A Magnetic-Based Microfluidic Device for On-Chip Isolation and

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A Magnetic-Based Microfluidic Device for On-Chip Isolation and Detection of Tumor-Derived Exosomes Huiying Xu, Chong Liao, Peng Zuo, Ziwen Liu, and Bang-Ce Ye Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b03272 • Publication Date (Web): 20 Sep 2018 Downloaded from http://pubs.acs.org on September 21, 2018

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Analytical Chemistry

A MagneticMagnetic-Based Microfluidic Device for OnOn-Chip Isolation and DeDetection of TumorTumor-Derived Exosomes Huiying Xu,†,⊥ Chong Liao,†,⊥ Peng Zuo,† Ziwen Liu,‡ and Bang-Ce Ye*,† † Lab of Biosystem and Microanalysis, State Key Laboratory of Bioreactor Engineering, East China University of Science & Technology, Shanghai, 200237, China ‡ Department of Nuclear Medicine, The First People’s Hospital of Shangqiu City, Shangqiu, 476000, China ABSTRACT: Exosomes are membrane-enclosed phospholipid extracellular vesicles which can act as mediators of intercellular communication. Although the original features endow tumor-derived exosomes great potential as biomarkers, efficient isolation and detection methods remains challenging. Here, we presented a two-stage microfluidic platform (ExoPCD-chip) which integrates onchip isolation and in situ electrochemical analysis of exosomes from serum. To promote exosomes capture efficiency, an improved staggered Y-shaped micropillars mixing pattern was designed to create anisotropic flow without any surface modification. By combining magnetic enrichment based on specific phosphatidylserine-Tim4 protein recognition with a new signal transduction strategy in a chip for the first time, the platform enables highly sensitive detection for CD63 positive exosomes as low as 4.39×103 particles/mL with a linear range spanning 5 orders of magnitude, which is substantially better than the existing methods. The reduced volume of samples (30 µL) and simpler affinity method also make it ideal for rapid downstream analysis of complex biofluids within 3.5 h. As a proof-of-concept, we performed exosomes analysis in human serum and liver cancer patients can be well discriminated from the healthy controls by the ExoPCD-chip. These results demonstrate that this microfluidic chip may serve as a comprehensive exosome analysis tool and potential non-invasive diagnostic platform.

Tumor cells secrete various types of humoral factors into their surrounding tumor stroma, among which exosomes can act as a transmission medium for intercellular communication in the tumor microenvironment.1-3 Exosomes are membraneenclosed phospholipid nanovesicles (30−150 nm in diameter) actively secreted by various normal and tumor cells through an endolysosomal pathway.4,5 During this plasma membrane fusion process, exosomes carry enriched tetraspanin proteins (CD9, CD63, CD81), cytosol proteins, mRNA, and microRNA from their parental cells.6,7 These original features endow cancer-specific exosomes with distinctive opportunities to serve as potential cancer biomarkers for identifying early-stage tumors and monitoring of treatment response non-invasively.8,9 However, the small size and low buoyant density of exosomes pose significant challenges to their isolation and quantification in the complex biofluids. Conventional ultracentrifugation isolation requires tedious steps and expensive instruments, which may result in low recovery with high expenditure.9,10 Standard methods widely used for exosome quantification such as enzyme-linked immunosorbent assay (ELISA) and western blot are limited by large sample demand and low sensitivity.11,12 Moreover, exosomes analysis during these separately multi-step workflows can be time-consuming and vulnerable to contaminations. Recent advances in microfluidic technologies offer incredible potential to construct comprehensive analysis platform of exosomes on a tiny chip. Microfluidic platforms based on acoustic nanofilter,13 viscoelastic flow sorting,14 lateral displacement15 or immunoaffinity16-20 have been reported to spe-

cifically isolate exosomes from biological fluids. As the most commonly used isolation approach, immunoaffinity has the ability to isolate specific subpopulations of exosomes based on the expression of specific surface proteins.21 Even so, the stronger antibody-antigen interaction makes it difficult to detach exosomes from the binding antibodies under mild elution conditions.22 Phosphatidylserine (PS) expressed on exosomes membranes leaflet can be well recognized by PS-binding receptors Tim4, which appears to be essential for exosome budding within the late endosomes.23-25 Capturing exosomes with magnetic beads immobilized with Tim4 is Ca2+-dependent and intact exosomes can be easily elution by chelating agent.22 Inspired by this novel affinity method, we presented a twostage microfluidic platform (ExoPCD-chip) which integrates on-chip isolation and in situ electrochemical analysis of exosomes from blood samples. The microchip is made up of an array of Y-shaped micropillars and a cascading ITO electrode. The PDMS micropillar array can perform cross mixing efficiently over and over again to enhance the opportunities of collisions between Tim4 modified magnetic beads and exosomes. The captured exosomes can be detected sensitively through magnetic enrichment on the surface of ITO electrode where a new signal transduction strategy was constructed. Taking into account the need for portable and sensitive, we designed a label-free and immobilization-free electrochemical aptasensor (named as LGCD) which contains a CD63 (an enriched marker in exosomes surface) aptamer26 and mimicking DNAzyme sequence27. The single-stranded DNA forms a hairpin structure and G-rich mimicking DNAzyme sequence in the strand is caged in the stem-loop structure. The target

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Scheme 1. Integrated exosomes isolation and analysis platform. (A) Schematic diagram of the ExoPCD-chip. (B) Schematic of the electrochemical sensor on the surface of ITO electrode. CD63-positive exosomes can open the original single-stranded DNA hairpin and form a G-quadruplex as a signal reporter with the aid of hemin. The hemin/G-quadruplex can be employed as the NADH oxidase and HRP-mimicking DNAzyme simultaneously.28 Hence, the newly generated H2O2 by NADH oxidation can be catalyzed continuously which accompanied with obvious signal enhancement. Unlike the reported aptamer-modified electrode for exosomes detection methods, the new proposed exosomes probe can be utilized in the microfluidic chip conveniently without expensive nucleic acid modification, complicated immobilization process and signal amplification. In this report, we showed that our ExoPCD-chip can efficiently capture tumor-derived exosomes and response rapidly within 3.5 h in a small-volume sample (30 µL) with a low detection limit than most currently report. Finally and most importantly, we analysed clinical blood samples from patients with confirmed hepatic carcinoma and compared with healthy controls. The positive correlated between exosomes and tumorigenesis further demonstrated a potential usability of the ExoPCD-chip for clinical application and cancer diagnosis. EXPERIMENTAL SECTION Reagents and Materials. Polydimethylsiloxane (PDMS) Elastomer were obtained from Dow Corning (Michigan, USA) and SU8-2010 was purchased from MicroChem (Newton, MA, USA). All oligonucleotides (Table S1) were synthesized by Sangon Biotechnology Co., Ltd. (Shanghai, China). The following antibodies were used in the experiments: biotinlabeled mouse anti-human CD63 antibody (anti-CD63) from Biolegend (USA), biotin-labeled mouse anti-human CD9 antibody (anti-CD9) from Allcells Biotech Co., Ltd. (Shanghai, China) and HRP-conjugated anti-mouse IgG from QF Biosciences Co., Ltd. (Shanghai, China). MagCaptureTM Exosome Isolation Kit PS and PS CaptureTM Exosome ELISA Kit were purchased from Wako Pure Chemical Industries, Ltd. ExoEasy Maxi Kit was purchased from Qiagen Translational Medicine

Co., Ltd. (Suzhou, China). Alexa Fluor 488-conjugated streptavidin was provided by Yeasen Biotech Co., Ltd. (Shanghai, China). Streptavidin-labeled HRP, TMB Substrate solution, RIPA lysis buffer, PMSF and BCA Protein Assay Kit were purchased from Beyotime Biotechnology Co., Ltd. (Shanghai, China). 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), hemin, NADH, thionine (Thi) were purchased from Sigma Aldrich, Inc. (Saint Louis, MO, USA). Instrumentation. Transmission electron microscopy (TEM) was performed on a JEOL JEM-1230 instrument (JEOL Ltd., Japan) using a working voltage of 120 kV and the sample was negatively stained with 2% aqueous uranyl acetate solution. Scanning electron microscopy (SEM) images were obtained using a Hitachi S-4800 filed emission scanning electron microscopy at an accelerating voltage of 1 kV. Fluorescence imaging was carried out using a Nikon inverted fluorescence microscope and a Nikon confocal scanning system. UVvis absorption spectra was measured by a microplate reader (Bio-Tek Instrument, Winooski, USA). The gels were visualized under a BioRad Molecular imager (Tanon-1600, China) and Gel Image System (Tanon, China). Flow cytometry was performed by Flow NanoAnalyzer (Xiamen Fuliu Biological Technology Co., China). Electrochemical data were obtained on a CHI 660A electrochemical workstation (Shanghai Chenhua Co.). The ITO electrode was designed by AutoCAD and etched precisely by laser on a 26-mm-width, 1.1-mm-thick ITO glass (Guluo Glass Co., Ltd. Luoyang, China) to form a three-electrode system. Cell Culture and Exosome Extraction. HepG2 was obtained from the Cell Bank of Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China) and cultured in DMEM (Invitrogen, Thermo Fisher Scientific) containing 10% (v/v) FBS and 1% (v/v) penicillin-streptomycin at 37 °C. After incubation for 48 h, cell culture supernatant was collected and centrifuged at 4 ℃ (300g for 10 min and 2000g for 20 min) to remove cells and cellular debris, followed by filtration through a 0.22 µm filter to thoroughly remove the large extra-


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Analytical Chemistry cellular vesicles. If necessary, exosomes can be enriched further through MWCO 10 kDa filters at 4 ℃, 3500g for 15 min. The separation of exosomes from the cell culture supernatant was performed strictly in accordance with the manufacturer’s instructions of ExoEasy Maxi Kit. The concentration and size distribution of exosomes were measured by qNano (Izon Science Ltd., Oxford, UK). Microfluidic Chip Design and Fabrication. The microfluidic chip is composed of a PDMS layer sealed with an ITO electrode. The PDMS layer contains a microchamber for exosome capture with an array of Y-shaped micropillars which has a uniform height of 70 µm. A hole at the end of the microchannel was designed to match the detection area. The microchips were fabricated using the standard soft lithography with SU8-2010 master mold on a silicon substrate. Cured SU-8 molds were protected from light and stored at 4-21°C. A 10:1 mass ratio of PDMS and curing agent were poured on the molds and then solidified in an oven at 65°C for 6 hours. After removing from the mold, the patterned PDMS was punched for holes at desired location and bonded to the ITO electrode through oxygen plasma activation. All chips and ITO glass electrodes were ultrasonically cleaned with isopropyl alcohol and ddH2O for 30 minutes before bonding. To prevent nonspecific adsorption and generation of bubbles, a surface treatment for the PDMS microchannels was applied with blocking buffer (2.5 w/w% BSA and 0.01 w/w% Tween-20 in 1×PBS) for 15 min at a rate of 5 µL/min. Finally, copper wires were connected to respective electrodes by conductive silver paste plus (SPI, USA) to make it convenient to link with the electrochemical workstation. Preparation of Tim4 Conjugated Magnetic Beads (Tim4 beads). 0.15 mg magnetic beads coated with streptavidin (Wako, Japan) and 0.25 µg biotinylated mouse Tim4-Fc (Wako, Japan) were suspended in 400 µL buffer (20 mM TrisHCl, pH 7.4, 2 mM CaCl2, 150 mM NaCl, 0.0005% Tween20) for 20 min to obtain Tim4 beads at 4℃. And then, the Tim4 beads were washed three times with washing buffer and resuspended with 200 µL buffer for further experiments. Exosomes Captured and Electrochemical Detection on the Chip. All liquids in the experiment were accurately injected at constant flow rates by a programmable LSP04-1A syringe pump. In the exosomes capture stage, Tim4 beads were pumped into the microchamber at a rate of 2 µL/min and retained in the Y-shaped micropillars area by a permanent magnet placed underneath the chip. Subsequently, the pretreated samples were injected at the same rate from the two injection ports to obtain better dispersion via two liquid flow disparity. Washing buffer (20 mM Tris-HCl, pH 7.4, 2 mM CaCl2, 150 mM NaCl, 0.0005% Tween20) was introduced to rinse the nonspecifically bounded vesicles gently at 3 µL/min for 10 min. In the detection stage, the permanent magnet was removed to the electrode area and captured exosomes were flushed by washing buffer at 10 µL/min into electrochemical detection area where they were immobilized again by magnetic force. A mixture of DNA probe (LGCD), Mg2+ and NADH in HEPES buffer (10 mM, pH 7.4) were injected from inlet #3 into the detection area. Meanwhile, another mixture of hemin, K+ and Thi in HEPES were injected from inlet #4 to keep the optimal concentration. After incubation for 45 minutes, DPV was performed within the applied potential range from 0.1 V to -0.5 V. When demonstrating the binding ability of LGCD, biotin-labeled LGCD and a random DNA were used to couple

streptavidin-labeled HRP. And the sequences of the two strands are shown in Table S1. Enzyme Linked Immunosorbent Assay (ELISA) for Quantifying Exosomes. In this experiment, PS CaptureTM Exosome ELISA Kit was used according to the manufacturer's instructions. In brief, 100 µL of purified exosomes were added to the wells of an Exosome Capture 96 Well Plate (Tim4 coated microplate) and incubated for 2 h at room temperature. Then, the well plate was washed three times with washing buffer and incubated with 100 µL of control primary antibody anti-CD63 for 1h at room temperature. After unbound antibodies were washed, 100 µL of secondary antibody HRPconjugated anti mouse IgG was added for 1 h incubation. Then, 100 µL of TMB solution were added to each well for 30 min followed by washing five times. The reaction was stopped by 100 µL of 1 M HCl, and absorbance was recorded at 450 nm. Western Blot (WB). Exosomes purified by Tim4 beads and ExoEasy Maxi Kit, and HepG2 cells were lysed by RIPA lysis buffer containing 1 mM PMSF at 4 ℃ for 45 min. Proteins lysates were resolved by SDS-PAGE before being transferred onto PVDF. PVDF was blocked for 2 h at room temperature with a blocking solution containing 5% (w/v) albumin bovine V in TBST (20 mM Tris-HCl, pH 7.6, 150 mM NaCl, and 0.1% Tween 20). Following incubation with primary antibody anti-CD63 (1:2000 dilution in TBST) for 12 h at 4 ℃ and another incubation with secondary antibody HRP-conjugated anti-mouse IgG (1:10000 dilution in TBST) for 1 h at room temperature, immunoreactive bands were visualized by Gel Imaging System. Clinical Samples. Human serum samples including healthy people and cancer patients were obtained from the First People's Hospital of Shangqiu City. Before the analysis of the sample, the serum was purified by the following centrifugation: (1) 1200g for 20 min at 4 °C; (2) 10000g for 30 min at 4 °C. After centrifugation, the resulting supernatant was analyzed quantitatively by ELISA and ExoPCD-chip. When quantified by flow cytometry, exosomes were preextracted from serums by ExoEasy kit. RESULTS AND DISCUSSION Design and Characterization of the Microfluidic Exosomes Analysis Platform. We devised a two-step cascade approach for cancer-specific exosomes analysis in the microfluidic device. The proposed ExoPCD-chip is composed of an array of Y-shaped micropillars with two sample inlet for magnetic bead-based exosomes capture and a microchamber with etched three-electrode system on the ITO glass substrate for exosomes detection (Scheme 1A, S1B). The magnetic beads were pretreated with phosphatidylserine (PS)-binding protein (Tim4 beads) and introduced through inlet #1 and #2 into the capture area where the well-distributed magnetic beads were retained by magnetic force (Figure 1A). Subsequently, samples were injected from inlet #1 and #2 again to obtain better dispersion via two liquid flow disparity. To promote exosomes capture efficiency, a staggered Y-shaped micropillars mixing pattern was designed to create anisotropic flow as the fluid travels through microchannels (Figure 1C, SEM). The PDMS Y-shaped micropillars array works as a chaotic micromixer in which a flow is bifurcated and cross mixing with adjacent streams periodically11 to enhance the opportunities of collisions between Tim4 beads and exosomes. The simulation results of the flow velocity profile confirmed this continuously



interaction within Y-shaped micropillar identically, and the asymmetric flow at the downstreams of every high velocity field brought more contact probability when they encounter next time (Supporting Information, Figure S1A).

Figure 1. (A) Bright-field microscope images of Tim4 beads manipulated in the microfluidic channel. (B) Fluorescence images of the DiI labeled exosomes in the microfluidic channel. (C) SEM image of the array of Y-shaped PDMS micropillars. (D) TEM image of exosomes captured by the ExoPCDchip. qNano analysis of the size distribution of exosomes purified by the ExoPCD-chip (E) and commercial exoEasy kit (F). Optimization of the Microfluidic Design. To visualize the distribution of exosomes in this chamber, sample pre-labeled with a fluorescence dye (DiI) which specifically stains only the membrane vesicles16 were flowed through the ExoPCDchip after injection of Tim4 beads. Figure 1B revealed bright red fluorescence was produced in a high density by the injected exosomes samples, indicating the device was functioning properly. In addition, the captured exosomes were eluted by chelating agent and maintained a round-cup morphology typical of exosomes (Figure 1D). Western blotting on isolated exosomes via Tim4-specific capture further confirmed expression of tetraspanins CD9 and CD63, which was in accordance with HepG2 cell lysates and exosomes isolated by ExoEasy kit (Figure S1C). We also characterized the size distribution of HepG2 exosomes extracted by ExoEasy Maxi Kit and our ExoPCD-chip. Compared to the commercial method, the ExoPCD-chip yielded a higher percentage of vesicles smaller than 150 nm (93% smaller than 150 nm, which is a common criterion to differentiate exosomes from larger microvesicles9) as well as a notably narrower size distribution (Figure 1E and F). Taken together, such a microchip could realize tumour-derived exosomes specifically and purify them efficiently in a manageable strategy. To validate the capture performance of the ExoPCD-chip, a suspension of exosomes harvested from HepG2 cells were used to optimize the micropillar spacing distance and flow rate. Capture efficiency was calculated by comparing total protein of the isolated exosomes of flows at the outlet and inlet of the capture chamber.29 As illustrated in Figure 2A, the capture efficiency at flow rate of 0.2 µL/min was decreased from 73.1% to 54.1% when spacing was increased from 25 to

100 µm. 50 µm was selected for following experiments, because magnetic beads may aggregate in a too narrow distance and thus block the microchannels during the actual operation. Different flow rates (from 0.2 to 1.6 µL/min) were tested, and the capture efficiency was decreased with the increase of flow rate which is presumably owing to the increased shear stress.30 0.2 µL/min was chosed as the optimal rate with final capture efficiency ≈ 68.5% (Figure 2B).

Figure 2. Capture performance of the ExoPCD-chip. (A) Optimization of micropillar spacing and (B) flow rate of the ExoPCD-chip. (C) Capture efficiency of Tim4, antibodies against CD63 and CD9 conjugated magnetic beads. (D) Comparison of capture efficincy between commercial Exosome Isolation Kit and the ExoPCD-chip. Error bars indicate standard deviation of measurements (n = 3).

The specific interaction between PS expressed on tumor cell-derived exosomes and the target cell surface receptor Tim4 contributes to efficient cell-to-cell spread as well as biomolecular information exchange in the cancer microenvironment.25,31 According to this relationship, the Tim4-PS can be brought into focus as a new candidate pair for enhanced membranes recognition. In order to evaluate the capture performance of Tim4 in our platform, magnetic beads conjugated with antibodies against CD63 and CD9 were also tested under the same conditions. Tim4 conjugated magnetic beads displayed the highest capture efficiency in the ExoPCDchip (Figure 2C), indicating the PS-recognition pathway is an appropriate anchorage site for exosomes capture. Meanwhile, compared with a commercial Exosome Isolation Kit, the ExoPCD-chip demonstrated excellent capturing specificity with more than 3-fold enrichment of exosomes in a shorter period of time (Figure 2D). Such a high capture efficiency are attributed to duration collisions between two particles on the micropillar surfaces with a gradient of hydrodynamic forces in the high surface-to-volume ratio microchannels. LGCD Design and Binding Ability Validation. To quantify the exosomes enriched in the ExoPCD-chip, LGCD was designed which could be transformed into a small Gquadruplex-contained hairpin configuration by CD63-positive exosomes (Scheme 1B). This label-free probe undertakes great responsibilities for efficient exosomes analysis, hence another five single-stranded probes (Table S1) were designed to


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Analytical Chemistry explore the optimal sequence in the absence and presence of exosomes. We found that too short stem or loop may threaten the stability of the original hairpin, thus leading to a high background. Conversely, an extended self-complementarity makes it harder to screen the target exosomes. When removing the five base pairing in the ultimate structure, the DPV signal decreased with an increased background which indicates it is an essential element to maintain structural stability. And the DNA probe failed to identify exosomes if we changed three base in the CD63 aptamer. The relative signal (F/F0-1; F0 presents the peak current without exosomes, while F was defined as the peak current with exosomes) was also calculated in Figure S2, and LGCD demonstrated the remarkably highest signal intensity among them. Meanwhile, we demonstrated the binding ability between LGCD and HepG2 exosomes through labeling the LGCD with HRP which can catalyze H2O2-mediated oxidation of TMB. Figure 3A showed a distinct absorption pattern with a maximum peak at 450 nm when exosomes were captured by anti-CD9 conjugated magnetic beads and incubated with HRPlabeled LGCD afterwards (Sample 4). The lack of any one of anti-CD9 (Samlpe 1), exosomes (Sample 2) and LGCD (Sample 3) will result in low absorbance, suggesting the signal was generated by HRP-labeled LGCD binding in exosomes.32 An HRP-labeled random DNA (Samlpe 5) was also used as a probe under the same condition. The absorbance of HRPlabeled random DNA was much smaller than that of HRPlabeled LGCD because of the specific recognition of CD63 aptamer in LGCD. In another validation, we introduced Alexa Fluor 488 instead of HRP to label the LGCD and took images by confocal fluorescence microscopy. As expected, colocalization images of Alexa Fluor 488 and magnetic beads can only be observed in the sample 1 (Figure S3), confirming that the green fluorescent dye labeled LGCD has been attached on exosomes surface uniformly through the inherent CD63 aptamer without nonspecific adsorption. Inspired by this, we were able to visualize the bright green fluorescence produced by Alexa Fluor 488-labeled LGCD binding in captured exosomes at Y-shaped micropillars area in a simple way. Exosomes were pretreated with Alexa Fluor 488-labeled LGCD to avoid complicated process to generate fluorescent exosomes (Figure 3B). Detection of Exosomes with the Probe DNA. Then feasibility of our designed strategy was elucidated by cyclic voltammogram (CV) and differential pulse voltammetry (DPV). The etched ITO slides exhibited a pair of well-defined redox peaks with a peak potential separation (∆Ep) less than 80 mV in 5 mmol L−1 K3Fe(CN)6 and 0.1 mol L−1 KCl solution (Figure S4A), suggesting its intrinsic electronic properties are suitable in fabricating electrodes. Subsequently, 0.1 µM LGCD were exposed to a reaction solution containing suspended HepG2 exosomes at an approximate concentration of 6×108 particles/mL. After incubation with hemin and K+, DPV was carried out in the absence and presence of unmodified magnetic beads on the ITO surface (Figure S4B). The electrochemical signal derived from the added thionine and both of them acquired enhanced cathodic current than background current similarly. Only slightly shift in peak potential can be observed, effectively proving the little influence by magnetic beads immobilized on electrode surface. In contrast, the cathodic current increased dramatically when Tim4 beads were employed beforehand as an alternative for exosomes capture.

Figure 3. (A) The contrast of absorption spectral of HRPlabeled LGCD binding in exosomes which were captured by anti-CD9 conjugated magnetic beads and four controls which lack of anti-CD9, exosomes and LGCD in the reaction system, respectively. Sample 1: Magnetic beads + Exosomes + LGCD + HRP. Sample 2: Magnetic beads + anti-CD9 + LGCD + HRP. Sample 3: Magnetic beads + anti-CD9 + Exosomes + HRP. Sample 4: Magnetic beads + anti-CD9 + Exosomes + LGCD + HRP. Sample 5: Magnetic beads + anti-CD9 + Exosomes + random DNA + HRP. (B) Confocal microscopy image of Alexa Fluor 488 labeled LGCD binding in exosomes which were captured by Tim4 beads at Y-shaped micropillars area of the ExoPCD-chip.

As the unique identifier, the concentration of LGCD would exert great effect on the performance of electrochemical biosensor. Figure S5A showed the distinct DPV responses with an increasing LGCD concentration. The relative signal increased with LGCD and reached a plateau over 0.1 µM (Figure S5B). Thus, 0.1 µM was chosen as the optimized LGCD concentration for the following detection. As the electron mediator, thionine is an important factor in the signal transduction process. From Figure S6, the addition of thionine could cause a gradual increased DPV response both in the absence and presence of exosomes simultaneously, while 1 mM thionine displayed the apparent advantage than others when compared the relative signal. Incubation time of HepG2 exosomes with the LGCD aptamer affected their binding ability seriously, and 45 min was chosen when cathodic current reached a plateau (Figure S7A). Hemin and K+ had great influence on the formation and stabilization of hemin/G-quadruplex-based DNAzyme. 2 µM was chosen as the optimized hemin concentration because of its highest relative signal and the optimum concentration of K+ (20 mM) was selected in the same way. Detection Performance of the Assay. In view of outstanding ability of the developed sensing platform to sensitively detect CD63-positive exosomes, the proposed LGCD was employed in the detection area after HepG2 exosomes enrichment by the Tim4 beads. The DPV curves in Figure 4A illustrated that increasing concentrations of exosomes in the range from 7.61×104 particles/mL to 7.61×108 particles/mL resulted in gradual current increase, which was ascribed to the liberation of the quadruplex-forming fragment. The calibration curve in Figure 4B showed that the peak currents were logarithmically related to the concentration of exosomes. The limit of detection (LOD) was calculated to be 4.39×103 particles/mL based on the 3 times the standard deviations of the background. Similar measurements by ELISA Kit which also based on Tim4 coated microplate yielded lower sensitivity as well as a more narrow linear range than the ExoPCD-chip (Figure 4C and D). Compared to



previous reports, the current ExoPCD-chip was superior in sensitivity, and the obtained LOD was reduced by nearly one order of magnitude than that of the most sensitive microfluidic (5×104)11 or electrochemical (2.09×104)34 methods with a wider linear range than that obtained by ac-EHD methodology1, B-Chol anchor assay with enzyme-linked HCR32 and g-C3N4 NSs-CD63 aptamer33 (Table S2). Evidently, the low LOD benefits not only from specific enrichment of the Tim4 bead but also from the continuous analysis by transducing the biorecognition into a sensitive electrochemical signal with the aid of a label-free and immobilization-free DNA probe.

Figure 4. (A) DPV responses on the ITO electrode with different concentration of HepG2 exosomes. (B) Calibration curve of the LGCD aptamer. The linear range of exosome concentration varied from 7.61×104 to 7.61×108 exosomes/mL. (C) Absorption spectral of ELISA with different concentration of HepG2 exosomes. (D) Calibration curve of ELISA. The linear range of exosome concentration varied from 7.64×106 to 1.53×108 exosomes/mL. Clinical Sample Analysis. Tumor-derived exosomes secreted into the blood stream may provide a more accessible and representative source of cancer biomarkers. To evaluate the diagnostic potential of the ExoPCD-chip, we first spiked different concentration of exosomes into 10% diluted supernatant of ultracentrifuged fetal bovine serum (10% diluted UC FBS) to simulate a biological sample. As shown in Figure S8A, in the absence of exosomes, the DPV response was quite low, suggesting neglectable unspecific binding in the chip. The DPV response increased gradually with increasing exosomes concentrations and showed linear related to the logarithm of exosomes concentration in the range of 5.98×105 to 5.98×108 particles/mL with a LOD of 2.23×104 particles/mL (Figure S8B). The calibration curve obtained in 10% diluted UC FBS were approximate to that obtained in Hepes indicating the little effect of biological matrix in the ExoPCD-chip except the small shifting of peak potential and baseline. Subsequently, we performed exosomes analysis in human serum from ten liver cancer patients and six healthy individuals. Obviously, patient samples have elevated number of CD63-positive exosomes isolated from serum (Figure 5A), which corroborates previous literatures of increased secretion of exosomes in cancer patients and higher expression of CD63 protein on the surface of exosomes secreted by tumor cells than normal cells.16,32,33 As displayed in Figure 5B, liver

cancer patients can be well discriminated from the healthy controls by the ExoPCD-chip (p = 0.0007). According to the DPV responses and diluting factor, we calculated the average quantity of exosomes and found an approximately 1.5 fold increase in serums of liver cancer patients (7.51×108 particles/mL) compared to healthy controls (3.02×108 particles/mL). Besides, the molecular profiles of CD63positive exosomes were highly correlated with their derived cells, especially the expression levels of several tumor marker.18 Notably, as cellular surrogates, CD63-positive exosomes are suitable for applying to the ExoPCD-chip and have the potential to be a convenient tool for preliminary screening in the diagnosis of liver cancer. The microfluidic results were also compared to parallel ELISA analysis by a commercial ELISA kits from the same serum samples without any exosomes extracting process. On the whole, the expression of CD63-positive exosomes have same tendency in most of serums from liver cancer patients. However, we observed lower concentrations of exosomes in serums according to ELISA data, especially liver cancer patients (Figure 5C). In view of its inadequacy for serum pointed out in the instruction book, we suppose this phenomenon is a result of lack of purification process which may lead to nonspecific reaction between antibody and impurities in the serum. As a consequence, the ELISA kits failed to distinguish healthy controls and liver cancer patients with a large signal overlap (p = 0.1081) (Figure 5D). Unlike the antibodies-based isolation and detection method, the ExoPCD-chip offers much simpler conjugation and signal transduction strategy, resulting in relatively small interference from the complex biofluids.

Figure 5. Quantitative detection of CD 63-positive exosomes directly from clinical serum samples. (A) The results of the ExoPCD-chip presented in the bar. (B) Boxplots overlaid with dot plots for clinical sample analysis by ExoPCD-chip. (C) The results of ELISA presented in the bar. (D) Boxplots overlaid with dot plots for clinical sample analysis by ELISA. In order to investigate the accuracy of the ExoPCD-chip, flow cytometry was also performed to quantify exosomes followed by separation from three liver cancer patients and three healthy individuals by ExoEasy kit. Exosomes isolated from serum were stained with phycoerythrin (PE) conjugated primary antibodies specific to CD63, therefore concentrations of CD63-positive exosomes can be obtained by Flow


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Analytical Chemistry NanoAnalyzer. As seen in Figure 6, the concentrations measured by the ExoPCD-chip are agreement with those obtained by this commercial method from the same samples. Compared with flow cytometry and ELISA, the ExoPCD-chip can accurately analyze CD63-positive exosomes with no need for extra exosomes extracting process within 3.5 h in a smallvolume sample (30 µL). In addition, the design of this labelfree and immobilization-free electrochemical aptasensor provided an advantage to recognize other exosome subpopulations expressing different surface proteins through altering corresponding aptamer conveniently.

* E-mail: [email protected] Tel/Fax no. 0086-21-64252094

ORCID Bang-Ce Ye: 0000-0002-5555-5359 Author Contributions ⊥

These authors contributed equally.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This work was supported by the National Natural Science Foundation of China (Grant No. 21335003, 21575089 and 21705047), the Fundamental Research Funds for the Central Universities (Grant No. 222201814030) and the Chinese PostDoctoral Fund (Grant No. 2017M621380).

REFERENCES

Figure 6. (A) Comparison of CD63-positive exosomes concentrations detected by ELISA, flow cytometry and ExoPCD-chip in serum samples from three healthy individuals (H1, H2 and H3) and three liver cancer patients (P1, P2 and P3).

CONCLUSIONS In summary, a two-step cascade microfluidic platform was developed for rapid and simple hepatocellular exosomes analysis. Compared to common antibody-antigen interaction, the novel Tim4-PS affinity method displayed enhanced capture efficiency and can detach exosomes under mild elution conditions, which benefits downstream analysis. After on-chip isolation, the electrochemical sensor could detect CD63 positive-exosomes at an extremely low concentration with the aid of a label-free and immobilization-free single-stranded DNA probe. In addition, we showed that the ExoPCD-chip could differentiate liver cancer patients from healthy controls, making this strategy applicable to analysis of clinical specimens. We believe that the ExoPCD-chip is a promising platform that should contribute to the development of exosomes-based cancer diagnostics.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Finite element simulations, western blotting images, assay optimization, confocal microscopy analysis, feasibility of our designed strategy, sequence of oligonucleotides and performance comparison

AUTHOR INFORMATION Corresponding Author

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A Magnetic-Based Microfluidic Device for On-Chip Isolation and

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