Metallic Fe–Au Barcode Nanowires as a Simultaneous T Cell

Research Article Cite This: ACS Appl. Mater. Interfaces 2019, 11, 23901−23908

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Metallic Fe−Au Barcode Nanowires as a Simultaneous T Cell Capturing and Cytokine Sensing Platform for Immunoassay at the Single-Cell Level Yoo Sang Jeon,†,# Hyun Mu Shin,§,∥,⊥,# Yu Jin Kim,‡,# Da Yeon Nam,† Bum Chul Park,† Eunmin Yoo,† Hang-Rae Kim,*,§,∥,⊥ and Young Keun Kim*,†,‡

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Department of Materials Science and Engineering and ‡Research Center for Biomedical Nanocrystals, Korea University, Seoul 02841, Republic of Korea § Department of Anatomy and Cell Biology, ∥Department of Biomedical Sciences, and ⊥Medical Research Institute, Seoul National University, Seoul 03080, Republic of Korea S Supporting Information *

ABSTRACT: Barcode nanowires (BNWs) composed of multiple layered segments of different materials are attractive to bioengineering field due to their characteristics that allow the adjustment of physicochemical properties and conjugation with two or more types of biomolecules to facilitate multiple tasks. Here, we report a metallic Fe (iron)−Au (gold) BNW-based platform for capturing CD8 T cells and the interferon-γ (γ) they secrete, both of which play key roles in controlling infectious diseases such as tuberculosis, at the single-cell level. We also describe an efficient approach for conjugating distinct antibodies, which recognize different epitopes to appropriate materials. The platform achieved detection even with 4.45−35.6 μg mL−1 of BNWs. The T cell capture efficiency was close to 100% and the detection limit for interferon-γ was 460 pg mL−1. This work presents a potential guideline for the design of single-cell immunoassay platforms for eliminating diagnostic errors by unambiguously identifying disease-relevant immune mediators. KEYWORDS: Fe−Au barcode nanowire, single-cell immunoassay, T cell capture, interferon-γ monitoring, tuberculosis

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INTRODUCTION Barcode nanowire (BNW), also called multilayered or multisegment nanowire,1−3 is appealing to researchers for the application of bioengineered materials such as cell immobilization and imaging, drug delivery systems, and biosensors.4−9 Since their structural characteristics allow facile arrangement of intrinsic properties and multiple tasks by tuning shapes and selectively modifying heteromaterial surfaces10 unlike nanoparticle engineering, which can reduce the working efficiencies of biomolecules, conjugation with two or more types of BNWs was attempted. Instead of exploiting the benefits of multifunctionality of BNWs, many studies and reviews have focused only on improving higher sensitivity or utilizing for multipurposes such as multiplexed detection.11−13 Therefore, many substantial and meaningful issues for a convergent purpose have not been vigorously addressed. In vitro evaluation of immune substances from whole blood or isolated peripheral blood mononuclear cells (PBMCs) is a way to quantitatively measure immune cells or their products (i.e., immune substances), representing the state of immunity of the human body as a whole.14,15 For example, interferon-γ (IFN-γ) is one of the most well-known and crucial immune substances produced by various immune cells including T cells, © 2019 American Chemical Society

natural killer (NK) cells, NK T cells, monocytes, and macrophages. Since T cells secrete IFN-γ upon stimulation by antigens presented from various infectious agents, such as Mycobacterium tuberculosis (MTB), latent viruses, and malignant cells, it is useful to determine immunity against a specific pathogen by measuring their IFN-γ production. Among them, tuberculosis is a highly contagious disease caused by MTB and the leading infectious cause of death worldwide. Diagnosis is a critical issue since only 5−10% of screen test-positive patients will develop active tuberculosis in the future.16 Thus, the quantitative analysis of immune substances has been recognized as an appropriate indicator for evaluating infection or immunity.17−19 In this regard, enzyme-linked immunosorbent assays (ELISA) and enzyme-linked immunospot are widely used to detect immune substances such as cytokines.20,21 However, rather than detecting the immune substance secreted by anonymous immune cells that cannot be identified, the system we have developed can simultaneously detect both specific Received: April 14, 2019 Accepted: June 12, 2019 Published: June 12, 2019 23901

DOI: 10.1021/acsami.9b06535 ACS Appl. Mater. Interfaces 2019, 11, 23901−23908

Research Article

ACS Applied Materials & Interfaces immune cells and their associated secreted immune modulator.22 For instance, the currently available ELISA-based IFNγ release assay for MTB infection does not allow for phenotypic discrimination of different subsets of antigenspecific T cells. Therefore, this assay is unable to distinguish between different clinical forms of the MTB infection, such as active, latent, and recent infections.23 In addition, studies that solely improve sensitivity not only require a large amount of blood from patients but also exhibit a high intrinsic risk of diagnostic errors by inadvertently detecting substances naturally present in the blood.24 Thus, we have decided to address the problems mentioned above without sacrificing accuracy and high sensitivity, even with a small number of nanowires. Herein, we describe a new concept consisting of a BNWbased single-cell immunoassay platform for the simultaneous capture of specific immune cells and their associated secreted immune substances. Nanoparticles have been a leading trend in the in vitro diagnostic field,25−28 but the processes of proposed immunoassays directly monitor specific substances released from targeted cells, thus providing the possibility of a homogeneous assay allowing measurement by a simple mixing and reading procedure without the need to process samples using a separation step. In addition, although BNW flexibility techniques are well-known and an essential element in applications, it has been difficult to conjugate different types of antibodies (Abs) or biomolecules while maintaining their active state, resulting in limitations in applicability. The platform we propose also includes an efficient method for immobilizing different types of Abs on suitable BNW materials. The BNWs were synthesized via template-assisted pulsed electrodeposition, where Fe and Au layers were alternately stacked under two types of regulated current densities and duration times. Then, BNWs were bio-functionalized with two different types of Abs. This protocol may be applied to any targeted biomolecule and therefore is suitable for various diagnostic purposes. Since we focused on the diagnosis of active and latent infectious diseases, we utilized BNWs for capturing CD8 T cells and secreted IFN-γ to evaluate the immunity state of patients without the risk of diagnostic errors, resulting in increased specificity. Our characterization aimed to assess the microstructural, magnetic, and optical properties of the BNWs and verify their suitability for the designed purposes. We also evaluated their competence as an advanced immunoassay platform through various fluorescence properties.

Figure 1. (a) Representative scheme of the chemical method employed for the surface treatment and immunoassay process. (1) Modification of the Fe segment with 11-aminoundecanoic acid to activate the initial functional group, (2) anti-human IFN-γ Abs pretreated with 2-iminothiolane (C4H7NS, Traut’s reagent) are mixed and shaken with the surface-modified BNW solution, (3) thiolated anti-human IFN-γ Abs are selectively conjugated to the Au segment, (4) selective conjugation of anti-human CD8 Abs amino-terminus to Fe segments, and (5) the platform for simultaneous capturing and detection of both T cells and secreted cytokines. (b) Magnified image of simultaneous detection.

First, modification of the Fe layer was performed with 11aminoundecanoic acid (NH2C10H20COOH) to activate the initial functional group (step 1). From the surface modification results of Fourier-transform infrared spectroscopy and ζpotential, we judged that the amine-terminus of the chemical was preferentially conjugated to the Fe surface in the phosphate-buffered saline (PBS) environment (see the Supporting Information for more details, Figures S1 and S2). This outcome could also be deduced by the formation of the native oxide layer on the Fe layer, as mentioned above. Second, the anti-human IFN-γ Ab pretreated with 2iminothiolane (C4H7NS, Traut’s reagent) was mixed and shaken with the surface-modified BNW solution. 2-Iminothiolane is a cyclic shaped chemical, which is highly water soluble and reacts with the amino-terminus of Abs by opening the ring and forming thiol residues at the end (step 2). This protocol has merits to form thiol groups minimizing the impediment of activity by retaining the divalent nature of biomolecules. Owing to the strong affinity between thiol and Au, the thiolated anti-human IFN-γ Abs were easily and selectively conjugated to the Au segment (step 3). Due to the large molecular size of Abs (about 150 kDa), which are composed of heavy and light chains, with weights of ∼50 and ∼25 kDa, respectively,37 the primarily attached anti-human IFN-γ Abs can generate steric hindrance as a result of the occupation of the reaction sites on the surface. The steric hindrance effect may be both beneficial and detrimental, depending on the purpose, e.g., antifouling38,39 and ELISA.40 In our case, however, the steric hindrance effect could be exploited as an approach for selective conjugation of different types of Abs. Finally, the amino-terminus of anti-human CD8 Abs can be selectively conjugated to Fe layers, which may be activated by a carboxyl group via two distinct strategies, i.e., the 1-ethyl-3-(3dimethylaminopropyl)carbodiimide hydrochloride/sulfo-N-hydroxysulfosuccinimide (EDC/NHS) reaction and pre-engaged anti-human IFN-γ Abs on the Au layers (step 4). Essentially, this platform operates using two kinds of fluorescence, and the properties of BNWs are endowed with Brilliant Violet 421 (BV421) conjugated to anti-human CD8 Abs and Alexa Fluor 488 (AF488) conjugated to secondary anti-human IFN-γ Abs

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RESULTS AND DISCUSSION Biofunctionalization and Platform Design. Several attempts to selectively functionalize a multisegment surface have exploited the high reaction affinities between the functional groups and their specific materials.5,6,29−31 For example, thiol and polyhistidine sequences can be easily and efficiently attached to gold (Au) and divalent metals such as nickel (Ni 2+ ), respectively. Moreover, the amine and isocyanide groups are conceived to stick to native oxide layers and platinum (Pt),32 whereas silane, phosphate, and catechol groups are commonly utilized to modify metal oxides.33−36 Despite this well-known flexibility, it is also important to adjust chemical reactions for conjugating different types of Abs or biomolecules while maintaining their active state. The overall process of biofunctionalization of Fe−Au BNWs and the design of the BNW-based platform are depicted in Figure 1. 23902

DOI: 10.1021/acsami.9b06535 ACS Appl. Mater. Interfaces 2019, 11, 23901−23908

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Figure 2. (a) Morphology of Fe−Au BNWs embedded in the AAO template, (b) single BNW with EDS line for comparing the relative contents of Fe and Au, and (c) size distribution of each segment. (d) Elemental mapping data of (e) K series of Fe and (f) L series of Au. (g) Magnetic hysteresis loop with an inset presenting a magnified range of −700 to 700 Oe. (h) XRD pattern corresponding to PDF no. 04-0748 (Au) and PDF no. 01-087-0722 (Fe). (i) HR-TEM image analyses and FFT patterns (insets) revealing lattice directions. (j, k) SAED patterns for each layer of Fe and Au, respectively.

nanomaterials.42 In this study, we minimized this problem by designing the aspect ratio of the magnetic layer as a disk shape.43 Thus, the evaluated remanence was below 5 emu g−1, which was low enough to circumvent this hurdle. The EDS line profile clearly revealed a sharp interface between Fe and Au owing to their large differences in standard reduction potential, structural features, and the control of precursor concentrations. The sharp boundary was a core point suitable for conjugation of different types of Abs to the respective material. The K series of Fe and the L series of Au were also mapped to identify intuitively where they are located and to display the definite boundaries of each layer (Figure 2d−f). The peaks of the X-ray diffraction (XRD) pattern were indicative of two reference patterns, PDF no. 04-0784 for Au and PDF no. 01-087-0722 for Fe (Figure 2h). Au and Fe were typical face-centered-cubic (fcc) and body-centered-cubic (bcc) structures, respectively. The crystallite size was determined by the (111) peak of Au, which was estimated to be 13.7 nm by Scherrer’s equation.44 In addition, the selective area electron diffraction (SAED) patterns and high-resolution TEM (HR-TEM) images around the Fe−Au interface suggested that both layers had polycrystalline states when they were viewed in sequential ring shapes (Figure 2i−k). Specifically, Fe was found to have relatively low crystallinity compared to Au. This finding seems to be due to the rapid growth rate of Fe. In contrast, the HR-TEM image from the Fe to the Au layer with their corresponding fast Fourier-transform (FFT) patterns clearly showed how fcc Au could be deposited on the bcc Fe. At the interface, Fe showed wide grains with a direction of [110], and its d-spacing value

(step 5). The procedure for a single-cell-based immunoassay is described by concurrently capturing CD8 T cells and monitoring the IFN-γ they secrete (Figure 1b). Because it preferentially detects the secreted immune substances from cells that are very closely immobilized to BNWs, it is expected to reduce not only diagnostic errors but also provide good detection performance with only a small amount of BNWs. Characterization of Barcode Nanowires. To obtain a comprehensive characterization, we analyzed the morphological, microstructural, and magnetic properties of BNWs using electron microscopes, diffraction patterns, and an M−H hysteresis loop (Figure 2). A bundle of BNWs embedded in the anodized aluminum oxide (AAO) (Figure 2a), and a line profile and atomic mapping of a single BNW by an energy dispersive X-ray spectrometer (EDS) are displayed (Figure 2b, d−f). BNWs were 200 nm in diameter, and 148 nm long Fe and 184 nm long Au segments with high uniformity were alternately deposited according to the size of the AAO pores (Figure 2c). The length of each segment could be easily adjusted through the elapsed time of electric field and each growth rate was calculated as 13.9 and 0.864 nm s−1 for Fe and Au, respectively (Figure S3, Supporting Information). We also measured the magnetic properties of BNWs in the powdered state (Figure 2g), considering the real environmental conditions for capturing biomolecules. An external magnetic field was applied from −19 to 19 kOe. Magnetic remanence and coercivity values are 4.71 emu g−1 and 124 Oe, respectively, as shown in the inset. Because most of the magnetic nanomaterials should present issues with selfaggregation according to their remanence,41 superparamagnetic behavior has been considered by decreasing the size of 23903

DOI: 10.1021/acsami.9b06535 ACS Appl. Mater. Interfaces 2019, 11, 23901−23908

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ACS Applied Materials & Interfaces was measured as 0.2020 nm, which was nearly identical to the reference value of 0.2022 nm. By analyzing another HR-TEM image in the thin surface area (not shown), we found that an Au crystallite size was in the range of 11−17 nm which is qualitatively in good agreement with the value found by XRD. The optical properties of BNWs with two distinct Abs attached by different dyes, i.e., BV421 and phycoerythrin (PE), were analyzed (Figure 3). The anti-human IFN-γ Abs attached

Figure 3. (a) Fluorescence properties of BNWs measured by photoluminescence. (b) Confocal microscopic analysis of selective Ab conjugation with the BV421 dye (blue), phase contrast, PE dye (yellow), and merged images. Original magnification, ×100. Due to the resolution limit of the confocal microscope, the length of each segment appears approximately 550 nm in clear images.

to the Au layers do not require specific optical properties for the IFN-γ detection platform, but the fluorescence dye is provided to ensure their conjugation. When BNWs were measured by photoluminescence (PL) and confocal microscopy (CM), the excitation wavelength was set at 405 nm for BV421 (blue) and 490 or 540 nm for PE (yellow), respectively. As a result, PL emission peaks were observed at 424 nm for BV421 and at 450 and 574 nm for PE, indicating successful conjugation of Abs on the surface of the BNWs (Figure 3a). In addition to the detection range obtained in PL spectra, CM clearly confirmed that the two different Abs were conjugated to each metallic layer as we designed (Figure 3b). It should be noted that we intentionally fabricated Fe−Au BNWs consisting of 558 nm thick segments for both Fe and Au with seven repetitions for the purpose of better optical imaging. Capturing Human CD8 T Cells. Furthermore, we investigated whether the Abs can perform their original activities after undergoing consecutive chemical reactions. Thus, to quantitatively analyze the ability of Ab-conjugated BNWs to capture CD8 T cells, isolated CD8 T cells from peripheral blood mononuclear cells (PBMCs) were incubated with BNW-immobilized BV421−anti-human CD8 Abs (i.e., CD8-BNWs, blue) at the indicated amounts for 1 h and counterstained with allophycocyanin (APC)−anti-human CD8 Abs (red). CM images showed a dose-dependent increase in attached CD8-BNWs (pink), but not in the control represented by CD8 T cells alone (Figure 4a).

Figure 4. (a) PBMCs were incubated with anti-human CD8 Abimmobilized BNW (blue) and counterstained with APC−anti-human CD8 Abs (red, a different clone) with capturing Ab-immobilized BNWs. Images were acquired using confocal microscopy. Original magnification, ×100. Scale bar = 10 μm. (b) Schematic description of the process where (c) PBMCs were incubated with anti-human CD8 (top panel) or/and anti-human IFN-γ Ab (bottom panel)immobilized BNW. Their capturing capability was measured by flow cytometry. The values on the axis indicate the mean fluorescence intensity.

The data indicated that the anti-human CD8 Abs immobilized on BNW were functional and bound to CD8 T cells after multiple conjugations. The capturing efficiency of BNW-immobilized with anti-human CD8 Abs was measured by flow cytometry (Figure 4b), demonstrating that both single and double Ab-immobilized BNWs were capable of capturing CD8 T cells in a dose-dependent manner (Figure 4c). For simplicity, the units for measuring the concentration of the BNW solution were arbitrarily defined (“arbitrarily defined unit”, ADU) and the value of 1 ADU was set equal to 0.89 μg mL−1 of BNW. Monitoring Immune Substances. Next, IFN-γ detection was carried out through a sandwich method using anti-human IFN-γ capturing Abs on the BNWs and AF488−conjugated 23904

DOI: 10.1021/acsami.9b06535 ACS Appl. Mater. Interfaces 2019, 11, 23901−23908

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concentrations over 3 × 105 pg mL−1 recombinant IFN-γ and the threshold range was confirmed to be less than 650 relative fluorescence units in all samples. Although 5 and 20 ADU samples exhibited similar limitations in IFN-γ detection, lowdensity samples exhibited relatively high standard errors and low detection range. In conclusion, the optimal limit of detection was estimated at 460 pg mL−1 with 40 ADU samples, which is suitable for the commercial application of immunoassay systems. To measure the capture efficiency of immobilized BNW with both anti-human CD8 and anti-human IFN-γ Abs, IFN-γproducing effector memory CD8 T cells and naive CD8 T cells that did not produce IFN-γ were isolated from PBMCs. Mixed pools of the two populations can provide accurate estimates as to which BNWs are capable of simultaneously capturing both CD8 T cells and IFN-γ secreted in the same cells. As a result, the increased proportion of IFN-γ-producing effector memory CD8 T cells in the mixed pools well reflected the BNWcaptured amount of secreted IFN-γ (Figure 5c). Therefore, Ab-immobilized BNWs efficiently and simultaneously captured both cells and their immune substances at the single-cell level, suggesting that the BNW platform could be a useful tool for various immunodiagnostic purposes.

anti-IFN-γ detecting Abs. Although the anti-human IFN-γ Abs were stably immobilized on the Au surface, as shown in Figure S4 in the Supporting Information, it was necessary to verify that the original activities of the thiolated anti-human IFN-γ Abs had been maintained. Therefore, 25 ADU samples of BNW were incubated in the presence or absence of human recombinant IFN-γ. Green fluorescence emission by the AF488−human IFN-γ detecting Abs was specifically observed in the presence of IFN-γ and not with the PBS control (Figure 5a).

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CONCLUSIONS In this study, we optimize the conditions for the simultaneous detection of a single target cell and its secreted immune substances by employing Fe−Au BNWs. The Fe−Au BNWs were synthesized via electrodeposition, and the boundaries between each segment were sharp. Next, the magnetic layer was adjusted to a disk shape to minimize the frequent aggregation phenomenon caused by residual magnetization. Subsequently, a chemical method capable of selectively conjugating different kinds of Abs to BNWs was devised, and the optical properties were successfully confirmed through PL and CM. The suitability of the prepared BNWs for immunoassay applications was analyzed qualitatively and quantitatively by capturing both CD8 T cells and IFN-γ secreted from them. Flow cytometric analysis indicated that the BNW-based platform can detect most immune cells, even at low concentrations. The tested concentrations, ranging from 5 to 100 ADU (1 ADU = 0.89 μg mL−1) are extremely low compared to the amounts used in previously reported nanomaterial-based immunoassays. Furthermore, the limit of detection for IFN-γ measured at an optimized BNW density of 40 ADU was 460 pg mL−1, which is sufficient for commercial applications. Finally, we succeeded in detecting cytokines secreted from the captured single cell, indicating that the BNW-based platform was an advanced immunoassay. Although this study was designed for the capturing of CD8 T cells and IFN-γ, we expect that the proposed immunoassay platform is generally applicable to various infectious and malignant diseases, owing to the flexibility in biofunctionalization according to the target biomolecules.

Figure 5. Human recombinant IFN-γ was added in solution to antihuman IFN-γ Ab-immobilized BNWs, and the BNW-bound IFN-γ was detected by AF488−anti-human IFN-γ Ab. (a) The fluorescence image (green) was acquired using confocal microscopy and (b) the relative fluorescence units were analyzed using a multidetection microplate reader measuring the sensitivity of the BNW-based platform; error bars represent the standard errors, and threshold points were acquired at a 20 ADU-concentration. Original ̈ and magnification, ×60. Scale bar = 10 μm. (c) Purified naive effector memory CD8 T cells were mixed with the proportions and then stimulated with PMA and ionomycin. Total 20 000 cells including effector memory CD8 T cells as the indicated proportions were incubated with anti-human CD8 and anti-human IFN-γ Abimmobilized BNW and analyzed by flow cytometry. The values on the axis indicate the mean fluorescence intensity.

Furthermore, the limit of IFN-γ detection was determined by testing various concentrations of IFN-γ. The excitation wavelength was set at 480 nm for all samples, and the relative intensity of fluorescence was estimated. Since the amount of the BNWs can be an important factor for the sensitivity of IFN-γ detection, BNW densities of 5, 20, and 40 ADU (Figure 5b) were employed, corresponding to concentrations of approximately 20−200 times lower compared to the previously reported nanomaterial-based immunoassay platforms.40,45,46 The relative intensity of fluorescence logarithmically increased with the concentration of IFN-γ. As to the effective detection range of the platform, fluorescence saturation was reached at

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EXPERIMENTAL SECTION

Preparation of Barcode Nanowires (BNWs). The Fe−Au barcode nanowires were synthesized by a pulsed electrodeposition method using a nanoporous anodized aluminum oxide (AAO) membrane with a nominal pore size of 200 nm. On the back side of commercial AAO membranes (Shenzhen TopMembranes Inc.), 300 nm Ag was deposited as a conductive layer by an e-beam evaporator. 23905

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ACS Applied Materials & Interfaces The iron sulfate hepta-hydroxide (FeSO4·7H2O, 0.16 M), potassium gold cyanide (KAu(CN)2, 0.01 M), and boric acid (H3BO3, 0.80 M) as a buffer solution were mixed in a precursor solution. This solution was added to the electrodeposition system and a Pt plate was used as a counter electrode. The Fe and Au segments were alternately deposited in a single bath by applying desired pulse current densities with regulated pulse durations to control the respective segmental lengths through the source meter (2612B; Keithley). The current densities are 10 and 1 mA cm−2, respectively. After electrodeposition, the Ag layer was removed by a Ag etchant (Transene Company) and then, the AAO membrane was removed using sodium hydroxide and rinsed with deionized water. Finally, Fe−Au barcode nanowires were dispersed in phosphate-buffered saline (PBS, pH 7.4; Thermo Fisher Scientific). The morphology of the Fe−Au BNW was analyzed by an ultra-high-resolution field emission scanning electron microscope (UHR-FESEM, SU-70; Hitachi) and a transmission electron microscope (TEM, JEM-2100F; JEOL). The microstructure was analyzed by a TEM-EDS (X-MAXn; HORIBA) and XRD (D/MAX2500V/PC; Rigaku). Moreover, vibrating sample magnetometry (EV9; Microsense) was measured. Antibody Immobilization. The EDC/NHS reactions were used to immobilize the Ab on the Fe layer. For the surface modification of the Fe layer, 1 mL of Fe−Au BNWs dispersed in PBS and 1 mL of 11aminoundecanoid acid (6 mM) were allowed to react for 12 h at room temperature (RT). Traut’s reagent (Thermo Fisher Scientific) was dissolved in PBS at a concentration of 2 mg mL−1. For the modification of mouse anti-human IFN-γ antibody (Ab) (IgG1, clone B-B1; Thermo Fisher Scientific) and PE-conjugated mouse antihuman IFN-γ Ab (IgG1, clone 4S.B3, BD Biosciences), 50 μL of antihuman IFN-γ Ab and 50 μL of Traut’s reagent stock solution were left to react for 2 h at RT. The thiolated anti-human IFN-γ Ab was purified from unreacted Traut’s reagent using columns (Zeba spin desalting columns, 7 K MWCO, 0.5 mL; Thermo Fisher Scientific). The purified anti-human IFN-γ Ab was added to the solution, followed by the dispersion of 0.5 mL of Fe−Au BNW and 0.5 mL of ethylenediaminetetraacetic acid (5 mM EDTA; Sigma-Aldrich) (all steps proceeded in the PBS buffer solution). The addition of EDTA to this solution helps to prevent oxidation of the sulfhydryl groups and the resulting disulfide formation. The latter reaction proceeded for 2 h at RT, after which the thiolated Ab was conjugated on the surface of the Au segment. The unconjugated Abs were washed several times by centrifugation and the Fe−Au BNWs were dispersed in 0.5 mL PBS. To immobilize the Brilliant Violet 421 (BV421)conjugated mouse anti-human CD8 Ab (IgG1, clone RPA-T8; BD Biosciences) on the Fe layer, 0.4 mL of N-ethyl-N′-(3(dimethylamino)propyl)carbodiimide (EDC; Sigma-Aldrich), 0.5 mL of N-hydroxysuccinimide (NHS; Sigma-Aldrich), and 30 μL of anti-human CD8 Ab were incubated with 0.5 mL of Fe−Au BNWs for 2 h at RT. Thereafter, the fluorescence properties of BNWs were assessed by PL (RF5301PC, Shimadzu) and CM (Leica Microsystems, TCS SP8). Human Subjects. This work was approved by the Institutional Review Boards of the Seoul National University Hospital (# 0905014-280). Peripheral blood samples were obtained from healthy volunteers. Individuals who were taking immunosuppressive drugs or had a disease potentially affecting the immune system, including autoimmunity, infections, and malignancies, were excluded. Written informed consent was obtained from all subjects in accordance with the Declaration of Helsinki. Capturing CD8 T Cells by BNWs. Human PBMCs were purified from heparinized peripheral blood using a Ficoll−Histopaque gradient (1.077 g mL−1; GE Healthcare Life Sciences). To measure the capture efficiency of CD8 T cells by the BNW-immobilized Abs, PBMCs (2 × 106) were incubated with BNW-immobilized antihuman CD8 Abs at the indicated concentrations for 1 h at RT, protected from light. The cells were counterstained with fluorescent dye-labeled mouse anti-human CD8 (IgG1, clone OKT8, e Biosciences) and mouse anti-human CD3 (IgG1, clone UCHT1, BD Biosciences) Abs, followed by confocal microscopic analysis (A1; Nikon) or flow cytometric analysis on a BD LSRII (BD Biosciences).

Measurement of IFN-γ by BNWs. To determine the sensitivity of IFN-γ detection, eight different concentrations of human recombinant IFN-γ were added in solution to BNWs, which thiolated anti-human IFN-γ Ab-immobilized on the Au layer. This reaction proceeded for 12 h at RT. The remaining unbound IFN-γ was washed three times with PBS and then the AF488−conjugated mouse antihuman IFN-γ Ab (IgG1, clone B27; BD Biosciences) was added. After incubation, the samples were rinsed several times, dispersed in 200 μL PBS, and added to wells of a 96-well microplate (Thermo Fisher Scientific) to measure the relative fluorescence using a multidetection microplate reader (SENSE; HIDEX). Simultaneous Detection of CD8 T Cells and IFN-γ by BNWs. To measure the capture efficiency of IFN-γ secreted from CD8 T cells, IFN-γ-producing CD8 T cells (CCR7−, effector memory) and nonproducing CD8 T cells (CCR7+CD45RA+, naive) were sorted with a BD FACSAria cell sorter (BD Biosciences) and the two populations were mixed with 20 000 total cells at the indicated ratios. The mixed cells were stimulated for 4 h with phorbol 12-myristate 13acetate (PMA; 100 ng mL−1) and ionomycin (1 μg mL−1) (both reagents from Sigma-Aldrich) and incubated with BNW-immobilized anti-human CD8 and anti-human IFN-γ Abs for 1 h, followed by flow cytometric analysis.

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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/acsami.9b06535. Surface modification analysis, growth control of each segment, internal strains from Fe layers, and fluorescence analysis (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (H.-R.K.). *E-mail: [email protected] (Y.K.K.). ORCID

Hang-Rae Kim: 0000-0002-3983-6193 Young Keun Kim: 0000-0002-0868-4625 Author Contributions #

Y.S.J., H.M.S., and Y.J.K. contributed equally to this work.

Notes

The authors declare no competing financial interest. Safety statement: no unexpected or unusually high safety hazards were encountered during the course of our studies.

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ACKNOWLEDGMENTS This work was supported in part by the National Research Foundation of Korea (2015R1A2A1A15053002 and 2014M3A7B4052192), funded by the Ministry of Science, ICT and Future Planning. H.-R.K. acknowledges the financial support provided by the Creative-Pioneering Researchers Program through Seoul National University. Y.S.J. acknowledges the financial support provided by the Global Ph.D. Fellowship Program through the National Research Foundation of Korea (NRF-2015H1A2A1034498), funded by the Ministry of Education.

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REFERENCES

(1) Park, B. C.; Kim, Y. K. Synthesis, Microstructure, and Physical Properties of Metallic Barcode Nanowires. Met. Mater. Int. 2017, 23, 413−425. (2) Lee, J. H.; Wu, J. H.; Liu, H. L.; Cho, J. U.; Cho, M. K.; An, B. H.; Min, J. H.; Noh, S. J.; Kim, Y. K. Iron−gold Barcode Nanowires. Angew. Chem., Int. Ed. 2007, 46, 3663−3667.

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DOI: 10.1021/acsami.9b06535 ACS Appl. Mater. Interfaces 2019, 11, 23901−23908

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ACS Applied Materials & Interfaces

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DOI: 10.1021/acsami.9b06535 ACS Appl. Mater. Interfaces 2019, 11, 23901−23908

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DOI: 10.1021/acsami.9b06535 ACS Appl. Mater. Interfaces 2019, 11, 23901−23908