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Sensitive Detection of Single-cell Secreted H2O2 by Integrating a Microfluidic Droplet Sensor and Au Nanoclusters Rui Shen, Peipei Liu, Yiqiu Zhang, Zhao Yu, Xuyue Chen, Lu Zhou, Baoqing Nie, Anna #aczek, Jian Chen, and Jian Liu Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b04798 • Publication Date (Web): 07 Mar 2018 Downloaded from http://pubs.acs.org on March 7, 2018
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Analytical Chemistry
Sensitive Detection of Single-cell Secreted H2O2 by Integrating a Microfluidic Droplet Sensor and Au Nanoclusters Rui Shen,† Peipei Liu,† Yiqiu Zhang,† Zhao Yu,† Xuyue Chen,† Lu Zhou,† Baoqing Nie,‡ Anna Żaczek,§ Jian Chen,*† Jian Liu*† † Institute of Functional Nano and Soft Materials (FUNSOM), Jiangsu Key Laboratory for Carbon-Based Functional Materials and Devices, Soochow University, 199 Ren Ai Road, Suzhou Industrial Park, Jiangsu Province 215123, China. ‡ School of Electronic and Information Engineering, Soochow University, Jiangsu Province 215123, China. § Medical Biotechnology Department, Intercollegiate Faculty of Biotechnology, University of Gdańsk and Medical University of Gdańsk, Dębinki 1, 80-211, Gdańsk, Poland.
Fax: 0512 65880820. E-mail:
[email protected];
[email protected]. ABSTRACT: As an important signaling molecule, hydrogen peroxide (H2O2) secreted externally by the cells influences cell migration, immunity generation, and cellular communications. Herein, we have developed a microfluidic approach with droplets in combination with Au nanoclusters for the sensitive detection of H2O2 secreted by a single cell. Isolated in the ultrasmall volume (4.2 nL) of a microdroplet, single-cell secreted H2O2 can initiate dramatic fluorescence changes of horseradish peroxidase-Au nanoclusters. We have demonstrated an ultrahigh sensitivity (200-400 attomole H2O2 directly measured from a single cell) with good specificity. It offers a useful research tool to study the cell-to-cell differences of H2O2 secretion at the single cell level.
Reactive Oxygen Species (ROS), including hydrogen peroxide (H2O2), hyperoxide anion radical (O2-), hydroxyl radical (·OH), and hypochlorite (OCl-), are critical signaling molecules. Depending on the concentration, ROS can promote cell proliferation, stem cell self-renewal and tumor metastasis.1-3 They can also induce cell apoptosis, cell necrocytosis and DNA damage.4-5 The breakdown of cellular ROS equilibrium will lead to significantly disordered metabolisms.5 Hydrogen peroxide (H2O2) is a particularly important representative of ROS, with well-established roles in DNA strand breaks,6-7 cell adhesion,8 endothelial tissue permeability,9 the release of cytokines,10 and many oxidative stress response associated diseases.11-12 Hydrogen peroxide is featured with relatively lower redox potential or molecular polarity, longer half time (halflife 10-3 s), and ability to diffuse farther away, in comparison with other radical-based ROS members.13 Its lifetime allows a diffusion distance of 1.5 mm, approximately 150 times longer than the radius of a mammalian cell.13-14 Once diffused out of the cell membrane, extracellular H2O2 influences cell migration,8, 15 immunity generation,16 or cellular communications.1718 The conventional methods to measure cellular H2O2 content include colorimetric,19 electrochemical method,14, 20 spectrophotometric method,21 chemiluminescence,22 fluorescence,23-24 and chromatography methods.25 These methods rely on the consumption of large populations of cells in the experiments, thus unable to reveal cell-to-cell heterogeneity in H2O2 secretion. As important advances, label-free methods have been developed with scanning electrochemical microscopy (SECM), providing quantitative extracellular profiles of ROS or H2O2 to monitor the physiological activities of a single cancer cell.26-28
Recently, gold nanoclusters (AuNCs) emerge for attractive applications in biosensing, molecule imaging, and nanomedicine. They are composed of a collection of gold atoms (ranging from several to hundreds) to form a stable cluster protected by ligand molecules, with advantages such as ultra-small sizes, highly fluorescent properties, and good biocompatibility.29,30 Varieties of fluorescent AuNCs have been developed with clever selections of different ligands.31-37 AuNCs have been proposed for dual-modal imaging and mitochondria-targeted radiosensitization of cancer cells.33, 38 Lu group has synthesized Lys-AuNCs for sensitive detection of Hg2+ with the detection limit of 10 nM.39 Choi et al. have discovered that NO2can react with human serum albumin in the presence of hydrochloric acid, resulting in the fluorescence quenching of HSAAuNCs.40 Zhu et al. have designed a fascinating fluorescence “off-on-off” detection method for the glutathione levels in blood samples using BSA-AuNCs.41-42 Interestingly, a horseradish peroxidase (HRP) templated synthesis of AuNCs (HRPAuNCs) has been reported through a biomineralization process, which allows for sensitive fluorescent detection of H2O2 in the test tube format.43 However, it remains a great challenge to detect single-cell secreted H2O2 quantitatively, especially in an enclosed, well-defined tiny volume without cross contamination. Herein we report a microfluidic approach to quantify the extracellular H2O2 secreted by different types of cells at the single cell level. Microfluidic droplets have become attractive for miniaturization of genetic or biochemical assays in a highthroughput fashion.44-47 They offer a platform to generate signals dependent on the analyte concentration within ultra-small, volume well-defined liquid droplets (from picoliters to nanoliters). Previously, we developed a microdroplet platform to measure single-cell secreted matrix metalloproteinase
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(MMP9).48 In this work (Scheme 1), we integrate fluorescent HRP-AuNCs with our microdroplet-based sensing platform to investigate single-cell secreted H2O2. Compared with protein enzymes such as MMP9, H2O2 is lack of the ability of enzymatic signal amplification. It is much easier to be annihilated by the reducing agents in the solution, and diffuses much faster. These characters make the detection of H2O2 more challenging. We demonstrate that the microdroplet-based method can achieve the detection limit nearly three orders of magnitude better than the traditional assays. Our results reveal that higher content of H2O2 may be produced under stimulation by the fast-growing cancer cells, in comparison with normal cell lines. Our approach provides a powerful research tool to study the cell-to-cell difference through measurements of extracellular H2O2 secreted by a single cell. Scheme 1. Schematic illustration of detecting hydrogen peroxide in the single cell-encapsulated droplets in combination with HRP-AuNCs.
EXPERIMENTAL SECTION Sensitive detection of H2O2 in the single-cell microdroplets. After trypsinization, the cells were washed by PBS (pH 7.4) twice, counted and diluted to three different concentrations (6×104, 6×105 and 6×106 cells mL-1) in cell culture medium (500 µL) without serum and phenol red. The cell suspensions were placed in an ice bath (4oC) to slow down cell metabolism, preventing H2O2 secretion before tests. After generation, the microdroplets were collected by a confocal dish. The fluorescence changes of AuNCs induced by cell-secreted H2O2 in the microdroplets were monitored at different time points through an inverted fluorescence micro-scope (Olympus IX71) coupled with a multispectral imaging CCD camera (Nuance, CRI) (Ex: 542/20 nm, Em: 620/52 nm). In the calibration experiments for cell counting after encapsulation, the cells were pre-stained by Hoechst dye for 15 minutes in the dark. The cell-encapsulated microdroplets were visualized by a confocal laser scanning microscope (CLSM, Leica TCS SP5) with z-axis scanning. The counting experiments verified that single-cell microdroplets became predominant among the cell-encapsulated ones after the cell loading density was reduced below 6×105 cells mL-1. Detailed experimental procedures are available in the Supporting Information. RESULTS AND DISCUSSION Synthesis and characterization of HRP-AuNCs. HRPAuNCs were synthesized by assembling gold nanoclusters with HRP molecules through a one-step biomineralization
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process, which yielded Au25 (4.7 nm) in the aqueous solution (Figure S1A, B). The synthesized HRP-AuNCs maintained the fluorescent properties (Figure S1C, Ex: 511 nm, Em: 650 nm) with excellent photostability in the absence of H2O2 (Figure S1D). Interestingly, the original absorption peak of HRP at 403 nm disappeared after conjugation with AuNCs (Figure 1A). In the presence of H2O2, the Au-S bonds between HRP residues and the gold atoms of AuNCs could take place of redox reactions, which would induce deterioration of AuNCs structure and subsequent aggregation. [2RS-Au → RS-SR + 2Au (low H2O2 concentration); RS-Au → RSO3H + HO-Au (high H2O2 concentration)]. Therefore, the fluorescence of AuNCs could be quenched quickly.43, 49-50 Our experiments verified that the cluster size was increased by 3 folds after mixing with 10 mM H2O2 solution (Figure S2). The fluorescent intensity of HRP-AuNCs decreased in response to H2O2 incubation in a dose dependent manner (Figure 1B-D). In Figure S3, we measured the enzyme activity of HRP-AuNCs in catalysis of H2O2 in comparison with the pure enzyme of HRP (10 mg mL-1). It suggested that the peroxidase activity was increased after the incorporation of HRP and AuNCs.
Figure 1. Responses of HRP-AuNCs to the H2O2 solution. (A) UV-vis absorption spectra of HRP (black), HRP-AuNCs in the absence (red) and presence of 100 mM H2O2 aqueous solution (blue). (B) Fuorescent changes of HRP-AuNCs by H2O2 in different concentrations over time using a FluoroMax 4 spectrometer. (C) Fluorescence spectra of bare HRP (10 mg/mL) (black), HRPAuNCs in the absence (red) and presence (blue) of H2O2 (100 mM). (D) Fluorescence spectra of HRP-AuNCs in the presence of H2O2 in different final concentrations, including 0, 100 nM, 200 nM, 550 nM, 3 mM, 15 mM, 60 mM, 300 mM, 5 mM and 10 mM.
Generation of microdroplets with tunable sizes. Different types of microdroplets, including water-in-oil and oil-in-water emulsions, can be produced in our microfluidic device by switching the dispersed and continuous phase. The microdroplets of water-in-oil emulsion were selected for the experiments of our assay. As shown in Figure S4A-D, the continuous phase was mineral oil with ABIL EM 90 (2 wt. %), while the dispersed phase was an aqueous solution of HRP-AuNCs. Microdroplets of HRP-AuNCs-in-oil were generated when the two immiscible phases met each other and changed the interface profile in the narrow gap region by the capillary orifices. The flow rate ratios of the two liquid phases were adjusted to tune the size of microdroplets. In Figure S4A-E, a series of flow rates of Qc including 5, 15, 30, 50 µL min-1 and the flow
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Analytical Chemistry rate of Qd: 5 µL min-1 were tested to generate the microdroplets in different sizes ranging from 85 to 380 µm. In each flow condition, the microdroplets were very uniform in size. The throughput of our microfluidic device was approximately 2.6×103 droplets per min (Qc: 15 µL min-1, Qd: 5 µL min-1). Single cell encapsulation in the droplets. Different cell concentrations (6×104, 6×105, 6×106 cells mL-1) were titrated to investigate cell encapsulation profiles in individual droplets. The cells were pre-stained by the Hoechst dye for enhanced imaging contrast. The collected droplet samples were analyzed by a Confocal Laser Scanning Microscopy (CLSM) coupled with a z-axis scanning. As shown by the images and histogram plots (Figure S5A-C), when the cell loading concentration was 6×104 cells mL-1, a portion of microdroplets encapsulated a single cell, but nearly 90% of microdroplets were empty. With the increase of the cell loading concentration to 6×105 cells mL-1, the microdroplets encapsulating 1-2 cells became predominant. However, when the cell loading concentration was further increased to 6×106 cells mL-1, there was a shift of the predominant microdroplets which encapsulated 4-7 cells each. Therefore, the primary cell loading concentration was chosen to be 6×105 cells mL-1 for single-cell experiments, by balancing the factors to promote the possibility of encapsulating one single cell in each droplet and to decrease the proportion of empty droplets. Our experiments allowed for generation of single-cell encapsulated droplets with a percentage of 33%, while the empty droplet percentage was 17% approximately. The frequency of droplet generation was 43 Hz. These parameters in performance evaluation were comparable to the representative reports in the literature.45, 51-53 Usually, the percentage of empty droplets would climb up in order to reduce the risks of generating droplets containing more than one cell. This issue could be alleviated by optimization of the microfluidic setting-up, such as fine-tuning the feature size of capillary nozzles, precise control of cell loading density, and careful pairing the flow rates of the liquid phases. Highly sensitive detection of H2O2 released from a single living cell. Although extracellular H2O2 molecules released from a single living cell were few, our approach of using ultrasmall (4.2 nL) microdroplets enabled highly sensitive detection by allowing the secreted molecules to rapidly reach the detectable concentration. Four different cell lines with various endogenous ROS levels were tested for the assay feasibility, including HUVEC, MCF7, U937, and Mut6 cells (Figure 2 and S7). The assay time of using microdroplets was determined on the basis of our cell experiments, which suggested that the fluorescence change became negligible after 90 minutes’ incubation (Figure S6). In Figure 2A, the fluorescence of microdroplets was monitored as a blank control, containing the identical components except for the loaded cells. There was almost no fluorescent change in the microdroplets due to the excellent photostability of HRP-AuNCs. In Figure 2B-C, HUVEC cells were tested by our microdroplet assay with or without stimulation of PMA (10 µL, 1 µg mL-1), a small molecular chemical compound which can trigger H2O2 secretion of cells in situ. Without stimulation of PMA, the fluorescence change of the HUVEC cell-encapsulated droplets was marginal (< 10%) because H2O2 secreted by normal cells was very limited. PMA stimulation indeed promoted HUVEC cell-secreted H2O2, leading to a nearly 40% decrease of the fluorescent intensity (Figure 2C). Distinct from the normal cells, the tumor cell lines (MCF7, U937 and Mut6)exhibited
obviously higher levels of H2O2 secretion even in the absence of PMA stimulation, resulting in a greater reduction of fluorescence signals (Figure 2D, and Figure S7A, C). The tumor cell lines under the PMA stimulation accelerated H2O2 secretion, reducing the fluorescent intensity of HRP-AuNCs by nearly 70% (Figure 2E, and Figure S7B, D). These malignant tumor cells tended to proliferate at a higher rate, and responded rapidly to the external stimuli for metastasis.54 Therefore, the H2O2 levels secreted by malignant tumor cells were higher than normal cells, which was consistent with the previous report.55
Figure 2. Fluorescence changes of single cell-encapsulated microdroplets at the time point of 0 and 90 min, including original fluorescent images and plots of quantitative analysis. (A) Blank control without cell loading. (B-E) The tests of using HUVEC or MCF7 cell line in the absence (-) or presence (+) of PMA for in situ stimulation. Error bar: standard deviation (n = 6). The bar with asterisks indicates a statistically significant difference (P***
