Immunomagnetic Separation Combined with Inductively Coupled

Jul 23, 2014 - Plasma Mass Spectrometry for the Detection of Tumor Cells Using. Gold Nanoparticle Labeling. Yuan Zhang, Beibei Chen, Man He, Bin Yang,...
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Immunomagnetic Separation Combined with Inductively Coupled Plasma Mass Spectrometry for the Detection of Tumor Cells Using Gold Nanoparticle Labeling Yuan Zhang, Beibei Chen, Man He, Bin Yang, Jing Zhang, 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: This work reports an efficient, specific, and sensitive immunoassay protocol for detection of tumor cells by using inductively coupled plasma mass spectrometry (ICPMS) with two probes. Magnetic nanobeads modified with antiCD3 were used as capture probes for efficient and fast magnetic separation of Jurkat T cells from a mixture of cells, and gold nanoparticles (Au NPs) conjugated with anti-CD2 were used as detection probes for ICP-MS measurement. The capture and detection probes target the Jurkat T cells with high affinity and specificity, while they do not target other CD2/CD3-negative cells such as 97L cells and A549 cells. On the basis of these results, we proposed a new immunoassay for specific detection of Jurkat T cells. The conditions for this immunoassay were carefully optimized, including the incubation time and temperature, the concentration of the labeling probe, and the elution conditions. Under the optimized conditions, the linear range of 300−30 000 and the limit of detection of 86 Jurkat T cells were obtained, and the relative standard deviation for seven replicate detection of Jurkat T cells was 5.2% (3000 Jurkat T cells). This method has numerous advantages, including ease of preparation, low sample consumption, and high sensitivity and selectivity. Importantly, the methodology could be extended to the simultaneous detection of other cells based on their cellular biomarkers.

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signaling, ion channels, transporters, enzymes, immune globulins, or adhesion molecules). Diagnostic precision and refinement of immunological classification enables selection of the most appropriate therapy for the patients.11 Analytical methods for detecting leukemias include flow cytometry (FC),12 real-time quantitative reverse-transcriptase polymerase chain reaction (RQ-PCR),13 a cluster of differentiation antibody microarray,14 electrochemical method,15 and immunofluorescence.16 FC is quantitative and rapid with a generally high predictive value, while the use of CD antigen expression (immune phenotyping) for identification of leukemias has been constrained by FC, which is expensive and requires plenty of fluorescently labeled cells, and thus may suffer from spectral overlapping. PCR, as a molecular method, is labor-intensive and time-consuming in the measurement process. The detection sensitivities of antibodies microarray are not satisfactory. To address these issues, much effort has been directed to enhance the analytical sensitivity and the facility of corresponding detection systems. Immunoassays17 are commonly used to detect cancer cells based on the overexpressed surface antigens. Immunoassays use

ancer is one of the most serious diseases that causes death worldwide.1 In blood, the number of circulating tumor cells (CTCs)2 is correlated very sensitively with the recurrence of cancer and relapse. The detection of CTCs especially at low concentration has a drastic effect on the accurate early diagnosis of cancer, and subsequently successful cancer treatment using contemporary therapeutic methods.3 Unfortunately, at the early stage of a tumor, disseminated cells circulate in the blood at extremely low concentrations,4 making the detection of lowfrequency cancer cells difficult. Therefore, novel approaches are required for highly sensitive, rapid, and specific detection of cancer cells at low frequency in real biological samples.5 Leukemia6 is known as blood cancer, and the routine clinic pathologic diagnosis is based on the morphology according to the WHO (World Health Organizations) and the FAB (French−American−British) criteria,7 with molecular methods and immunophenotyping as supplemented alternatives. In immunophenotyping,8 cells are classified by the identification of cell membrane receptors. Different leukemias9 express on their plasma membranes particular subsets of cluster of differentiation (CD) antigens, which may resemble those of precursor cells along the lineages of differentiation to myeloid and lymphoid leukocytes. CD antigens10 associated with the plasma membranes of leukocytes may be involved in various cell functions (cell−cell interactions, cytokine receptors, cell © 2014 American Chemical Society

Received: March 17, 2014 Accepted: July 23, 2014 Published: July 23, 2014 8082

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different label probes, such as radioactive,18 enzymatic,19 and fluorescent20 labels, for this purpose. Unfortunately, these labels often restrict the biomedical applications of immunoassays, which possess inherent limitations, such as the spectral overlap of different dye, significant reduction in signal-to-ratios caused by autofluorescence of substratums, radio hazard, and so on. To overcome these problems, metal-based labels, including nanoparticles (NPs)21 and semiconductor crystals (quantum dots),22 have attracted considerable interest. In biotechnological systems, these metal-based labels improve the sensitivity because of their unique physical and chemical properties, and they have good biocompatibility. As an ideal biological tag, Au NPs17,23−25 have been extensively applied as detection and imaging probes to label a broad range of biological receptors for surface-enhanced Raman scattering26 and cell imaging.27 The biological activities of proteins can be retained after binding to Au NPs, and a high-sensitivity detector can be used to detect the proteins indirectly.28 Inductively coupled plasma mass spectrometry (ICP-MS),29 as an element-specific detector, is one of the most sensitive techniques providing a wide dynamic range, low detection limits, low matrix effects, rapid analysis capabilities, and extremely high sensitivity and element specificity. Previous work has shown that Au NPs can be quantified by ICP-MS, and that the atomization efficiency of ICP-MS for NPs is the same as that for ions in solution.23 In principle, with selection of appropriate elemental labels for biological molecules, elemental-tagging-based ICP-MS in combination with an immunoassay may allow sensitive detection of tumor cells.30 Cancer cells are extremely rare in a complex mixture such as whole blood, and to achieve acceptable detection sensitivity, appropriate sample preparation prior to ICP-MS detection is required for the identification, isolation, and enrichment of specific target cells from the heterogeneous suspension.31 The separation of CTCs from whole blood usually relies on the difference between CTCs and other cells in either physical characteristics, such as density, cell size, and electrical properties, or biological characteristics, such as expression of protein markers, cancer-specific antigen−antibody interactions. Currently, immunomagnetic separation32 is routinely used for target cells separation from a blood sample. In this method, biochemically functionalized paramagnetic beads are coated with specific antibodies that are targeted to specific cancer cells and can recognize the surface antigens expressed on the surface of cells. The cancer cells are then isolated from other cells using a magnetic field. These immunomagnetic beads33 are ideal for separation of cancer cells because of their stability and large surface to volume ratios. Immunomagnetic separation is rapid and has the ability of performing in parallel. After the cancer cells are captured and isolated, the cells−MBs (magnetic beads) complexes can be further labeled with Au NPs and then analyzed by ICP-MS. To the best of our knowledge, Au NPs labeling combined with ICP-MS for the detection of Jurkat T cells based on their overexpressed surface antigens has not been reported. One of the main types of leukemia is acute lymphoblastic leukemia (ALL), derived from immature T or B-lymphocytes. ALL is the most frequent hematologic malignancy of children, comprising 25% of all cancers, with biologically and clinically distinct subsets divided into B and T lineages. A T-cell phenotype is present in 15% of childhood ALL. Jurkat T cell (human peripheral blood leukemia T cells) was used as a model for the study of T-ALL. CD2 and CD3 are members of the

immunoglobulin super family, containing an extracellular portion, cytoplasmic domain, and transmembrane domain, which are highly expressed by Jurkat T cells. Monoclonal antibodies can specifically recognize the extracellular portion, making them ideal target molecules for the detection of Jurkat T cells. Herein, we describe a novel and effective strategy for detecting tumor cells present at low levels in biological fluids (blood samples) based on coupling of immunomagnetic separation with an element-tagged ICP-MS detection with Jurkat T cells as model. In this method, immunomagnetic beads (MB-anti-CD3) bind to the Jurkat T cells based on antigen− antibody specificity, and then Au NPs coupled to anti-CD2 are used to label the magnetically captured Jurkat T cells. Also, the interference of A549 and 97L cells on the capturing and detection of Jurkat T cells was investigated. The proposed method was demonstrated to be simple, rapid, and sensitive for the detection of cancer cells.

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EXPERIMENTAL SECTION Material and Reagents. Monoclonal mouse antihuman CD2 antibodies and antihuman CD3 conjugated MBs were purchased from BD Pharmingen (BD Biosciences, Franklin Lakes, NJ). Gold chloride tetrahydrate (HAuCl4·4H2O) was obtained from Shanghai Chemical Reagent Co. (Shanghai, China). Bovine serum albumin (BSA) and formic acid were obtained from Aladdin Reagent Inc. (Shanghai, China). The phosphate buffer saline (PBS, pH 7.4) contained 137 mmol L−1 NaCl, 2.7 mmol L−1 KCl, 1.5 mmol L−1 KH2PO4, and 8 mmol L−1 K2HPO4. DMEM and RPMI 1640 were purchased from Gibco. All reagents used were Specpure or at least of analytical reagent grade. High-purity deionized water (18.25 MΩ cm, Milli-Q Element, Millipore, Billerica, MA) was used throughout this work. An Agilent 7500a ICP-MS system (Agilent Technologies, Tokyo, Japan) was used for the detection of 197Au. An inverted fluorescence microscope (TE2000-U, Nikon, Tokyo, Japan) coupled with a charge-coupled device camera (Retiga 2000R, QImaging, Surrey, BC) was used for observations of the cells. An XS105 Dual Range microbalance (Mettler Toledo Instruments Co., Ltd., Shanghai, China) and BS110S electronic balance (Beijing Sartorius Instrument Systems, Inc., Beijing China) were used for weighing the reagents. The pH values were adjusted with a Mettler Toledo 320-S pH meter (Mettler Toledo). The operating conditions for ICP-MS are listed in Supporting Information Table S1. Cell Culture. A549 and 97L cells were cultured in cell culture flasks at 37 °C with DMEM supplemented with 10% (v/v) fetal bovine serum and 100 U mL−1 penicillin− streptomycin. Cells were maintained under standard culture conditions at a temperature in a humidified atmosphere of 5% CO2. Suspension lymphoblast cells (Jurkat T cells) were grown with RPMI 1640 supplemented with 10% (v/v) fetal bovine serum and 100 U mL−1 penicillin−streptomycin. The culture medium was replaced once every 2 days. After a cell density of approximately 106 cell mL−1 was reached, A549 and 97L cells were detached by trypsinization using a 0.25% trypsin-EDTA solution, and then collected by centrifugation at 1500 rpm for 5 min. Jurkat T cells were collected by centrifugation at 1000 rpm for 5 min. After being washed with 0.01 mol L−1 PBS (pH 7.4) three times, the cells were dispersed in PBS. The cell density was counted using a hemocytometer (Counting chamber 8083

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Figure 1. Principle for the detection of Jurkat T cells by using Au NPs as detection probes and MBs as capture probes. (a) Preparation of Au NPs− anti-CD2 conjugate; (b) immunomagnetic separation of Jurkat T cells with anti-CD3-conjugated MBs, then incubation of the captured Jurkat T cells with Au NPs−anti-CD2 conjugate, and at last ICP-MS measurement.

medium by placing the tubes on a permanent magnet for 8 min. The supernatant fraction containing possible unbound Jurkat T cells was removed by an additional washing step with 200 μL of PBS. Finally, MB-anti-CD3−Jurkat T cell conjugates were resuspended in 200 μL of PBS. Both the fraction containing possible unbound cells and the fraction containing the magnetically trapped cells were collected separately, and a hemocytometer was used for counting captured Jurkat T cells and Jurkat T cells not bound to the MB-anti-CD3. Incubation of the Captured Jurkat T Cells with Au NPs−Anti-CD2 Conjugate. To minimize nonspecific binding and aggregation, 200 μL of 1% skim milk was added to 5 μL of Au NPs−anti-CD2 conjugate, and the solution was shaken for 30 min. The blocked Au NPs−anti-CD2 conjugates then were immuno-incubated with MB-anti-CD3−Jurkat T cells at 4 °C for 45 min to form MB-anti-CD3−Jurkat T cells−anti-CD2-Au NPs conjugates. Washing with 3 × 200 μL of PBS removed unbound Au NPs−anti-CD2 conjugate by magnetic separation. The MB-anti-CD3−Jurkat T cells−anti-CD2-Au NPs then were resuspended in 200 μL of PBS. ICP-MS Measurement. Antigen−antibody complexes can be dissociated under some extreme physiochemical conditions such as high temperature, low pH, and strong ionic strength.33 The immunocomplex of MB-anti-CD3−Jurkat T cells−antiCD2-Au NPs was dissociated in 30 μL of 1 mol L−1 formic acid, which released the captured Au NPs labels. After the immunomagnetic separation, 28 μL of the eluent containing the Au NPs was collected in a 0.2 mL centrifuge tube and directly introduced into ICP-MS to detect the signal of 197Au, and detection of the corresponding target tumor cells based on their overexpressed surface antigens could be obtained accordingly. Immunoassay Protocol. As shown in Figure 1, all Jurkat T cells (target) are designed to be detected using their two biomarkers of CD2 and CD3 simultaneously from a mixture with other cells, which did not express CD2 or CD3 biomarkers. First, to prevent nonspecific binding, MB-anti-CD3 conjugates (10 μL) were transferred into a 0.5 mL centrifuge tube and blocked for 30 min by incubating with 200 μL of PBS containing of 1% skim milk. Jurkat T cells then were added to the solution and incubated for 30 min at 4 °C with gentle mixing. The CD3 biomarkers on the Jurkat T cells coupled with the MB-anti-CD3 by the antigen−antibody reaction, and this formed the conjugates of MB-anti-CD3−Jurkat T cells. After

0610030 with Neubauer improved bright-line, Marienfeld, Lauda-Konigshofen, Germany). Synthesis of Citrate-Stabilized Au NPs and Au NPs− Anti-CD2 Conjugate. Colloidal Au NPs were prepared via a reduction of HAuCl4 solution with trisodium citrate according to the method reported previously with a slight modification.24 All glassware was soaked in aqua regia (HNO3:HCl = 1:3, by volume), then rinsed thoroughly with ultrapure water and dried before use. Briefly, HAuCl4 solution (0.1%, 50 mL) was heated to boiling with vigorous magnetic stirring, and then trisodium citrate (1%, 1.5 mL) was added quickly. The solution was boiled for another 10 min with stirring, and changed from pale yellow to deep red. The solution then was cooled to room temperature with continuous stirring. After filtering through a 0.45 μm membrane filter, the prepared Au NPs (50 mg L−1, the number of Au NPs was ∼1.5 × 1012 /mL) were stored in a brown glass bottle at 4 °C. The Au NPs prepared by this method were approximately 15 nm in diameter (Supporting Information Figure S-1). Monoclonal antibody anti-CD2 labeled with Au NPs was prepared according to the literature with slight modification. The minimum amount of anti-CD2 was determined by using a flocculation test, and 10% more than this amount was added to an appropriate volume of an Au NPs solution. The pH of the Au NPs solution was adjusted to 9.0 with 0.1 mol L−1 K2CO3. After incubation at 4 °C overnight, the Au NPs−anti-CD2 conjugate was stabilized with 240 μL of a 10% BSA solution for 1 h. The solution then was centrifuged at 12 000 rpm for 15 min at 4 °C to separate the AuNPs-labeled anti-CD2 from the unlabeled anti-CD2. The supernatant was discarded, and the obtained sediment containing the Au NPs−anti-CD2 conjugate was resuspended in 200 μL of PBS (pH 7.4). After being washed with 200 μL of PBS three times, the Au NPs−anti-CD2 conjugate was resuspended in 400 μL of PBS and stored at 4 °C. Immunomagnetic Separation of Jurkat T Cells with Anti-CD3-Conjugated MBs (MB-anti-CD3). Anti-CD3 conjugated MBs (10 μL, ø 200 nm) were transferred into a 0.5 mL centrifuge tube and washed with 200 μL of PBS. To prevent nonspecific adsorption, the MB-anti-CD3 was blocked for 30 min by incubating with 200 μL of PBS containing 1% skim milk. The Jurkat T cells (human CD3 positive selection cocktail) were incubated with MB-anti-CD3 for 30 min at 4 °C with gentle mixing. This produced MB-anti-CD3−Jurkat T cells complexes, which were magnetically separated from the 8084

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CD2 conjugate, transmission electron microscopy showed a cloud of protein around the AuNPs particles, which suggested that anti-CD2 was bound to the Au NPs. UV−vis absorption spectra of Au NPs in the absence and presence of anti-CD2 as well as UV−vis absorption spectra of anti-CD2 are shown in Supporting Information Figure S-2. As can be seen, no obvious absorption peak was observed for anti-CD2 in the wavelength range of 400−700 nm (curve c). On the contrary, the Au NPs exhibited a sharp absorption band with maximum absorbance at 519 nm (curve a). After addition of anti-CD2, the absorption band displayed a red shift to 522 nm (curve b) because of the interaction of anti-CD2 with the Au NPs. This shift indicated that the Au NPs−anti-CD2 conjugate was formed. Optimization of Anti-CD2 Labeling of the Au NPs. The interaction forces between the Au NPs and proteins were electrostatic interaction and van der Waals forces, but the exact forces involved in this binding were not fully understood.17,23−25 The reversible adsorption was dependent on pH, ionic strength, molecular weight of the protein, size of the Au NPs, and the protein loading. The following experiments were performed to optimize the conditions for preparation of the anti-CD2−Au NPs conjugate. The optimum pH for preparation of the anti-CD2−Au NPs conjugate was investigated. At pH values below the pI of the protein, the protein carries a net positive charge, and spontaneous flocculation will occur with the negatively charged Au NPs. Therefore, for labeling of most proteins with Au NPs, the pH of the Au NPs solution should be slightly higher than the pI of the protein. We chose pH of 9.0 (pH adjustment with 0.1 mol L−1 of K2CO3) for the labeling of anti-CD2 (pI = 8.0) with Au NPs. The most suitable loading amount of anti-CD2 onto the Au NPs was studied by a simple flocculation experiment using UV−vis spectroscopy. The Au NPs prepared in this study consisted of a gold core surrounded by a negative ionic double layer of charges, which prevented aggregation of the Au NPs. With NaCl addition, the double layer is destroyed, and the Au NPs will aggregate. However, antibody on the surface of the Au NPs can stabilize the gold against this aggregation in the presence of NaCl. In this experiment, various quantities of antiCD2 (1.5, 2, 2.5, 3, 3.5, 4, and 4.5 μg) were added to 100 μL of Au NPs solution (50 mg L−1) at pH 9.0, and the resulting mixture was incubated for 2 h at room temperature. Next, 20 μL of aqueous 10% NaCl was added to each solution, and after 1 h the UV−vis absorption spectrum of the mixture was recorded. Supporting Information Figure S-3 shows that as the quantity of anti-CD2 increased, the Au NPs displayed a red shift. As shown in Supporting Information Figure S-4, the absorbance increased with the increase of the spiked quantity of anti-CD2 when the spiked quantity was lower than 3.5 μg, which shows that the quantity of anti-CD2 was not enough to eliminate the flocculation of the Au NPs caused by NaCl. By contrast, the absorbance was constant when the quantity of anti-CD2 was higher than 3.5 μg, which shows that the Au NPs were stabilized. Therefore, 2.5 μg of anti-CD2 was selected for the loading of Au NPs in 60 μL of solution. The anti-CD2−Au NPs conjugate was prepared under the optimized conditions. Anti-CD2 (5 μL, 500 μg mL−1) was added to 60 μL of the Au NPs solution at pH 9.0, and then incubated overnight at 4 °C. BSA then was added into the above solution, followed by incubating for 1 h. The resulting mixture was centrifuged at 12 000 rpm for 15 min to separate any unbound anti-CD2 from that bound to the Au NPs. Finally,

attachment of the MBs, the target cells could be separated from other cells in the mixture using an external magnet. Following the magnetic separation and washing of the separated cells, the conjugates of MB-anti-CD3−Jurkat T cells were resuspended in 5 μL of the Au NPs−anti-CD2 conjugate, and diluted to 200 μL with PBS containing 1% skim milk. After incubation for 45 min at 4 °C, the resulting mixture of MB-antiCD3−Jurkat T cells-anti-CD2-Au NPs was separated using an external magnet and washed with PBS as the above. Au NPs− anti-CD2 associated with the MB-anti-CD3−Jurkat T cells by immunoreaction with their CD2 biomarkers, and this produced an immunocomplex of Au NPs labeled Jurkat T cells (MB-antiCD3−Jurkat T cells−anti-CD2-Au NPs). The Jurkat T cells captured by the MBs were labeled with Au NPs successfully. Finally, the Jurkat T cells (Au NPs−Jurkat T cells-MB) were detected according to the intensity of Au. A 30 μL aliquot of 1 mol L−1 formic acid solution was added to the immunocomplex of MB-anti-CD3−Jurkat T cells−anti-CD2-Au NPs for 10 min to release the AuNPs from the labeled MB-Jurkat T cells. After magnetic separation, 28 μL of the suspension containing the released Au NPs was transferred into a 0.2 mL centrifuge tube and then analyzed by ICP-MS for 197Au. Blank experiments were carried out using the same procedures with Au NPs alone rather than AuNPs−anti-CD2. All analyses were performed in triplicate. Real Sample Analysis. Fresh whole blood of healthy people was collected from the hospital of Wuhan University (Wuhan, China) according to the rules of the local ethical committee. These fresh whole blood samples were centrifuged at 1500 rpm for 5 min, the resulting faint yellow supernate was discarded, and the residue was mixed gently with a certain amount of PBS solution, of which the volume is equal to that of the discarded supernate. The obtained mixed solution was then employed for sample analysis to evaluate the application potential of the proposed method. To investigate the interference of blood matrix on the Au NPs labeling immunoassay system, 20 μL of the above obtained sample solution was spiked with 250, 1000, 4000, and 16 000 Jurkat T cells, respectively, and subjected to the immunoassay procedure and subsequent ICP-MS detection. For real sample analysis, 5, 10, and 20 μL of whole blood sample were subjected to the immunoassay procedure and subsequent ICP-MS detection, respectively, after the removal of the supernate by centrifugation. It should be noted that all handling and processing was performed carefully, and all tools in contact with blood specimens and immunoreagents were disinfected after use.

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RESULTS AND DISCUSSION The immunoassay (Figure 1) depicted the principles of rapid and sensitive detection of low-frequency cancer cells by a typical cell capture and labeling procedure following ICP-MS detection. The key steps in this assay include immunomagnetic separation of the Jurkat T cells using MB-anti-CD3, conjugation of anti-CD2 to the Au NPs, specific binding of these conjugates to the Jurkat T cells, and sensitive detection of the Au NPs using ICP-MS. The experimental parameters for each of these steps were optimized to improve the sensitivity and reproducibility of the method. Characterization of the Au NPs and the Au NPs−AntiCD2 Conjugate. The average diameter of the Au NPs prepared by trisodium citrate reduction was about 15 nm (Supporting Information Figure S-1). For the Au NPs−anti8085

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the precipitate was resuspended in 60 μL of PBS and stored at 4 °C. The most suitable loading amount of anti-CD2 onto the Au NPs was studied by a simple flocculation experiment using UV−vis spectroscopy. However, this method could not be exactly quantified. To get the precise ratio of anti-CD2 to Au NPs in the conjugate of Au NPs−anti-CD2, immunofluorescence staining experiment was performed. The Au NPs−antiCD2 (mouse-antihuman) was first blocked by 5% BSA, and then incubated with dylight 539 conjugated goat antimouse IgG for 1 h, followed by fluorescence spectroscopy detection. On the basis of the reduced fluorescence signal (quenched by Au NPs), we could calculate the amount of IgG, and thus the amount of anti-CD2 loaded onto the Au NPs. In our experiment, it was found that in 1 mL of Au NPs (1.5 × 1012 /mL) solution, the maximum loading amount of anti-CD2 was 8 μg. In addition, the anti-CD2 antibody used in this work was mouse IgG1 with the molecular weight of about 160 kDa, which means that the number of anti-CD2 (8 μg) was [(8 × 10−6)/(160 × 103)] × 6.02 × 1023 = 3 × 1013. Therefore, the number of anti-CD2 molecules labeled on one Au NP was 3 × 1013/(1.5 × 1012) = 20. Optimization of the Immunoassay Conditions. To achieve optimal detection performance, various parameters in the Au NPs labeled immunomagnetic assay and the ICP-MS detection were evaluated. This included the incubation time and temperature for MB-anti-CD3 and Jurkat T cells, incubation time for the captured cells and Au NPs probe, and the elution conditions. To optimize binding of the Jurkat T cells to the MB-antiCD3, incubation temperatures of 0, 4, 25, and 37 °C were investigated. Effective immunoreaction and low endocytosis are a benefit for antibody recognizing cells. As shown in Supporting Information Figure S-5, the maximum signal intensity for Au was obtained at 4 °C, indicating an effective immunoreaction with lower endocytosis at this temperature. Therefore, 4 °C was used in subsequent experiments for optimal cell capture efficiency. Supporting Information Figure S-6 shows the Au signal intensities obtained for optimization of the incubation time (10−60 min) for incubation of MB-anti-CD3 with Jurkat T cells at 4 °C. The immunoreactions were completed in 30 min. Consequently, this incubation time was selected as 30 min for the following experiments. Supporting Information Figure S-7 shows the Au signal intensities obtained with different incubation times (10−60 min) for the immunoreactions between the Au NPs−anti-CD2 probe and Jurkat T cells that were captured by MB-anti-CD3. The experiments were conducted at 4 °C to reduce cell uptake by the Au NPs probe. The immunoreactions could be completed in 30 min. This incubation time is much shorter than that typically required with plastic plate supports (approx 3 h).25 To ensure good reproducibility, an incubation time of 40 min was used in subsequent experiments for the immunoreactions between captured Jurkat T cells and the Au NPs probe. The dependence of the Au signal on the volume of Au NPs− anti-CD2 is shown in Figure 2. The response increased rapidly as the volume of Au NPs−anti-CD2 was increased from 0.5 to 2 μL, and then leveled off above 2 μL. This indicated that the interaction of Jurkat T cells with anti-CD2 had reached equilibrium after 2 μL, and a volume of 3 μL was selected for further investigations.

Figure 2. Effect of the volume of Au NPs−anti-CD2. The volume of Au NPs−anti-CD2 was 0.5, 0.8, 1.5, 2, 2.5, 3, 4, and 5 μL, respectively. MB-anti-CD3, 10 μL; Jurkat T cells, 2.5 × 104; anti-CD2-Au NPs, 3 μL.

Optimization of the Elution Conditions. To completely dissociate the immunocomplex of MB-anti-CD3−Jurkat T cells−anti-CD2-Au NPs and release Au, the type of eluent, concentration of eluent, elution volume, and elution time were optimized. The effect of the eluent (1 mol L−1, nitric acid, hydrochloric acid, formic acid, acetic acid, and citric acid) was studied. As shown in Figure 3, formic acid was the most effective for Au removal from the captured Au NPs labels. Therefore, formic acid was selected as the eluent in this study. The concentration of formic acid used for the release of Au was also optimized, and the experimental results are shown in Supporting Information Figure S-8. Gold was quantitatively released from the immunocomplex when the concentration of formic acid was higher than 0.5 mol L−1. Accordingly, 1.0 mol L−1 of formic acid was chosen for subsequent experiments.

Figure 3. Effect of different eluents (1 mol L−1, including nitric acid, hydrochloricacid, formic acid, acetic acid, and citric acid) on the signal intensity of released Au. MB-anti-CD3, 10 μL; Jurkat T cells, 2.5 × 104; anti-CD2-Au NPs, 3 μL. In the control group, Au NPs−anti-CD2 were replaced by Au NPs. The left in each group represented the blank group, and the right represented the experiment group. 8086

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The cross-reactivity of MB-Jurkat T cells−Au NPs was examined using A549 and 97L cells (Figure 5). Jurkat T cells

To study the effect of the elution volume, Au was removed from the immunocomplex with four successive volumes of formic acid (1.0 mol L−1, 30 μL each, total volume 120 μL). Gold was completely eluted with the first 30 μL volume (Supporting Information Figure S-9), and this volume was selected for the optimized elution protocol. Elution times between 2 and 30 min were investigated using 30 μL of formic acid (1.0 mol L−1) as the eluent. There were no obvious fluctuations in the Au signal intensity after 2 min, and 10 min was selected as the elution time for subsequent experiments. Specificity and Cross-Reactivity. The specificity for the analysis of tumor cells based on the overexpressed surface antigens was mainly dependent on the antigen−antibody recognition reactions that occurred simultaneously with two biomarkers. One of these reactions was between MB-anti-CD3 and the CD3 marker on the Jurkat T cells, and the other was a coupling reaction between Au NPs−anti-CD2 and the CD2 marker on the Jurkat T cells surface. In the experiment, a strong Au signal was obtained after sequential incubation of 6 × 104 Jurkat T cells with MB-anti-CD3 and Au NPs−anti-CD2, while incubation of Jurkat T cells with MB-anti-CD3 and then with Au NPs gave a weak Au signal in control experiments (Figure 4). This result shows that the background signal produced by the nonspecific binding or adsorption of Au NPs on Jurkat T cells is negligible.

Figure 5. Cross-reaction tests of the developed gold tagged-ICP-MS method for Jurkat T cells. Column A presents signal intensity from 6 × 104 Jurkat T cells. Column B presents signal intensity from 6× 105 A549 cells mixed with 6× 104 Jurkat T cells. Column C presents signal intensity from 6× 105 97L cells mixed with 6× 104 Jurkat T cells. Column D presents signal intensity from 6× 105 A549 cells and 6× 105 97L cells mixed with 6× 104 Jurkat T cells.

were mixed with A549 and 97L cells, respectively, incubated with MB-anti-CD3 and Au NPs−anti-CD2, magnetically separated, and analyzed by ICP-MS. In each case, 6 × 105 A549 cells, 6 × 105 97L cells, and 6 × 104 Jurkat T cells were used. Almost the same signal intensities were observed for the Jurkat T cells when they were mixed with only A549 cells or only 97L cells, or both A549 and 97L cells. Jurkat T cells were observed because both CD3 and CD2 were used to elucidate the coupling of MB-anti-CD3 and Au NPs−anti-CD2 labels. These results confirmed that cross-reactivity of MB-anti-CD3 and Au NPs−anti-CD2 on Jurkat T cells with A549 and 97L cells was prevented. MB-anti-CD3 could specifically immune magnetically separated Jurkat T cells from a mixture of cells, and Au NPs−anti-CD2 acted as a reliable label probe for the detection of Jurkat T cells by ICP-MS. Analytical Performance. Under the optimized experimental conditions, the sensitivity and dynamic linear range of the developed method was evaluated for the detection of Jurkat T cells on the basis of their surface antigens. The relationship between the Au signal intensity and cell density of the Jurkat T cells is shown in Figure 6, and a good linear relationship was obtained in the range of 300−30 000 Jurkat T cells (y = 3.95x + 326, R2 = 0.9977). On the basis of the Au signal intensity, the limit of detection (3 times the signal-to-noise ratio) of the developed immunoassay was calculated to be 86 cells (3σ, n = 7), and the relative standard deviation (RSD) for sevenreplicate detection of 3000 Jurkat T cells was 5.2%. The incubation time in the developed method is shorter than that typically required with plastic plate supports. A comparison of the LODs obtained in this work and those obtained by several other approaches was shown in Supporting Information Table S2. As can be seen, the LOD obtained by this method was comparable with that obtained by other approaches. The detection limit is 86 cells, which is lower than that of 6000 cells

Figure 4. Specificity test of the developed gold tagged-ICP-MS method for Jurkat T cells. Column A presents signal intensity from MB-anti-CD3 incubated with Au NPs−anti-CD2, without adding cells. Columns B, C, and D present signal intensity from 6 × 105 A549 cells, 6 × 105 97L cells, and 6 × 104 Jurkat T cells, respectively, incubated with MB-anti-CD3 and then Au NPs. Columns E, F, and G represent signal intensity obtained from 6 × 105 A549 cells, 6 × 105 97L cells, and 6 × 104 Jurkat T cells, respectively, incubated with MB-anti-CD3, followed by Au NPs−anti-CD2.

A549 and 97L cells do not react with anti-CD2 or anti-CD3; we tested these cells as interference, and the results are also presented in Figure 4. The test was performed using the same experimental procedures as for the Jurkat T cells. At a cell density of 6 × 105 cells, the signal intensities for these cells were similar to the background signal and much lower than that for the Jurkat T cells (cell density = 6 × 104 cells). The results showed that the developed method exhibited good specificity for Jurkat T cells. 8087

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rapidly and specifically. Neither A549 cells nor 97L cells caused significant interference. The application potential had been demonstrated by comparing the analytical results for analysis of the spiked blood sample and blood sample obtained from healthy people. The proposed method combining magnetic immunoassay with elemental mass spectrometry is efficient for highly sensitive detection of tumor cells, independent of whether detectable elements are naturally incorporated or not. Also, it can be applied for the simultaneous detection of various low-abundance tumor cells based on multiple-element tags, demonstrating a great potential for bioassays and clinical diagnosis.

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ASSOCIATED CONTENT

S Supporting Information *

Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

Figure 6. Analytical performance of the proposed gold-tagged immunomagnetic separation coupled with ICP-MS detection for the analysis of JurkatT cells.

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AUTHOR INFORMATION

Corresponding Author

with an EIS cytosensor, 8000 cells with a quartz crystal microbalance cytosensor or 100 cells with electrochemical detection based on signal amplification. The developed method provided high sensitivity and selectivity, along with a very low detection limit for the detection of tumor cells. Real Sample Analysis. To evaluate the application potential of the developed method for real sample analysis, fresh human whole blood obtained from healthy people was spiked with 0, 250, 1000, 4000, and 16 000 Jurkat T cells after the removal of the supernate by centrifugation, respectively, and subjected to the immunoassay procedure and subsequent ICP-MS detection. With a deduction of the blank signal that was presented by the blood sample without the addition of Jurkat T cells, the obtained linear equation was y = 3.93x + 370, which is consistent with that linear equation (y = 3.95x + 326) obtained for the cells in PBS solution without blood matrix addition. This result revealed that the components coexisting in the blood sample matrix did not interfere with the proposed immunoassay procedure and Au NPs labeling ICP-MS detection. After the supernate was removed by centrifugation, the blood sample was mixed with a certain volume of PBS solution, and 5, 10, and 20 μL of the obtained sample solution were subjected to the proposed immunoassay procedure and Au NPs labeling ICP-MS detection. On the basis of the fact that there are approximately 5 × 109 /mL of leukocyte in human whole blood, and about 30% of leukocyte was lymphocytes, we assumed that there are 7500 T cells in 5 μL of blood sample, and linear equations of y = 0.132x + 1057, y = 0.367x + 943, y = 0.422x + 873, and y = 0.256x + 944 were obtained for four blood samples offered by healthy people. From the slope difference obtained by the linear equation for Jurkat T cells (3.95) and T cells (0.132) in blood, an obvious high-expression CD2 on Jurkat T cells was observed over T cells in lymphocytes of blood.

*Tel.: 0086-27-68752162. Fax: 0086-27-68754067. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work is financially supported by the National Basic Research Program of China (973 Program, 2013CB933900), the National Nature Science Foundation of China (nos. 21375097, 21175102, 21075095, 21205090), the Science Fund for Creative Research Groups of NSFC (nos. 20621502, 20921062), SRFDP (20110141110010), and the Fundamental Research Funds for the Central Universities (114009) funded by the Ministry of Education of China.

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CONCLUSION In summary, a novel immunoassay was developed for the detection of tumor cells at low concentrations. Magnetic nanobeads were used as capture probes and Au NPs were used as detection probes for sensitive, selective, time-saving, and precise detection of cancer cells. With this immunomagnetic separation, Jurkat T cells were isolated from complicated matrix 8088

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