Quantum Dots Labeling Strategy for “Counting and Visualization” of

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Quantum Dots Labeling Strategy for “Counting and Visualization” of HepG2 Cells Bin Yang, Beibei Chen, Man He, and Bin Hu* Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), Department of Chemistry, Wuhan University, Wuhan 430072, China S Supporting Information *

ABSTRACT: We report a sensitive, selective, simple, and reliable magnetic immunoassay protocol for detection and imaging of HepG2 cells. After being captured by Cs-doped multicore magnetic nanoparticles (MMNPs), HepG2 cells were labeled by CdSe/ZnS quantum dots (QDs), which could be visualized by fluorescence imaging using the photoluminescence property of QDs, and subsequently, they can be counted by inductively coupled plasma mass spectrometry (ICP-MS) with Cd/Cs as elemental tag. Because of the superior photoluminescence properties and the large quantities of detectable Cd atoms contained in the QDs core, QDs play a dual function role in this assay, making the method easier and more comprehensive than other similar approaches. Under the optimal conditions, the limit of detection of 61 HepG2 cells and the relative standard deviation of 5.4% (800 HepG2 cells, n = 7) were obtained. The linear range was 200−30 000 cells, and the recoveries in human whole blood were in the range of 86−104%. The proposed method enables us not only to count but also to see the cancer cells with the same labeling process, opening a promising avenue for research and clinical application.

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detection of cells with appropriate elemental tags.11,13,14 The accuracy of using ICP-MS to count the cells was largely dependent on the element-labeling efficiency, and the reported elemental tags for ICP-MS bioanalysis include naturally occurring elements,15−18 metal chelates,19−22 metal-containing polymers,13,23−25 and metal-containing nanoparticles.26−34 In our previous work,11 an ICP-MS-based magnetic immunoassay protocol was proposed for the detection of Jurkat T cells using magnetic nanoparticles (MNPs)-anti-CD3 as capture probes, Au NPs-anti-CD2 as reporter probes. The method was demonstrated to be sensitive, selective, and capable of counting tumor cells in the real blood sample with the LOD of 86 cells. However, as a destructive detector, ICP-MS is challenging to use for presenting visualized images which would contain some details such as the location of labeling tags in cells. It can be expected that a combination of ICP-MS-based quantitative bioanalysis with fluorescence imaging will provide more comprehensive and valuable information. Several strategies for meeting these requirements have been reported. Zhang et al.35 reported a trifunctional probe for visualizing and counting cancer cells. The probe comprises a guiding cyclic Arg−Gly− Asp peptide to target integrin αVβ3 overexpressed on the surface of cancer cells, and loading Eu for subsequent

ith increasing incidence and mortality, cancer is becoming the leading cause of death worldwide. According to the worldwide cancer statistics (GLOBOCAN 2012) released by the International Agency for Research on Cancer (IARC), there were estimated to be 14.1 million new cancer cases and 8.2 million cancer deaths occurring in 2012 worldwide.1 In China, liver cancer has become the fourth common incident cancer, with mortality below only lung and stomach cancer.2 The metastasis of the primary cancer tumors is regarded as the major reason to cause cancer death.3 It is well known that an early and accurate diagnosis of cancer is essential for effective treatment.4 Therefore, novel approaches for highly sensitive, rapid, reliable, and accurate detection of rare cancer cells in real biological samples are in great demand.5 To date, there are a variety of analytical methods for the detection of cancer cells including flow cytometry,6 reversetranscriptase polymerase chain reaction (RT-PCR),7 microarray,8 electrochemical sensor,9 and fluorescence spectroscopy.10 However, these methods more or less suffered from falsepositive/negative results, complicated operations, lack of efficiency, and so on.11 In recent years, inductively coupled plasma mass spectrometry (ICP-MS) as an element-specific detector has become a revolutionary technique for quantitative bioanalysis by introducing inorganic element-labeling strategies.12 The advantages of ICP-MS include low limits of detection (LOD), low matrix effects, wide dynamic ranges, rapid analysis capabilities, high sensitivity, and high spectral resolution for elements and isotopes, which is commendable for © 2017 American Chemical Society

Received: November 3, 2016 Accepted: January 12, 2017 Published: January 12, 2017 1879

DOI: 10.1021/acs.analchem.6b04314 Anal. Chem. 2017, 89, 1879−1886

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Analytical Chemistry quantification using ICP-MS, and a fluorescent dye for fluorescence imaging. More recently, Zhai et al.5 demonstrated visualization of integrin αIIbβ3 by confocal fluorescence microscope through an Au cluster probe and subsequent quantification of the number of integrin αIIbβ3 in a human erythroleukemia cell line on single cell level by laser ablation ICP-MS. However, the Au cluster contains a smaller amount of ICP-MS-detectable atoms than common Au NPs. Therefore, simpler and more straightforward approaches are expected for cancer cells’ quantification as well as visualization. The reported metal-containing nanoparticle-based elemental tags include Au NPs,26−28 Ag NPs,29,30 Pt NPs,31 PbS NPs,32 HgS NPs,33 TiO2 NPs,34 and so on. Among these NPs, quantum dots (QDs) have good biocompatibility, low cytotoxicity, high sensitivity in ICP-MS, and low background in biological samples, which endowed QDs with excellent performance as elemental tags for ICP-MS-based immunoassays. On the basis of our previous study36 and another reported work,37 QDs were employed as elemental tags for determination of human IgG in human serum and progesterone in cow milk, respectively, which has demonstrated the feasibility of the QDs-tagged bioanalysis by ICP-MS. In addition, QDs are widely used in fluorescence immunoassays38 and fluorescence imaging39 due to the unique optical properties including high quantum yields, large extinction coefficients, pronounced photostability, and more importantly, broad absorption spectra coupled to narrow size-tunable photoluminescent emission spectra. Magnetic nanoparticles (MNPs) offer a unique superparamagnetic property that allows rapid separation of target analytes from a complex biological matrix under mild conditions.40 However, the magnetic separation inherently suffered from particle loss during several unavoidable washing steps, which inevitably caused signal fluctuation, measurement errors, and poor reproducibility.41 Lim’s group reported several works41,42 to settle this problem by introducing a ratiometric measurement method with an internal standard which took advantage of the multiplex capability of ICP-MS, and significantly improved the calibration linearity and reproducibility. The aim of this work was to develop a novel method involving QDs tags for both ratiometric measurement for cell counting by ICP-MS and visualization for cells via fluorescence imaging. Multicore MNPs doped with Cs (Cs-doped MMNPs) were synthesized and conjugated with anti-EpCAM. The asprepared MMNPs-anti-EpCAM probes were used to capture HepG2 cells in lysed human blood specifically, and then the HepG2 cells were labeled with commercially available CdSe/ ZnS QDs which were not only used as fluorophore to visualize HepG2 cells but also used as elemental tags for ICP-MS detection due to their photoluminescence property and containing large quantities of ICP-MS-detectable Cd atoms in each QD. In addition, the doped Cs was used as an internal standard for the ratiometric measurement, and the measurement error and signal fluctuation caused by particle loss were compensated and suppressed by using the signal ratio of 114 Cd/133Cs instead of 114Cd for quantification.

scanning microscope (LCSM, Zeiss LSM 710, Germany) coupled with a 561 nm DPSS laser was used for cell observation and imaging. The self-prepared MMNPs were characterized by a Fourier transform infrared spectrometer (FT-IR, Thermo, Nicolet iS10, U.S.A.), a transmission electron microscope (TEM, JEOL, JEM-2010 HT, Tokyo, Japan) and dynamic light scatting (DLS, Nano ZS ZEN3600 Malvern Instruments, UK). Materials and Reagents. Monoclonal mouse anti-EpCAM antibody, N-(3-(Dimethylamino)propyl)-N′-ethylcarbodiimide hydrochloride (EDC·HCl), N-hydroxysulfosuccinimide (SulfoNHS), cesium chloride (CsCl), and docusate sodium salt were purchased from Sigma-Aldrich (St. Louis, MO, U.S.A.). Monoclonal mouse anti-ASGPR antibody purchased from BD Pharmingen (BD Biosciences, CA) was conjugated with CdSe/ ZnS QDs (QDs 605) by Wuhan Jiayuan Quantum Dots Co. Ltd. (Wuhan, China). The number of Cd atoms in each QD and the anti-ASGPR anchored on each QD was calculated as 2394 Cd atoms and 6.7 anti-ASGPR molecules. The details on the calculations were illustrated in Supporting Information. Tetraethoxysilane (TEOS) and 3-aminopropyltriethoxysilane (APTES) were obtained from Organic Silicon Material Company of Wuhan University (Wuhan, China). Ferric chloride hexahydrate (FeCl3·6H2O), ferrous chloride tetrahydrate (FeCl2·4H2O), ammonium hydroxide (NH3·H2O), nheptane, acetone, and ethanol were obtained from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China). The red cell lysis buffer was obtained from Tiangen Biotech Co. Ltd. (Beijing, China). The phosphate buffer solution (PBS, pH 7.4) consists of 137 mmol L−1 NaCl, 2.7 mmol L−1 KCl, 1.9 mmol L−1 KH2PO4, and 8.1 mmol L−1 Na2HPO4. All reagents used were at least analytical reagent grade. High-purity deionized water obtained from a Milli-Q system (18.2 MΩ·cm, Millipore, Molsheim, France) was used throughout this work. Sub-boiled nitric acid (HNO3) was used in all experiments. Cell Culture. HepG2, HeLa, MCF-7, and Cal-27 cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% (v/v) fetal bovine serum (FBS), 100 U mL−1 penicillin, and 100 U mL−1 streptomycin in 5% CO2 at 37 °C, respectively. The culture medium was replaced once every 2 days. Cells were detached by trypsinization with 0.25% trypsin and 0.02% EDTA in PBS buffer when reaching the cell density of approximately 106 cell mL−1. The cell density was determined using a hemocytometer before the following experiments. Synthesis of Cs-Doped MMNPs and MMNPs-antiEpCAM Conjugate. The MMNPs were synthesized by two steps according to the method reported previously with a slight modification.41,43 A schematic diagram of the synthesis is shown in Figure S1. First, two types of cores were synthesized separately. Cs-doped silica cores were used for the internal standard, and Fe3O4 cores were used for magnetic separation. For the synthesis of Cs-doped silica cores, 5 mg of CsCl was dispersed in a mixture of 1 mL of high purity deionized water and 7 mL of ethanol and subjected to ultrasonication for 5 min. Then, 35 μL of APTES was added and stirred for 24 h. Fe3O4 cores were synthesized by a conventional coprecipitation method. Briefly, 1.35 g of FeCl3·6H2O and 0.5 g of FeCl2· 4H2O were dissolved in 25 mL of high-purity deionized water and heated to 85 °C under argon atmosphere and stirring. Then 12.5 mL of NH3·H2O was added with vigorous stirring for 0.5 h. The obtained Fe3O4 MNPs were washed three times with ethanol. Then, the MMNPs were synthesized by the reverse microemulsion method. Docusate sodium salt (2.2 g)

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EXPERIMENT Apparatus. An Agilent 7500a ICP-MS system (Agilent Technologies, Tokyo, Japan) with a Babington nebulizer was used for the measurement of 114Cd and 133Cs. The operating parameters for ICP-MS are listed in Table S1. A laser confocal 1880

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Figure 1. Scheme for the detection of HepG2 cells by using QDs-anti-ASGPR as detection probes and MMNPs-anti-EpCAM as capture probes.

and 860 μL of high purity deionized water was added to 48 mL of n-heptane and stirred for 10 min. Then 300 μL of Fe3O4 core solution was added and stirred for 30 min, followed by the addition of 300 μL of silica core solution slowly. After stirring for 10 min, 430 μL of TEOS and 224 μL of NH3·H2O were added. Then the materials were stirred at room temperature for 24 h. Subsequently, 100 μL of TEOS was added and stirred for 20 min, followed by the addition of 100 μL of APTES. After stirring at room temperature for 24 h for the functionalization of the MMNPs with amino groups, the obtained MMNPs-NH2 were washed with acetone, ethanol, and high-purity deionized water in sequence for several times. The obtained MMNPs were then stored in high-purity deionized water. For coupling MMNPs with the monoclonal antibody antiEpCAM, 5 mg of MMNPs-NH2 and 20 μg of anti-EpCAM were dispersed in 1 mL of HEPES (10 mmol L−1, pH 7.2), followed by the addition of 50 mmol L−1 EDC·HCl and 50 mmol L−1 Sulfo-NHS. After incubation for 2 h with shaking at room temperature, the prepared MMNPs-anti-EpCAM conjugates were separated by a permanent magnet and washed with 1 mL of PBS (10 mmol L−1, pH 7.4) for three times in order to remove any unreacted antibodies. Lastly, the MMNPsanti-EpCAM was blocked with 1% BSA in PBS (10 mmol L−1, pH 7.4) for 1 h and then stored at 4 °C for further use. Immunoassay Procedure. The magnetic immunoassay procedure for HepG2 cells is illustrated in Figure 1. First, 2 μL of MMNPs-anti-EpCAM was dispersed in 200 μL of PBS buffer (10 mmol L−1, pH 7.4) containing 1% skim milk and incubated with HepG2 cells for 60 min with gentle shaking. Due to the antigen−antibody specific binding, the HepG2 cells would be selectively captured by MMNPs-anti-EpCAM, and they were separated from numerous normal cells and the matrix by an external magnetic force. After washing three times with PBS buffer, 200 μL of 1% skim milk in PBS buffer was added and incubated for 30 min with gentle shaking to block the nonspecific binding site. Then, the MMNPs-HepG2 complexes were washed three times with PBS buffer. After that, 0.25 μL of

QDs-anti-ASGPR was added to the complexes and diluted to 200 μL with PBS buffer containing 1% skim milk, followed by incubation for 40 min with gentle shaking. QDs-anti-ASGPR conjugated with HepG2 cells by immunoreaction with the ASGPR expressed on cell surface, which produced a sandwichtype immunocomplex of MMNPs-HepG2-QDs. The sandwichtype immunocomplexes were separated by external magnetic force and washed three times with PBS buffer. Confocal Fluorescence Imaging and ICP-MS Determination. For confocal fluorescence imaging, the sandwich-type immunocomplexes were fixed to the glass-bottom cell-culture dishes with 4% paraformaldehyde for 20 min, and the confocal fluorescence imaging was observed under 561 nm laser excitation. For quantification, the sandwich-type immunocomplexes were diluted to 100 μL with 1 mol L−1 sub-boiled HNO3 and sonicated for 10 min, and the dispersed solution of complexes was directly introduced into the ICP-MS with a Babington nebulizer. Because the signal ratio of Cd to Cs is proportional to the number of target cells, the number of target cells could be obtained according to the Cd/Cs ratio. Blank experiments were carried out by using the same procedures with QDs-BSA rather than QDs-anti-ASGPR. All analyses were performed in triplicate.

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RESULTS AND DISCUSSION Characterization of the As-Prepared MMNPs-NH2. FTIR spectroscopy, DLS, and TEM were used to characterize the chemical composition of the as-prepared MMNPs-NH2. The FT-IR spectrum of the MMNPs-NH2 is shown in Figure S2. The peak at 583 cm−1 corresponding to the Fe−O bond stretching vibration was observed in Fe3O4 MNPs and became weaker in MMNPs-NH2, indicating that the Fe3O4 MNPs were encapsulated inside of the MMNPs-NH2. Furthermore, the broad and strong absorption around 1086 cm−1 was attributed to the stretching vibration of Si−O bond, indicating a successful 1881

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Analytical Chemistry coating with a silica shell. Hydrated particle sizes and ζpotential of MMNPs-NH2 were characterized by DLS to demonstrate the functionalization of MMNPs with amino groups. Compared with the MMNPs, the hydrated particle size of MMNPs-NH2 was found to change from 523 to 577 nm, while the ζ-potential varied from −39.4 to 34.2 mV (Table 1).

that the immunoreaction could be completed in 60 min. Hence an incubation time of 60 min was chosen for the following experiments, which is shorter than the typical incubation time with plastic plate supports (approximately 3 h). In addition, the QDs-anti-ASGPR played important roles in “counting and visualization” of HepG2 cells. To ensure a constant labeling proportion of QDs to HepG2 cells, it was essential to add excessive QDs-anti-ASGPR to the reaction system. Nevertheless, when too much QDs-anti-ASGPR was involved, the endocytosis by HepG2 cells and nonspecific adsorption could not be negligible, which may lead to a high blank and low reproducibility. Therefore, the effect of both the volume of QDs-anti-ASGPR and incubation time on cell labeling was optimized. By fixing the concentration of QDsanti-ASGPR at 1 μmol L−1, the effect of the volume of QDsanti-ASGPR was investigated from 0.05 to 0.30 μL. As can be seen in Figure S5, the signal ratio of Cd/Cs increased along with the increase of volume of QDs-anti-ASGPR from 0.05 to 0.20 μL and then remained constant from 0.20 to 0.30 μL. When we considered increasing the blank signal and the cost of the method, 0.25 μL of QDs-anti-ASGPR was employed for labeling of HepG2 cells. Figure S6 shows the signal ratio of Cd/ Cs obtained with different incubation times (10 to 100 min) for the immunoreaction between the QDs-anti-ASGPR and HepG2 cells captured by MMNPs-anti-EpCAM. The signal ratio of Cd/Cs increased rapidly from 10 to 40 min and remained constant after incubation for 40 min, indicating that the immunoreaction could be completed in 40 min. To reduce the time for entire analytical process, an incubation time of 40 min was employed in subsequent experiments. Optimization of the Concentration of HNO3. For the determination of Cd and Cs by ICP-MS, the QDs-labeled sandwich-type immunocomplexes were dispersed in diluted sub-boiled HNO3 before being introduced into ICP-MS. It should be mentioned that the signal response of the QDs in the ICP-MS might be different when QDs were dispersed in different media. Therefore, the signal response of QDs in different concentration of HNO3 (0−2.0 mol L−1) was studied by ICP-MS, and the results are presented in Figure S7. The optimal signal response of Cd/Cs was obtained when the concentration of HNO3 was within 0.5−1.5 mol L−1. Accordingly, 1.0 mol L−1 of diluted HNO3 was chosen for the further study. Comparison of the Quantification with/without Ratiometric Method. The washing procedure is one of the critical factors in achieving good reproducibility and calibration linearity, while the sandwich-type products may be lost during magnetic separation or even by careless treatment. To avoid/ decrease the error caused by the loss of MNPs, the doped Cs in

Table 1. Hydrodynamic Size and Zeta Potential of Fe3O4, MMNPs, and MMNPs-NH2 Fe3O4 MMNPs MMNPs-NH2

size (d, nm)

zeta potential (mV)

179 523 577

40.6 −39.4 34.2

The results suggested that APTES was functionalized on the MMNPs successfully. As can be seen from TEM images in Figure 2, Fe3O4 MNPs (Figure 2a) had a uniform size of about 10 nm, while MMNPs-NH2 (Figure 2b) had a uniform size of about 100 nm. Figure 2b clearly showed that several Fe3O4 cores were embedded in the microspheres with a typical core− shell structure. Optimization of the Immunoassay Conditions. To achieve the best analytical performance, several parameters affecting the QDs-labeled magnetic immunoassay procedure were examined and optimized at 25 °C, including incubation time for capturing and labeling cells, amount of MMNPs-antiEpCAM and QDs-anti-ASGPR. In this immunoassay, the target HepG2 cells were captured by MMNPs-anti-EpCAM. To achieve high cell-capturing efficiency, both the volume of MMNPs-anti-EpCAM and incubation time were optimized. By fixing the concentration of MMNPs-anti-EpCAM at 25 mg mL−1, the effect of the volume of MMNPs-anti-EpCAM ranging from 0.5 to 4.0 μL on the immunoreaction between MMNPs-anti-EpCAM and HepG2 cells was investigated. As shown in Figure S3, the signal intensity of Cd increased rapidly along with the increase of volume of MMNPs-anti-EpCAM from 0.5 to 2.0 μL and then remained constant from 2.0 to 4.0 μL. Considering that large amounts of MMNPs introduced into ICP-MS for a long time may cause instrument polluted and that using excessive MMNPs may result in too low Cd/Cs ratio (0.2−0.8 is suitable) to obtain superior sensitivity, 2.0 μL of MMNPs-antiEpCAM was employed for the capture of HepG2 cells. The effect of incubation time on the immunoreaction between MMNPs-anti-EpCAM and HepG2 cells in the range of 20 to 100 min was investigated, and the result is shown in Figure S4. The signal ratio of Cd/Cs increased from 20 to 60 min and remained constant after incubation for 60 min, which indicated

Figure 2. TEM images of the synthesized Fe3O4 (a) and MMNPs-NH2 (b). 1882

DOI: 10.1021/acs.analchem.6b04314 Anal. Chem. 2017, 89, 1879−1886

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Figure 3. Comparison of ratiometric (left) to conventional (right) measurement with the increase of washing times.

MMNPs was used as an internal standard for the ratiometric measurement of the QDs tags by taking the signal ratio of 114 Cd/133Cs instead of the 114Cd signal for quantification. Figure 3 shows the comparison of ratiometric and conventional measurement methods which were carried out with increasing washing times. As can be seen, both the signal ratio of 114 Cd/133Cs and 114Cd signal were decreased rapidly after the first two times of washing, which was probably attributed to the removal of massive excess or nonspecific adsorption probes. When the washing times were further increased, the signal ratio of Cd/Cs decreased slightly with better reproducibility, while 114 Cd signal unabatedly decreased with worse reproducibility. In the whole assay, there were three washing processes, and each washing process included three times washing. Therefore, the ratiometric measurement resulting in better reproducibility was applied throughout this experiment. Specificity and Cross-Reactivity. To label and analyze the HepG2 cells accurately, the specificity of the QDs-based probe needed to be verified. The specific recognition of QDs-based probe toward HepG2 cells was achieved by antigen−antibody immunoreaction. The human cervical cancer cell line HeLa and the human oral cancer cell line Cal-27, which do not express the target ASGPR, were chosen as interferences. The test was performed using the same experimental procedures as for the HepG2 cells. As presented in Figure 4, at the same cell density of 1 × 104 cells, the signal ratio of Cd/Cs for HeLa and Cal-27 cells was similar to the background signal and much lower than that for HepG2 cells. Besides, the cross-reactivity of the immunoassay was tested by mixing 1 × 104 HepG2 cells with 1 × 105 HeLa or Cal-27 cells, and the results are shown in Figure 4 as well. Almost the same signal ratio of Cd/Cs was observed for HepG2 cells and that mixed with HeLa or Cal-27 cells. All the above results indicate a remarkable specificity and selectivity of the method for HepG2 cells. Moreover, the specificity of the QDs-based probe toward HepG2 cells was further verified using fluorescence imaging. Confocal Fluorescence Imaging. Though ICP-MS determination can provide quantitative results of cell numbers, it fails in presenting visualized images for excluding falsepositive/negative results. Images are visualized and can be the compensation to ICP-MS results. Therefore, with the help of

Figure 4. Specificity and cross-reaction tests of the developed QDsbased ICP-MS method for HepG2 cells. Column A represents 114 Cd/133Cs ratio from MMNPs-anti-EpCAM incubated with QDsanti-ASGPR without adding cells. Columns B, C, and D represent 114 Cd/133Cs ratio from 1 × 104 HeLa cells, 1 × 104 Cal-27 cells, and 1 × 104 HepG2 cells, respectively. Columns E and F represent 114 Cd/133Cs ratio from 1 × 105 HeLa cells and 1 × 105 Cal-27 cells, respectively, mixed with 1 × 104 HepG2 cells.

the QDs-anti-ASGPR probe, HepG2 cells can be observed visually by LCSM. Figure 5 shows the confocal fluorescence images of HepG2 cells incubated with QDs-anti-ASGPR (a) and with QDs-BSA (b), and the fluorescence image of MCF-7 cells incubated with QDs-anti-ASGPR (c), respectively. As can be seen from Figure 5a, noticeable red fluorescence was observed, and the fluorescence image agreed well with the bright field image. While when HepG2 cells were incubated with QDs-BSA, no fluorescence was observed (Figure 5b). As expected, the human breast cancer cell line MCF-7 (ASGPRnegative) did not show any noticeable fluorescence (Figure 5c). These results support the specificity of the probe (QDs-antiASGPR) for HepG2 cells, which was demonstrated by the ICPMS measurement mentioned above. Furthermore, all these results reveal that it is feasible to utilize QDs to bridge the barrier between living cell imaging and destructive detector ICP-MS, ascribing to the superior photoluminescent property and abundant Cd atoms inside of QDs. 1883

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Figure 5. Fluorescence (a−c), bright field (a′−c′), and merged (a″−c″) images of the ASGPR-positive HepG2 cells and ASGPR- negative MCF-7 cells captured by MMNPs-anti-EpCAM incubated with 10 nmol L−1 QDs-anti-ASGPR (a, a′, a″; c, c′, c″) and QDs-BSA (b, b′, b″) for 40 min, respectively. λex = 561 nm, scale bar = 50 μm.

metric,9 photoluminescence,46 or square-wave voltammetric,46 the established method exhibited superior analytical performance, especially in low-cell-number detection. It is undeniable that the methods based on fluorescent analysis with a dual signal amplification strategy10 or electrochemical biosensor utilizing aptamers47 have already achieved LODs as low as 8 and 2 cells, respectively. However, both of these methods suffered from complicated biological matrix (e.g., whole blood) interference. Besides high selectivity, high sensitivity, and good reproducibility, the proposed method offers the “counting and visualization” of cells with one QDs label, which make the method simpler. Results obtained by ICP-MS and fluorescence imaging are complementary and comprehensive. Sample Analysis. Fresh whole blood of healthy people was collected from the Hubei Cancer Hospital (Wuhan, China) according to the rules of the local ethical committee. To evaluate the application potential of the developed method for real sample analysis, the spiking experiment was performed by adding different numbers of HepG2 cells into lysed human blood, and the recoveries for the spiked samples are listed in Table 2. As can be seen, the recoveries of HepG2 cells in the spiked lysed human blood at five concentration levels were in the range of 86−104%. These results reveal that the coexisting components in the lysed blood sample matrix hardly interfere with the proposed immunoassay.

Analytical Performance. Under the optimized experimental conditions, the analytical performance of the developed method based on ICP-MS detection was evaluated. The LOD (3σ) of the developed method was calculated to be 61 cells (3σ) based on the signal ratio of 114Cd/133Cs. Furthermore, the relative standard deviation (RSD) for seven replicate determinations of 800 HepG2 cells was 5.4%. As shown in Figure S8, a good linear relationship was obtained in a wide dynamic range of 200−30 000 HepG2 cells, and the linear regression equation was y = 5.8 × 10−5x + 0.029 (R2 = 0.9978). For the comparison with ICP-MS results, the fluorescence images of different numbers of cells were shown in Figure S9. The QDs-labeled HepG2 cells could be visualized under LCSM; however, only a few parts of the cells appeared in our range of vision, and much effort had to be done on searching the targeted cells when the cell number decreased to the level of thousands and even less. Under such a circumstance, ICPMS was much more sensitive and capable to count the cells. A comparison of the proposed method with other methods for the analysis of cancer cells is shown in Table S2. Generally, the LOD of the proposed method is lower or comparable than other reported methods based on ICP-MS11,14 except one using a trifunctional probe.35 Additionally, compared with that obtained by other approaches using magnet-quartz crystal microbalance,44 differential pulse voltammetry,45 cyclic voltam1884

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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.

Table 2. Analytical Results of Spiking Tests in Lysed Human Blood added (102 cells)

found (102 cells)

recovery (%)

5.0 10 20 50 100

4.9 ± 0.5 8.6 ± 3.0 18 ± 3.2 47 ± 9.2 104 ± 11

98 86 90 94 104

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.6b04314. Additional information on calculation of the number of Cd atoms in each QD and the amount of anti-ASGPR anchored on each QD, schematic diagram for the synthesis of MMNPs-NH2 (Figure S1), FT-IR spectra of Fe3O4 and MMNPs-NH2 (Figure S2), effect of the volume of MMNPs-anti-EpCAM (Figure S3), effect of incubation time for capturing HepG2 cells (Figure S4), effect of the volume of QDs-anti-ASGPR (Figure S5), effect of incubation time for labeling cells (Figure S6), effect of HNO3 concentration (Figure S7), calibration curves of HepG2 cells obtained by signal ratio of 114 Cd/133Cs in ICP-MS (Figure S8), confocal fluorescence images of different numbers of HepG2 cells (a−h) (Figure S9), operating parameters of ICP-MS (Table S1), and comparison of different methods for the analysis of cancer cells (Table S2) as noted in text (PDF)

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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) Rasooly, A.; Jacobson, J. Biosens. Bioelectron. 2006, 21, 1851− 1858. (4) Della Corte, C.; Triolo, M.; Iavarone, M.; Sangiovanni, A. Liver Int. 2016, 36, 166−176. (5) Zhai, J.; Wang, Y. L.; Xu, C.; Zheng, L. N.; Wang, M.; Feng, W. Y.; Gao, L.; Zhao, L. N.; Liu, R.; Gao, F. P.; Zhao, Y. L.; Chai, Z. F.; Gao, X. Y. Anal. Chem. 2015, 87, 2546−2549. (6) Paredes-Aguilera, R.; Romero-Guzman, L.; Lopez-Santiago, N.; Burbano-Ceron, L.; Camacho- Del Monte, O.; Nieto-Martinez, S. Am. J. Hematol. 2001, 68, 69−74. (7) Ghossein, R. A.; Bhattacharya, S. Eur. J. Cancer 2000, 36, 1681− 1694. (8) Belov, L.; de la Vega, O.; dos Remedios, C. G.; Mulligan, S. P.; Christopherson, R. I. Cancer Res. 2001, 61, 4483−4489. (9) Arya, S. K.; Wang, K. Y.; Wong, C. C.; Rahman, A. R. Biosens. Bioelectron. 2013, 41, 446−451. (10) Han, E.; Ding, L.; Ju, H. Anal. Chem. 2011, 83, 7006−7012. (11) Zhang, Y.; Chen, B. B.; He, M.; Yang, B.; Zhang, J.; Hu, B. Anal. Chem. 2014, 86, 8082−8089. (12) Liu, R.; Wu, P.; Yang, L.; Hou, X.; Lv, Y. Mass Spectrom. Rev. 2014, 33, 373−393. (13) 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. (14) Zhang, X.; Chen, B.; He, M.; Zhang, Y.; Peng, L.; Hu, B. Analyst 2016, 141, 1286−1293. (15) Wind, M.; Wegener, A.; Eisenmenger, A.; Kellner, R.; Lehmann, W. D. Angew. Chem., Int. Ed. 2003, 42, 3425−3427. (16) Navaza, A. P.; Encinar, J. R.; Sanz-Medel, A. Angew. Chem., Int. Ed. 2007, 46, 569−571. (17) Edler, M.; Jakubowski, N.; Linscheid, M. Anal. Bioanal. Chem. 2005, 381, 205−211. (18) Joo, J. Y.; Lim, H. B. J. Anal. At. Spectrom. 2012, 27, 1069−1073. (19) Zhang, C.; Wu, F. B.; Zhang, Y. Y.; Wang, X.; Zhang, X. R. J. Anal. At. Spectrom. 2001, 16, 1393−1396. (20) Yan, X. W.; Yang, L. M.; Wang, Q. Q. Angew. Chem., Int. Ed. 2011, 50, 5130−5133. (21) Peng, H. Y.; Chen, B. B.; He, M.; Zhang, Y.; Hu, B. J. Anal. At. Spectrom. 2011, 26, 1217−1223. (22) Liu, R.; Lv, Y.; Hou, X. D.; Yang, L.; Mester, Z. Anal. Chem. 2012, 84, 2769−2775. (23) Lou, X.; Zhang, G.; Herrera, I.; Kinach, R.; Ornatsky, O.; Baranov, V.; Nitz, M.; Winnik, M. A. Angew. Chem., Int. Ed. 2007, 46, 6111−6114. (24) Abdelrahman, A. I.; Dai, S.; Thickett, S. C.; Ornatsky, O.; Bandura, D.; Baranov, V.; Winnik, M. A. J. Am. Chem. Soc. 2009, 131, 15276−15283. (25) Peng, H. Y.; Jiao, Y.; Xiao, X.; Chen, B. B.; He, M.; Liu, Z. R.; Zhang, X.; Hu, B. J. Anal. At. Spectrom. 2014, 29, 1112−1119. (26) Zhang, C.; Zhang, Z. Y.; Yu, B. B.; Shi, J. J.; Zhang, X. R. Anal. Chem. 2002, 74, 96−99. (27) Baranov, V. I.; Quinn, Z.; Bandura, D. R.; Tanner, S. D. Anal. Chem. 2002, 74, 1629−1636.

CONCLUSIONS In this work, the synthesized Cs doped MMNPs combined with commercial available QDs were applied in element-tagging magnetic immunoassay and enable us to count and see cancer cells via ICP-MS and fluorescence imaging. Anti-ASGPR conjugated QDs tags play two essential roles in this immunoassay. As elemental tags, high sensitivity was achieved because of the large quantities of detectable Cd atoms in each QD particle. As fluorescence probes, QDs enable cells to be visualized under a fluorescence microscope. In addition, measurement error and signal fluctuation caused by particle loss were compensated and suppressed by using the doped Cs as an internal standard for ICP-MS ratiometric determination. A combination of ICP-MS quantification and fluorescence imaging with the same QDs tag provides more valuable information and makes the methods much more simple and robust.

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AUTHOR INFORMATION

Corresponding Author

*Fax: +86-27-68754067. Tel: +86-27-68752162. E-mail: [email protected]. ORCID

Bin Hu: 0000-0003-2171-2202 Notes

The authors declare no competing financial interest. 1885

DOI: 10.1021/acs.analchem.6b04314 Anal. Chem. 2017, 89, 1879−1886

Article

Analytical Chemistry (28) Li, F.; Zhao, Q.; Wang, C.; Lu, X.; Li, X.-F.; Le, X. C. Anal. Chem. 2010, 82, 3399−3403. (29) Liu, J. M.; Yan, X. P. J. Anal. At. Spectrom. 2011, 26, 1191−1197. (30) Zhang, X.; Chen, B. B.; He, M.; Zhang, Y. W.; Xiao, G. Y.; Hu, B. Spectrochim. Acta, Part B 2015, 106, 20−27. (31) Zhang, S. X.; Han, G. J.; Xing, Z.; Zhang, S. C.; Zhang, X. R. Anal. Chem. 2014, 86, 3541−3547. (32) Chen, B. B.; Hu, B.; Jiang, P.; He, M.; Peng, H. Y.; Zhang, X. Analyst 2011, 136, 3934−3942. (33) Liu, X.; Liu, R.; Tang, Y. R.; Zhang, L. C.; Hou, X. D.; Lv, Y. Analyst 2012, 137, 1473−1480. (34) Cho, H. K.; Lim, H. B. J. Anal. At. Spectrom. 2013, 28, 468−472. (35) Zhang, Z.; Luo, Q.; Yan, X.; Li, Z.; Luo, Y.; Yang, L.; Zhang, B.; Chen, H.; Wang, Q. Anal. Chem. 2012, 84, 8946−8951. (36) Chen, B. B.; Peng, H. Y.; Zheng, F.; Hu, B.; He, M.; Zhao, W.; Pang, D. W. J. Anal. At. Spectrom. 2010, 25, 1674−1681. (37) Montoro Bustos, A. R.; Trapiella-Alfonso, L.; Encinar, J. R.; Costa-Fernandez, J. M.; Pereiro, R.; Sanz-Medel, A. Biosens. Bioelectron. 2012, 33, 165−171. (38) Goldman, E. R.; Medintz, I. L.; Mattoussi, H. Anal. Bioanal. Chem. 2006, 384, 560−563. (39) Wegner, K. D.; Hildebrandt, N. Chem. Soc. Rev. 2015, 44, 4792− 4834. (40) Borlido, L.; Azevedo, A. M.; Roque, A. C. A.; Aires-Barros, M. R. Biotechnol. Adv. 2013, 31, 1374−1385. (41) Ko, J.; Lim, H. B. Anal. Chem. 2014, 86, 4140−4144. (42) Choi, H. W.; Lee, K. H.; Hur, N. H.; Lim, H. B. Anal. Chim. Acta 2014, 847, 10−15. (43) Ko, J. A.; Lim, H. B. J. Anal. At. Spectrom. 2013, 28, 630−636. (44) Pan, Y.; Guo, M.; Nie, Z.; Huang, Y.; Pan, C.; Zeng, K.; Zhang, Y.; Yao, S. Biosens. Bioelectron. 2010, 25, 1609−1614. (45) Zhang, X.; Teng, Y.; Fu, Y.; Xu, L.; Zhang, S.; He, B.; Wang, C.; Zhang, W. Anal. Chem. 2010, 82, 9455−9460. (46) Hua, X.; Zhou, Z. X.; Yuan, L.; Liu, S. Q. Anal. Chim. Acta 2013, 788, 135−140. (47) Kashefi-Kheyrabadi, L.; Mehrgardi, M. A.; Wiechec, E.; Turner, A. P.; Tiwari, A. Anal. Chem. 2014, 86, 4956−4960.

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DOI: 10.1021/acs.analchem.6b04314 Anal. Chem. 2017, 89, 1879−1886