer immobilized on MBs. Therefore, the density of Apt-FAM immobilized on the surface of MBs directly affects recognition capability of the dual-functional probe to target cells which would reflect in signal

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response of Au, and may further influence the sensitivity of the method. When the density of Apt-FAM immobilized on MBs was too low, the amount of Au NPs anchored would be insufficient, which could lead to low efficiency of Au NPs release as well as low signal response of Au; whereas, when the density of Apt-FAM immobilized on MBs was too high, more than one hybrid double-strands could be formed between each Au NP and MB, which may increase the resistance for Au NPs release and lower signal response of Au accordingly. The density of Apt-FAM immobilized on MBs was optimized by adjusting the ratio of MBs to Apt-FAM. The effect of the ratio of MBs to Apt-FAM on Au signal was investigated in the range of 1/10 -1/500 g nmol-1. As the results shown in Figure S1, the signal intensity of Au increased along with the increase in density of Apt-FAM when the ratio of MBs to Apt-FAM was lower than 1/200 g nmol-1, and the signal intensity of Au started to decrease thereafter when the ratio of MBs to Apt-FAM was higher than 1/200 g nmol-1. Therefore, a ratio of 1/200 g nmol-1 (MBs/Apt-FAM) was employed in the subsequent experiments. In addition, the amount of MB-Apt-FAM-Au NP conjugates was one of the key factors in counting and imaging of target cells. It was essential to add excessive of MB-Apt-FAM-Au NP conjugates to ensure a constant proportion of released Au NPs to the number of target cells. However, too many MB-Apt-FAM-Au NP conjugates involved may cause a high blank signal and low reproducibility, which may attribute to incomplete magnetic separation. Hence, the effect of the amount of MB-Apt-FAM-Au NP conjugates on Au signal was studied from 2.0 to 40 µg and the results are shown in Figure S2. As can be seen, the signal intensity of Au increased rapidly when the amount of MB-Apt-FAM-Au NP conjugates increased from 2.0 to 30 µg, and then remained constant with further increasing the amount of MB-Apt-FAM-Au NP conjugates from 30 to 40 µg. Therefore, 35 µg of MB-Apt-FAM-Au NP conjugates was employed in this method. Furthermore, the effect of incubation time for the MB-Apt-FAM-Au NP conjugates and target MCF-7 cells on Au signal in the range of 20-180 min was also investigated to ensure a complete reaction. As the results presented in Figure S3, the signal intensity of Au increased along with the increase in incubation time and kept constant after 120 min, indicating that the reaction could be completed in 120 min. Therefore, an incubation time of 150 min was chosen in this work. Specificity and cross-reactivity

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Since the detection of target cells may be affected by the co-existing numerous of normal and/or benign cells in complex biological samples, the selectivity and specificity of the proposed method need to be verified. For this purpose, the human hepatocellular carcinoma cell line HepG2 and squamous cell carcinoma cell line SCC-7 were chosen as control. The test was subjected to the same analysis procedures as for the MCF-7 cells. As the results shown in Figure 3, the signal intensity of Au for HepG2 and SCC-7 cells was much lower than that for MCF-7 cells at the same cell number of 2×103 cells. In addition, the cross-reactivity of the method was also verified by mixing 2×103 MCF-7 cells with 2×104 HepG2 or SCC-7 cells, and almost identical signal intensity of Au was observed for MCF-7 cells and other two mixed groups. All the above results demonstrate an excellent specificity and selectivity of the proposed method. Moreover, the selectivity and specificity of the method was further testified by fluorescence imaging. Fluorescence response of the dual-functional probe to MCF-7 cells To verify that the “off-on” fluorescence of the dual-functional probe could be triggered by target MCF-7 cells, the fluorescence response of the as-prepared dual-functional probe to MCF-7 cells was then investigated, and the results are displayed in Figure 4. As expected, the fluorescence intensity of the dual-functional probe increased along with increasing number of MCF-7 cells from 200 to 10,000. In the absent of MCF-7 cells, only a very weak fluorescence intensity of the dual-functional probe was observed with same procedures. The above results demonstrate the successful establishment of FRET-based “off-on” fluorescence and high selectivity of the dual-functional probe, and the developed system can be further applied to cell fluorescence imaging. Fluorescence imaging Despite providing quantitative results of cell numbers, ICP-MS cannot present visualized images due to its inherent limitation. Living cell imaging can provide visualized images, locations, as well as some other detail information, which can also be a supplement to ICP-MS results for further excluding false positive/negative results. In this work, with the help of MB-Apt-FAM-Au NP conjugates, fluorescence images of target cells were achieved by inverted fluorescent microscope. Fluorescence images of target and control cells are presented in Figure 5a and Figure 5b, respectively. As can be seen, the noticeable green fluorescence signal was generated from the

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surface of target cells, and the fluorescence image was in good agreement with the corresponding bright field image. By contrast, the control group HepG2 cells displayed only negligible fluorescence as expected after incubation with the MB-Apt-FAM-Au NP conjugates, indicating that the FRET-based “off-on” fluorescence of the dual-functional probe was hardly activated by non-target cells. These results demonstrated the specificity of MB-Apt-FAM-Au NP conjugates for target MCF-7 cells, and were in agreement with the results obtained by ICP-MS determination mentioned above. Furthermore, all these results reveal that the barrier between living cell imaging and destructive detector ICP-MS measurement can be bridged with the aptamer-based dual-functional probe, owing to the excellent fluorescence quenching properties and abundant ICP-MS detectable Au atoms composition of Au NPs. Analytical performance The analytical performance of the developed method of MB-Apt-FAM-Au NP conjugates based assay with ICP-MS measurement for cell counting was evaluated under the optimized conditions. Figure 6 is the dependence of signal intensity of 197Au on MCF-7 cell number. As can be seen, the 197

Au signal response in ICP-MS for varying amounts of MCF-7 cells presented a trend similar to

the results of fluorescence assay. Moreover, a good linear relationship was obtained within the cell number ranging from 2×102 to 1.2×104 cells, and the linear correlation equation was IAu=495.7[cell number]+322810 with a correlation coefficient (R2) of 0.9944. Based on the signal intensity of 197Au, the LOD (3σ) of the proposed method for the MCF-7 cell was calculated to be 81 cells, and the relative standard deviation (RSD) was 5.6% for seven replicate determinations of 8×102 MCF-7 cells. A comparison of the analytical performance of this work with those obtained by several other reported approaches for the detection of cancer cells is shown in Table 1. As can be seen, the LOD of the established method is comparable with most of the reported ICP-MS based methods, and much lower than that obtained by other approaches. Besides, this work takes the advantages of each detection technology by integrating fluorescence imaging detection modalities and ICP-MS measurement with single probe, which makes the method simpler and more reliable. It must be stressed that the analysis time is greatly shortened in the developed method compared to traditional cell analysis techniques due to the reduction of incubation times and avoiding of washing or elution operation involved. As well known, analytical time is usually

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one of the restraining factors against the development of rapid and early diagnosis. The success of this study provides a rapid, robust and reliable approach for the early clinical diagnosis of breast cancer. Blood sample analysis In order to evaluate the applicability of the developed method to real biological samples, a spiking experiment was carried out by adding different numbers of target MCF-7 cells into the lysed human blood, and the analytical results are listed in Table 2. As can be seen, this method has a robust resistibility to the complex biological matrix with a satisfactory recovery ranging from 98 to 110%. These results clearly indicate that the proposed method can be applied to detect trace amount of target MCF-7 cells in human blood with a good accuracy.

Conclusion In this paper, we demonstrate a simple, rapid, sensitive, and specific method using an aptamer-based dual-functional probe (MB-Apt-FAM-Au NP conjugates) for ICP-MS counting and fluorescence imaging of cancer cells. Here, Au NPs can not only serve as elemental tags for ICP-MS-based quantitative bioanalysis but also serve as efficient quenchers to construct FRET-based “off-on” fluorescence probe for living cell imaging. This simple and low-cost design of the dual-functional probe can take the advantages of each detection technology and offer more comprehensive and valuable information on cancer cells. It is greatly expected to be a powerful platform for the rapid and accurate clinical diagnosis of cancer at early stage. Even more importantly, this approach is not limited to the breast cancer cells studied here, it can also extend to detect various types of cancer cells, ssDNA, even nucleases if an appropriate primer DNA (aptamer) is selected.

Associated Content Supporting Information Additional information on effect of the ratio of MBs to aptamer DNA (Figure S1), effect of the amount of the probe (Figure S2), effect of incubation time for the probe and target MCF-7 cells

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(Figure S3), and operating parameters of ICP-MS (Table S1) as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

Author Information Corresponding Author *Tel.: 0086-27-68752162. Fax: 0086-27-68754067. E-mail: [email protected]. Notes The authors declare no competing financial interest.

Acknowledgments This work is financially supported by the National Nature Science Foundation of China (Nos. 21575107, 21575108, 21375097, 21175102), the National Basic Research Program of China (973 Program, 2013CB933900), the Science Fund for Creative Research Groups of NSFC (No. 20921062), the MOE of China, and the Large-scale Instrument and Equipment Sharing Foundation of Wuhan University (LF20170799).

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References (1) Torre, L. A.; Bray, F.; Siegel, R. L.; Ferlay, J.; Lortet-Tieulent, J.; Jemal, A. CA Cancer J. Clin. 2015, 65, 87-108. (2) Chen, W.; Zheng, R.; Baade, P. D.; Zhang, S.; Zeng, H.; Bray, F.; Jemal, A.; Yu, X. Q.; He, J. CA Cancer J. Clin. 2016, 66, 115-132. (3) Joosse, S. A.; Gorges, T. M.; Pantel, K. EMBO Mol. Med. 2015, 7, 1-11. (4) Ho, K. F.; Gouw, N. E.; Gao, Z. Q. Trac-Trends in Anal. Chem. 2015, 64, 173-182. (5) Dunphy, C. H.; Orton, S. O.; Mantell, J. Am. J. of Clin. Pathol. 2004, 122, 865-874. (6) Guo, Y.; Shu, Y.; Li, A.; Li, B.; Pi, J.; Cai, J.; Cai, H.-h.; Gao, Q. J. Mater. Chem. B 2017, 5, 5532-5538. (7) Koyanagi, K.; O'Day, S. J.; Boasberg, P.; Atkins, M. B.; Wang, H. J.; Gonzalez, R.; Lewis, K.; Thompson, J. A.; Anderson, C. M.; Lutzky, J.; Amatruda, T. T.; Hersh, E.; Richards, J.; Weber, J. S.; Hoon, D. S. B. Clin. Cancer Res. 2010, 16, 2402-2408. (8) Yang, W.; Xi, Z.; Zeng, X.; Fang, L.; Jiang, W.; Wu, Y.; Xu, L.; Fu, F. J. Anal. At. Spectrom. 2016, 31, 679-685. (9) Tang, N.; Li, Z.; Yang, L.; Wang, Q. Anal. Chem. 2016, 88, 9890-9896. (10) Liu, R.; Liu, X.; Tang, Y.; Wu, L.; Hou, X.; Lv, Y. Anal. Chem. 2011, 83, 2330-2336. (11) Ko, J.; Lim, H. B. Anal. Chem. 2014, 86, 4140-4144. (12) Luo, Y. C.; Yan, X. W.; Huang, Y. S.; Wen, R. B.; Li, Z. X.; Yang, L. M.; Yang, C. J.; Wang, Q. Q. Anal. Chem. 2013, 85, 9428-9432. (13) Iglesias Gonzalez, T.; Espina, M.; Sierra, L. M.; Bettmer, J.; Blanco-Gonzalez, E.; Montes-Bayon, M.; Sanz-Medel, A. Anal. Chem. 2014, 86, 11028-11032. (14) Zhang, S.; Liu, R.; Xing, Z.; Zhang, S.; Zhang, X. Chem. Commun. 2016, 52, 14310-14313. (15) Bendall, S. C.; Simonds, E. F.; Qiu, P.; Amir, E.-a. D.; Krutzik, P. O.; Finck, R.; Bruggner, R. V.; Melamed, R.; Trejo, A.; Ornatsky, O. I.; Balderas, R. S.; Plevritis, S. K.; Sachs, K.; Pe'er, D.; Tanner, S. D.; Nolan, G. P. Science 2011, 332, 687-696. (16) Giesen, C.; Waentig, L.; Mairinger, T.; Drescher, D.; Kneipp, J.; Roos, P. H.; Panne, U.; Jakubowski, N. J. Anal. At. Spectrom. 2011, 26, 2160-2165. (17) Zhang, C.; Wu, F. B.; Zhang, Y. Y.; Wang, X.; Zhang, X. R. J. Anal. At. Spectrom. 2001, 16, 1393-1396. (18) Yang, B.; Chen, B.; He, M.; Hu, B. Anal. Chem. 2017, 89, 1879-1886. (19) Yang, B.; Zhang, Y.; Chen, B.; He, M.; Yin, X.; Wang, H.; Li, X.; Hu, B. Biosens. Bioelectron. 2017, 96, 77-83. (20) Zhang, Z. B.; Luo, Q.; Yan, X. W.; Li, Z. X.; Luo, Y. C.; Yang, L. M.; Zhang, B.; Chen, H. F.; Wang, Q. Q. Anal. Chem. 2012, 84, 8946-8951. (21) Yeh, Y. C.; Creran, B.; Rotello, V. M. Nanoscale 2012, 4, 1871-1880. (22) Peng, H. Y.; Tang, H.; Jiang, J. H. Sci. China-Chem. 2016, 59, 783-793. (23) Liu, R.; Wu, P.; Yang, L.; Hou, X.; Lv, Y. Mass Spectrom. Rev. 2014, 33, 373-393. (24) Liu, Z.; Li, X.; Xiao, G.; Chen, B.; He, M.; Hu, B. TrAC Trends Anal. Chem. 2017, 93, 78-101. (25) Li, F.; Zhao, Q.; Wang, C.; Lu, X.; Li, X.-F.; Le, X. C. Anal. Chem. 2010, 82, 3399-3403. (26) Meng, H. M.; Liu, H.; Kuai, H. L.; Peng, R. Z.; Mo, L. T.; Zhang, X. B. Chem. Soc. Rev. 2016, 45, 2583-2602. 15



(27) Tan, W.; Donovan, M. J.; Jiang, J. Chem. Rev. 2013, 113, 2842-2862. (28) Zhang, H. M.; Zhou, L. J.; Zhu, Z.; Yang, C. Y. Chem.-Eur. J. 2016, 22, 9886-9900. (29) Zhang, X.; Xiao, K.; Cheng, L.; Chen, H.; Liu, B.; Zhang, S.; Kong, J. Anal. Chem. 2014, 86, 5567-5572. (30) Hua, X.; Zhou, Z.; Yuan, L.; Liu, S. Anal. Chim. Acta 2013, 788, 135-140. (31) Xie, Q.; Tan, Y.; Guo, Q.; Wang, K.; Yuan, B.; Wan, J.; Zhao, X. Anal. Methods 2014, 6, 6809-6814. (32) Liu, R.; Zhai, J.; Liu, L.; Wang, Y. L.; Wei, Y. T.; Jiang, X. L.; Gao, L.; Zhu, H. R.; Zhao, Y. L.; Chai, Z. F.; Gao, X. Y. Chem. Commun. 2014, 50, 3560-3563. (33) Ferreira, C. S.; Matthews, C. S.; Missailidis, S. Tumour Biol. 2006, 27, 289-301. (34) Pan, Y.; Guo, M.; Nie, Z.; Huang, Y.; Pan, C.; Zeng, K.; Zhang, Y.; Yao, S. Biosens. Bioelectron. 2010, 25, 1609-1614. (35) Zhang, L. N.; Deng, H. H.; Lin, F. L.; Xu, X. W.; Weng, S. H.; Liu, A. L.; Lin, X. H.; Xia, X. H.; Chen, W. Anal. Chem. 2014, 86, 2711-2718. (36) Han, E.; Ding, L.; Ju, H. Anal. Chem. 2011, 83, 7006-7012. (37) Xie, Q.; Tan, Y. Y.; Guo, Q. P.; Wang, K. M.; Yuan, B. Y.; Wan, J.; Zhao, X. Y. Anal. Methods 2014, 6, 6809-6814. (38) Chen, X. J.; Pan, Y. G.; Liu, H. Q.; Bai, X. J.; Wang, N.; Zhang, B. L. Biosens. Bioelectron. 2016, 79, 353-358. (39) Lv, S.; Guan, Y.; Wang, D.; Du, Y. Anal. Chim. Acta 2013, 772, 26-32. (40) Arya, S. K.; Wang, K. Y.; Wong, C. C.; Rahman, A. R. Biosens. Bioelectron. 2013, 41, 446-451. (41) Yang, B.; Zhang, Y.; Chen, B.; He, M.; Hu, B. Talanta 2017, 167, 499-505.

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Figure captions Figure 1. Schematic illustration of the experimental principle for counting and imaging of cancer cells based on the dual-functional probes. Figure 2. TEM images of the as-prepared dual-functional probes (a), and detail with enlarged scale of bare MBs (b) and Apt-FAM-modified MBs (c) after they reacted with c-Apt-modified Au NPs, respectively. Figure 3. Specificity and cross-reaction tests for MCF-7 cells. Column A, B, C present the relative intensity of 197Au in ICP-MS for detecting 2×103 of SCC-7 cells, 2×103 of HepG2 cells, and 2×103 of MCF-7 cells, respectively; Columns E and F represent the relative intensity of 197Au in ICP-MS for detecting 2×103 of MCF-7 cells mixed with 2×104 of SCC-7 cells and 2×104 of HepG2 cells, respectively. Figure 4. Fluorescence spectra of the dual-functional probes in the presence of different numbers of target MCF-7 cells. Figure 5. Fluorescence (a1-b1), bright field (a2-b2), and merged images (a3-b3) of MCF-7 cells and HepG2 cells incubated with the dual-functional probes, respectively. Figure 6. Calibration curve of MCF-7 cells obtained using signal intensity of 197Au in ICP-MS.

17



Figure 1

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Figure 2

19



1.2 MCF-7

MCF-7+SCC-7 MCF-7+HepG2

197

Au

1.0

Relative intensity of

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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0.8

0.6

0.4 SCC-7

HepG2

A

B

0.2

0.0 C

D

Figure 3

20


E

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10×103 8.0×103 6.0×103 4.0×103 2.0×103 1.0×103 0.5×103 0.2×103 0

160

Fluorescence Intensity (a.u.)

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


140 120 100 80 60 40 20 0 500

550

600

Wavelength (nm)

Figure 4

21


650


Figure 5

22


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2.0x106

6

7x10

1.5x106

Signal intensity of 197Au (counts)

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


6x106

1.0x106

5.0x105

5x106

0.0 0

400

800

1200

1600

2000

6

4x10

3x106

y=495.7x+322810 R2=0.9944

2x106 1x106 0 0

2000

4000

6000

8000

Numbers of MCF-7 cell

Figure 6

23


10000

12000


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Table 1 Comparison of different methods for the analysis of tumor cells

Detection method QCM Colorimetric Fluorescence Fluorescence Photoluminescence Atomic force microscope Electrochemical detection Electrochemical detection ICP-MS ICP-MS ICP-MS ICP-MS ICP-MS ICP-MS

Target cell CCRF-CEM MCF-7 BGC-823 SMMC-7721 MCF-7 HepG2 MCF-7 MCF-7 HT-29 SMMC-7721 HepG2 HepG2 HepG2 MCF-7

Linear range (cell mL-1) 4

5

1×10 -1.5×10 2.5×102-8×103 5×102-1×107 2×102-2×104 5×102-1×105 1×103-1×105 1×102-1×106 1×105-1×108 4×102-4×105 2×102-8×103 2×102-3×104 8×102-4×104 4×102-3×104 2×102-1.2×104

24


LOD (cell mL-1) 3

8×10 125 210 200 201 300 100 1×105 44 100 61 282 100 81

Ref. 34 35 36 37 30 38 39 40 20 8 18 41 19 This work

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Table 2 Recovery test for whole blood sample Added (×102 cells) 0 2.0 5.0 10 20 40 a

Found (×102 cells)

Recovery (%)

a

N.D. 2.2±0.2 5.4±0.2 10.9±1.0 19.5±1.7 41.9±1.7

Not detected.

25


110 108 109 98 105


For TOC only

26


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An aptamer-based dual-functional probe for rapid and specific

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