Rapid Isolation and Visible Detection of Tumor-derived Exosomes

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Rapid Isolation and Visible Detection of Tumor-derived Exosomes from Plasma Junge Chen, Youchun Xu, Ying Lu, and Wanli Xing Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b03031 • Publication Date (Web): 29 Oct 2018 Downloaded from http://pubs.acs.org on October 29, 2018

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Analytical Chemistry

Rapid Isolation and Visible Detection of Tumor-derived Exosomes from Plasma Junge Chen,† Youchun Xu,†,‡ Ying Lu,†,‡ Wanli Xing*,†,§ †

School of Medicine, Tsinghua University, Beijing 100084, China

‡ National Engineering Research Center for Beijing Biochip Technology, Beijing 102206, China § Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Hangzhou 310003, China

ABSTRACT Exosomes are nano-sized extracellular vesicles (ranging from 30-120 nm) released from many cells, which provide promising biomarkers for the non-invasive diagnosis of cancer. However, traditional exosome isolation methods are tedious, non-standardized, and require bulky instrumentation, thus limiting its clinical applications. In this paper, an anion-exchange (AE)-based isolation method was first proposed to isolate exosomes directly from plasma and cell culture medium by AE magnetic beads within 30 min. Exosomes isolated by AE magnetic beads had higher recovery efficiency (>90%) and less protein impurities than by UC. Then, prostate cancer (PCa) exosomes in plasma were detected in a visual, label-free and quantitative manner with aptamer-capped Fe3O4 nanoparticles for the first time. The linear range of PCa exosomes was estimated from 0.4×108 to 6.0×108 particles/mL with a detection limit of 3.58×106 particles/mL. The present study provides an efficient and practical approach for the rapid isolation and visible detection of exosomes, which is promising for the early diagnosis of PCa. Keywords: Exosomes; Anion-exchange; Fe3O4 nanoparticles; Prostate cancer

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INTRODUCTION Prostate cancer (PCa) is the most common solid malignant cancer for men throughout the world.1 PCa is curable at the early stages, however, it becomes castration resistant at the advanced stages and difficult to treat.2 Therefore, it is important to diagnose PCa in its early stages. Though prostate-specific antigen (PSA) is the most widely used biomarker for the early diagnosis of PCa,2 it is not specific to PCa. For example, PSA may increase in benign prostatic hyperplasia3, leading to a high rate of false-positive in PCa diagnosis. Thus, there is an urgent need to discovery novel PCa biomarkers to improve diagnostic accuracy. Among various new biomarkers, exosomes are a promising category. Exosomes are membrane-bound vesicles (ranging from 30-120 nm) of endocytic origin released from a variety of cells.4 Exosomes are involved in tumorigenesis, tumor metastasis, and chemotherapeutic resistance.5-7 Cancer-derived exosomes contain tumor-specific proteins, mRNAs and microRNAs.6,8,9 PCa patients have higher levels of exosomes in plasma compared to healthy individuals.2,10 Moreover, exosomes are common in many bodily fluids, such as blood, saliva, and cerebrospinal fluid.4,11 Therefore, exosomes are regarded as promising biomarkers for early diagnosis of PCa.12 In spite of the wide application of exosomes as non-invasive tumor markers, current exosome isolation methods suffer from time-consuming, low purity and exosome damage.13-17 For example, ultracentrifugation (UC) requires multi-step centrifugation, resulting in time-consuming (>8 h) and tedious operations.14 Immuno-affinity capture has also been widely used for exosome isolation, but it may exclude important subpopulations of exosomes. Apart from these methods, several precipitation reagents have been introduced into cell supernatants to enable exosome sedimentation into pellets at lower PEG concentrations.18 However, precipitation methods lead to a large number of protein contaminants. Anion-exchange (AE) chromatography is a feasible solution to address these problems.19,20 AE chromatography is a form of adsorption chromatography that separates molecules based on their charge using an ion-exchange resin containing positively charged groups. In current researches, AE magnetic beads have also been used for the rapid isolation of bacteria and virus due to their rich ion exchange capacity, high binding capacity and fast magnetic response.21 In this study, the potential of an AE-based exosome isolation method was explored. Exosomes exposed negative charged phosphatidylserine (PS) to the outer leaflet of the membrane.22 Exosomes may bind with AE magnetic beads. The performance of AE magnetic beads for exosome isolation was comprehensively studied. Apart from exosome isolation, appropriate quantitative detection methods for exosomes are necessary for the better study of exosomes.23-26 Despite numerous qualitative studies of exosomes, quantification of exosomes remains challenging.27,28 For example, flow cytometry detection is limited by weak light scattering.29 Nanoparticle-tracking analysis ( NTA) fail at detection with specificity.15 Moreover, given the tedious steps required to isolate exosomes from bodily fluids, it is not easy to collect abundant samples for continuous analysis using current method.30 Hence, the development of a simple exosome detection method which has high sensitivity and a low cost would be significantly important. To address these problems in exosome detection, nano-enzymes including Fe3O4 NPs, gold nanoparticles, and carbon-based materials are feasible solutions.31-34 Among these nanozymes, Fe3O4 NPs are particularly interesting because have characteristics of low cost, bulk preparation, resistance to pH and temperature varieties.34 DNA-capped Fe3O4 NPs are nearly 10-fold more active as a peroxidase mimic for TMB oxidation


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than naked nanoparticles.33 Most exosomes expressed epithelial cell adhesion molecule (EpCAM) on the surface. EpCAM aptamer-capped Fe3O4 NPs may also have higher peroxidase-like activity. EpCAM aptamer-capped Fe3O4 NPs have the potential for rapid and sensitive detection of exosomes. To overcome the limitations of conventional exosome isolation and detection methods, AE-based isolation and aptamer-capped Fe3O4 NPs were combined. An AE-based exosome isolation method was first proposed to isolate exosomes directly from plasma and cell culture medium. Exosomes isolated by AE magnetic beads had high recovery efficiency (>90%) and high purity (14.42× 1010 particles/mg). Subsequently, Aptamercapped Fe3O4 NPs were applied for visual and label-free detection of PCa exosomes from plasma. Though Fe3O4 NPs have weak peroxidase-like activity, the DNA aptamer is able to significantly increase the peroxidase activity of Fe3O4 NPs, catalyzing TMB- H2O2 for a visual change in color (from colorless to moderate blue), which is simple, rapid and intuitive. To the best of our knowledge, this is the first study to use aptamer-capped Fe3O4 NPs for label-free detection of PCa exosomes from plasma. With the AE-based isolation and the aptasensor combined, our method provides a powerful tool for the early diagnosis of PCa.

EXPERIMENTAL SECTION Materials and reagents. AE magnetic beads (particle size, 200 nm) were purchased from Chemicell (Berlin, Germany). PC3 and HeLa cells were purchased from the American Type Culture Collection. F12K medium was purchased from GIBCO (MA, USA). RPMI-1640, T75 flasks, FBS and penicillin-streptomycin solution were purchased from Hyclone (Logan, Utah, USA). Primary antibodies, such as anti-CD81, antiHSP70, anti-EpCAM, anti-PSA were purchased from Cell Signaling Technology (Danvers, United States). Secondary antibodies, such as anti-rabbit HRP and anti-mouse HRP were purchased from Jackson laboratories (Pennsylvania, USA). Enhanced chemiluminiscence (ECL) kits were purchased from Thermo Fisher Scientific (Massachusetts, USA). BCA kit were purchased from Beyotime (Shanghai, China). And ELISA Kits were purchased from Feimobio (Beijing, China). All oligonucleotides were synthesized by Sangon (Shanghai, China), and their sequences were illustrated in Table S1. The sequence of EpCAM aptamer and PSA aptamer were designed according to relevant reference29,35. Fe3O4 NPs (particle size: 50 nm) were purchased from Sigma (St. Louis Missouri, USA). The TMB was purchased from Tokyo Chemical Industry (Shanghai, China). All the other chemical reagents were purchased from Sigma. Cell culture. PC3 and HeLa cells have passed the test for mycoplasma contamination. PC3 cells were cultured in F12K, supplemented with 20% of FBS and 1% of penicillin-streptomycin solution (Hyclone). HeLa cells were cultured in RPMI 1640, supplemented with 10% of FBS and 1% of penicillin-streptomycin solution. All cells were cultured in an incubator under 5% CO2 at 37°C. Collection, storage and preparation of plasma samples and cell culture medium. This study was approved by the Institutional Review Board (IRB) of Tsinghua University (20180026). All plasma samples were collected following IRB guidelines. Consent was obtained for any experimentation with human subjects. Whole blood samples (5 mL) were collected from healthy donors and immediately centrifuged at 1000 g for 10 min at 4°C to obtain plasma samples. Then plasma samples were stored at -80℃ until use. Preparation of simulated plasma samples. 5.0× 106 cells/mL of PC3 and HeLa cells were grown in T75 flasks for 2 days until they reached a confluency of 70-80%. Then, the culture medium was replaced with serum-free medium for 48 h. For exosome isolation, the cell medium was collected and centrifuged at 3000



g for 10 min. Next, the supernatant was centrifuged at 10,000 g for 30 min to discard cellular detritus. Afterward, the supernatant was filtered with a 0.22 μm filter. A total of 100 mL of supernatant was captured and continuously centrifuged at 160,000 g and 4 °C for 70 min. Following that, the exosome pellets were washed with PBS centrifuged at 160,000 g and 4 °C for 70 min. the exosome pellets were suspended in PBS and stored at -20 °C until use. PC3 exosomes were spiked into exosome-deleted plasma to generate simulated plasma samples. Isolation of exosomes by the AE magnetic beads. AE bead suspension in tubes (80 μL) were vortexed for 30 s to fully resuspend the AE magnetic beads. Then, AE magnetic beads were separated from the store buffer with a magnetic stand. The positive charge on the surface of the AE magnetic beads was equilibrated with equilibration buffer (1 mL of 25 mM NaCl and 25 mM HEPES aqueous solution, pH = 6.2) in the above tube for 2 min. Following that, a magnetic stand was used to collect the AE magnetic beads at the bottom of the tube and discard the supernatant. For exosome isolation, simulated samples (500 μL)/ plasma samples (500 μL) or cell culture medium (20 mL) were added to the aforementioned tube containing the AE magnetic beads mentioned before and incubated for 5-30 min. The beads were drawn at the bottom of the EP and the impurities such as positively charged proteins and uncharged biological macromolecules were washed away with washing buffer (1 mL of 25 mM NaCl and 50 mM HEPES aqueous solution, pH = 6.2). After that, exosomes on AE magnetic beads were eluted by 100 μL of a certain concentration of NaCl, 50 mM HEPES aqueous solution. Finally, a magnetic stand was applied to collect the AE magnetic beads at the bottom of the tube. The purity of exosomes could be calculated by “Concentration of exosomes (particles/mL)/ Concentration of total protein (mg/mL)”. The exosome suspensions were collected and stored at -20°C. Immunogold transmission electron microscopy (IG-TEM). Exosomes isolated by UC/AE were mixed with an equal volume of 4% paraformaldehyde. For immune staining, 7μL of the mixture were placed onto formvar-carbon coated grids and allowed to absorb to the formvar for 2 min. Then PBS were dripped on a sheet of Parafilm. The grids were transferred to drops of PBS with clean forceps and washed twice for 3 min. Then grids were transferred to 50 mM glycine for 3 min and repeated for 3 times. Then grids were transferred to a drop of blocking buffer (PBS/5% (w/v) BSA) for 10 min. The grids were immediately placed into the primary antibody at the appropriate dilution (1: 100 anti-CD81) overnight at 4℃. After that, all the grids were rinsed with PBS then floated on drops of the anti-mouse secondary antibody attached with 5-nm gold particles for 2 h at room temperature. Grids were washed with PBS and were placed in 1% glutaraldehyde for 2 min. After rinsing in PBS and distilled water the grids were allowed to dry and stained for contrast using uranyl acetate. Finally, the samples were viewed with a FEI Tecnai F20 TEM D545. AE chromatography. Chromatographic experiments were conducted on an AKTA system (GE, America) consisting of a well plate automatic liquid sampler for injection, a degasser, a quaternary pump, and a diode array detector (DAD). Absorbance at 280 was monitored simultaneously. Exosomes were purified with a DEAE column (GE). The column was equilibrated with equilibration buffer A (50 mM HEPES, pH 8.2). 500μL of plasma were loaded. A wash step of 15 bed volumes was introduced before linear stepwise elution. Exosomes were eluted by a step gradient of 0–5–10-15-50% of elution buffer (50 mM HEPES, 1 M NaCl, pH 8.2) with a hold volume of 10 bed volumes) at the flowrate of 1 mL/min. Elution fractions were collected manually and pooled according to the chromatograms. Isolation of exosomes from plasma by UC. For exosome isolation, plasma samples (500 μL) were


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centrifuged at 3000 g for 10 min to discard blood cells. Next, the supernatant was centrifuged at 10,000 g for 30 min to discard cellular detritus. Afterwards, the supernatant was diluted with PBS and filtered by 0.22 μm filter. Then, the plasma diluent was centrifuged at 160,000 g for 70 min at 4°C. Subsequently, the exosome pellets were resuspended in 24 mL of PBS and centrifuged at 160,000 g for 70 min at 4°C. Finally, the exosome pellets were resuspended in 100 μL of PBS and stored at -20°C. Flow cytometry analysis of exosome-bound beads. Exosomes isolated by UC/AE beads were bound with CD9+ beads (Invitrogen) by mixing 10 μg exosomes with CD9+ beads (100 μL) at 4°C overnight. The suspension was diluted to 400 μL with PBS. Then the tube was placed in magnetic separator for 1-2 min before removing all supernatant. Exosome-bound beads were washed by 300 μL PBS and resuspended by 100 μL of PBS. Anti-human CD9-RPE was added into 100 μL of exosome-bound beads and the mixture was incubated at room temperature for 45 min on an orbital shaker at 1000 rpm. Mouse IgG1-RPE was added into 100 μL of exosome-bound beads to be negative control. Then exosome-bound beads were washed by 300 μL of PBS for two times. Finally, exosome-bound beads were resuspended in 300 μL of PBS and used for flow cytometry analysis. The percentage of positive beads were calculated relative to the total number of beads analyzed per sample (100,000 events). This percentage was therein referred to as the percentage of beads with CD9+ exosomes. Visible detection of exosomes. Exosomes were isolated from healthy plasma samples by AE. Then, exosomes were detected by a visible detection method. The EpCAM aptamer (1 μL, 100 μM) and 5 μL of 0.5 mg/mL Fe3O4 NPs were mixed for 10 min. Then exosomes solution was spiked into the mixtures and then thoroughly mixed by vortex. After 5 min, 5.0 μL of TMB (10 mM) and 1.0 μL of H2O2 (200 mM) were added to the above mixtures. HAc-NaAc buffer was then added into the above mixtures to get the final volume of 100 μL. The resultant solutions were incubated at 37 °C in the dark for 20 min, and the OD450 nm was measured with a microplate reader. Total number of aptamers modified on Fe3O4 NPs was calculated by “△Conc. of aptamer× Volume of reaction×6.02×1023 ×10-9/ (330× Number of bases)”. Where △Conc. of aptamer was detected by UV spectrophotometer, Number of particles of Fe3O4 NPs was detected by NTA. Finally, the copies of aptamers modified on each Fe3O4 NP was calculated by “Copies of aptamers modified on Fe3O4 NPs/ Number of particles of Fe3O4 NPs”. Visible and quantitative detection of PCa exosomes. PCa exosomes were quantitatively detected by PSA aptamer-capped Fe3O4 NPs. The PSA aptamer (1 μL, 100 μM) and 5 μL of 0.5 mg/mL Fe3O4 NPs were mixed by vortex for 10 min. Then different concentrations of PCa exosome solution were spiked into the mixtures and then thoroughly mixed with vortex. After 5 min, 5.0 μL of TMB (10 mM) and 1.0 μL of H2O2 (200 mM) were added into the mixtures. Then HAc-NaAc buffer was added to get the final volume of 100 μL. The resultant solutions were incubated at 37 °C for 20 min dark, and D450 nm was measured by a microplate reader. Then HeLa exosomes were used to replace PCa exosomes to test the selectivity of the aptasensor. Nanoparticle-tracking analysis. A nanoparticle-tracking analysis (NTA) system (NS500, Nanosight, England) was used to measure the concentration of exosomes. Exosomes isolated by UC and AE were diluted 50-fold with PBS before NTA measurements. All particle size analyses used the same set of parameters to ensure comparable results. Western blotting. 20 μL of exosome suspension was loaded per well and then separated by SDS-PAGE. Proteins were transferred onto polyvinylidene fluoride membranes and blocked with 5% skim milk. Primary antibodies such as anti-CD81, anti-CD9, anti-PSA, and anti-HSP70 were used, followed by secondary



antibodies, anti-rabbit HRP and anti-mouse HRP. Target proteins were detected with enhanced ECL kits.

RESULTS AND DISCUSSION Isolation of exosomes by AE magnetic beads from simulated samples. In order to evaluate the AEbased exosome isolation, AE magnetic beads were used to isolate exosomes from simulated plasma samples. The surface Zeta potential value of the exosomes was -27.64 mV (Fig. S1). The principle and experimental procedure are shown in Figure 1A. Negatively charged exosomes could

bind with positively charged AE magnetic beads. Impurities such as positively charged/uncharged proteins, cell debris, large particles couldn’t bind with AE magnetic beads and were washed away by the washing buffer. Then, the optimized elution buffer was used to elute exosomes off AE magnetic beads. Proteins and nucleic acid with more negative charge than that of exosomes would stay on the surface of AE magnetic beads. Finally, a magnetic stand was used to separate the exosomes from the AE magnetic beads and wipe off protein, nucleic acid and large particles. The AE magnetic beads were characterized by transmission electron microscopy (TEM). As displayed in Figure 1B, AE magnetic beads had uniform particle sizes (~ 200 nm). AE magnetic beads were added to simulated samples (500 μL) to capture exosomes and then washed by washing buffer. Following that, the AE magnetic beads that captured exosomes were characterized by TEM, showing that exosomes were found on the surface of the AE magnetic beads (Fig. 1C). Finally, the exosomes were eluted by high concentration of NaCl and characterized by TEM. As displayed in Figure 1D, exosomes were repelled from AE magnetic beads after elution. As displayed in Figure 2, the exosomes appeared in TEM image as well-defined membrane-bound vesicles (Fig. 2A). Nanoparticle tracking analysis (NTA) of the exosomes in simulated samples before AE-based isolation (mean size: 143.03 nm; concentration: 2.10 × 109 particles/mL) and the exosome suspension retrieved from the collection chamber after AE-based isolation (mean size: 122.46 nm; concentration: 1.19 × 1010 particles/mL) (Fig. 2B, C). To obtain the best recovery efficiency, different concentrations of AE magnetic beads were incubated with simulated samples for 10 min. The recovery efficiency of exosomes was higher than 80% when the concentration of AE magnetic beads≥ 1.25 μg/mL (Fig. 2D). Next, the incubation time was optimized by incubating the AE magnetic beads (1.25 μg /mL) with simulated samples for 10-30 min. The recovery efficiency of exosomes was higher than 90% when the incubation time ≥15 min (Fig. 2E). In the following experiments, AE magnetic beads (1.25 μg/mL) were incubated with simulated samples for 15 min. Then the elution condition was optimized by eluting exosomes with different concentration of NaCl and 50 mM HEPES. The elution efficiency of exosomes was higher than 90% when the concentration of NaCl≥ 200 mM (Fig. 2F). Therefore, AE-based isolation method can effectively enrich exosomes from simulated samples

and wipe off protein, nucleic acid and large particles. Isolation of exosomes by AE magnetic beads from plasma samples. In order to detect and analysis exosomes in PCa plasma samples, we need to isolate exosomes directly from plasma samples. So AE magnetic beads were applied to isolated exosomes directly from plasma samples (500 μL). As displayed in Figure 3, the exosomes isolated from plasma samples were characterized by TEM and NTA. Exosomes isolated by AE magnetic beads were well-defined membrane-bound vesicles (Fig. 3A). NTA results of plasma before AE-based isolation (mean size: 278.84 nm; concentration: 5.08 × 1010 particles/mL) and exosomes after AE-based isolation (mean size: 120.47 nm; concentration: 7.01 × 1010 particles/mL)


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confirmed that AE-based isolation method effectively enriched exosomes and wipe off large particles (such as cell debris, large vesicles) in plasma (Fig. 3B, C). Following a similar optimization procedure, different concentrations of AE magnetic beads were incubated with plasma samples for 10 min. The concentration of eluted exosomes was higher than 5.35× 1010 particles/mL when the concentration of AE magnetic beads≥ 1.875 μg/mL (Fig. 3D). Next, the incubation time was optimized by incubating the AE magnetic beads (1.875 μg/mL) with plasma for 2 -40 min. The concentration of eluted exosomes was higher than 14.96× 1010 particles/mL when the incubation time was 20 min (Fig. S2A). The AE magnetic beads were next washed with washing buffer for a variable number of washes, and the influence of the number of washes on the concentration of exosomes was examined by NTA. The concentration of eluted exosomes was the highest when AE magnetic beads were washed only once (Fig. S2B), and protein impurities in the exosome suspension were greatly reduced after one time washing (Fig. S2C). Subsequently, the pH and concentration of the elution buffer were optimized. An aqueous solution containing 150 mM NaCl, 50 mM HEPES was adjusted to different pH values (5.2, 6.2, 7.2, 8.2, and 9.2) to elute exosomes. The concentration of exosomes was the highest (8.20 ×1010 particles/mL) when the pH of the elution buffer was 8.2 (Fig. 3E). Then the concentration of NaCl was optimized by eluting exosomes with different concentration of NaCl and 50 mM HEPES (pH = 8.2). The concentration of exosomes was higher than 11.10 ×1010 particles/mL when the concentration of NaCl≥ 200 mM (Fig. 3F). Higher concentration of NaCl could bring in more protein impurities. Therefore, the optimized elution buffer contained 200 mM NaCl, 50 mM HEPES (pH= 8.2). The entire process of AE-isolation takes less than 30 min. Comparison to conventional exosome isolation methods. The enrichment efficiency of AE-based exosome isolation was compared to a commonly used exosome-isolation method, i.e., UC. Exosomes were isolated from 500 μL of plasma samples（N= 3）using AE magnetic beads and UC. Exosomes enriched by these two methods were characterized by NTA, immunogold TEM (IG-TEM), flow cytometry, western blotting, BCA and mass spectrometry (MS). NTA analysis showed that the mean size of exosomes isolated by UC was 139.64 nm and the mean size of exosomes isolated by UC was 108.52 nm (Fig. 4A, B). Exosome isolated by AE had a more uniform particle size distribution than those isolated by UC. And the concentration of exosomes yielded by AE was 2.53-fold higher compared to that of exosomes yielded by UC (Fig. 4C). Flow cytometry analysis displayed that the concentration of CD9+ exosomes yielded by AE was 2.49-fold higher compared to those yielded by UC (Fig. S3A). Which is consistent with NTA results. IG-TEM analysis of exosomes isolated by UC and AE both revealed lipid bilayer and CD81 positivity (Fig. 4D, E). There are many protein aggregates in the IG-TEM image of exosomes isolated by UC (Fig. 4D), while there are few protein aggregates in the IG-TEM image of exosomes isolated by AE (Fig. 4E). The BCA results demonstrated that the total protein concentrations of exosomes isolated by UC, AE was (0.20 ± 0.006) mg/mL and (0.16 ± 0.002) mg/mL (Fig. S3B). The purity of exosomes isolated by AE is (4.36 ± 0.10) ×1011 particles/mg protein, which was 2.79-fold higher than that of exosomes isolated by UC (Fig. 4F). The most highly abundant plasma protein-albumin is the major protein impurity in exosomes isolated from plasma.13 The concentrations of albumin in the exosome suspension isolated by AE is lower than by UC (Fig. S3C). These results displayed that the AE-based isolation provided a higher yield of enriched exosomes with higher purity than UC. Further, the cellular components of proteins in exosomes isolated by AE and UC were analyzed by HPLC/MS, and the similar results were achieved (Fig. 4G). Western blotting results



demonstrated that the levels of CD81 and HSP70 in exosomes isolated by AE (right) were higher than by UC (left) (Fig. 4H). Moreover, the entire process of AE-isolation takes less than 30 min, while the entire process of UC-isolation takes more than 6h. Overall. the AE-based exosome isolation method proposed in this study displayed better performance compared to UC. We have also isolated exosomes directly from plasma samples by the standard AE chromatography. The results were showed in Figure S4. We could see that the concentration of exosomes isolated by AE chromatography was (3.02 ± 0.89) ×109 particles/mL, which was 23.38-fold lower than that of exosomes isolated by AE magnetic beads (Fig. S4C, D). The BCA results demonstrated that the total protein concentration of exosome solution isolated by AE chromatography, AE magnetic beads was (1.52 ± 0.006) mg/mL and (0.16 ± 0.002) mg/mL, separately (Fig. S4E). The purity of exosomes isolated by AE magnetic beads was 257.75-fold higher than that of exosomes isolated by AE chromatography (Fig. S4F). Therefore, the AE magnetic beads-based exosome isolation method displayed better performance compared to AE chromatography. Isolation of exosomes by AE magnetic beads from cell culture medium. AE magnetic beads were also applied to isolated exosomes directly from cell culture medium (20 mL) of HeLa cells. NTA results of cell culture medium before AE-based isolation (mean size: 201.87 nm; concentration: 2.83 × 109 particles/mL) (Fig. S5A) and HeLa exosomes after AE-based isolation (mean size: 115.93 nm; concentration: 2.82 × 1010 particles/mL) (Fig. S5B) confirmed that the AE-based isolation method could effective enrich HeLa exosomes in cell culture medium. Exosomes isolated by AE magnetic beads appeared in TEM image as welldefined membrane-bound vesicles (30-120 nm) (Fig. S5C). The enrichment efficiency of AE-based exosome isolation was compared with commonly used exosome-isolation method- UC. Exosomes were isolated from 20 mL of cell culture medium（N= 3）using AE and UC. As displayed in Figure S5D, the concentration of exosome biomarker-CD81 in exosomes isolated by AE was 2.36-fold higher than that in exosomes isolated by UC. Thus, AE-based exosome isolation method proposed in this study could be used for isolation of exosomes from cell culture medium. Visible detection of exosomes. Exosomes were isolated from healthy plasma samples by AE magnetic beads and used for visible detection. In this study, an aptasensor was constructed by using EpCAM aptamer and Fe3O4 NPs to detect exosomes. The principle of the visible detection of exosomes was displayed in Fig. 5A. TMB was used as the substrate of peroxidase. Aptamer-capped Fe3O4 NPs were found to have higher peroxidase activity than untreated Fe3O4 NPs, which can catalyze a visual color change in TMB for from colorless to blue in the presence of H2O2 (Fig.5 B, C). In the presence of exosomes, the catalytic activity of Fe3O4 NPs decreased, and thus they produced lower UV-vis absorbance and a moderate blue color (Fig.5 C).

A negative control (without exosomes, curve c) was used to exclude the impact of the released aptamer caused by the nonspecific adsorption on the aptamer- Fe3O4 NPs. The DNA aptamer for EpCAM was absorbed onto the surface of Fe3O4 NPs through noncovalent reactions. The quantity of EpCAM aptamer modified on each Fe3O4 NP was displayed in in Figure S6. We could see that 341 copies of EpCAM aptamer were modified on each Fe3O4 NP (Fig.S6B). Due to the high affinity between aptamer-capped Fe3O4 NPs and TMB, the peroxidase activity could be improved. Meanwhile, in the presence of exosomes, the EpCAM aptamer modified on Fe3O4 NPs could bind to EpCAM on the surface of exosomes via conformational changes, leading to the desorption of aptamer from the Fe3O4 NP surface. Consequently, some Fe3O4 NPs


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returned to their original state, resulting in a decrease in catalytic activity and color change. The decreased color change and absorbance depended on the concentration of the exosomes. The color changes could also be directly observed by the naked eye or quantified using as the absorbance change using a UV-vis spectrometer. Next, the feasibility of our aptasensor was examined. Sensitivity of the aptasensor. Exosomes were isolated from healthy plasma samples by AE magnetic beads. Different concentrations of exosomes were then detected by aptasensor. As displayed in Figure S8, the solutions’ color was getting lighter with increasing concentration of exosomes (Fig. S8A). The OD562 nm was 2.79 (RSD=1.26%), 2.48(RSD=0.93%), 2.14(RSD=0.27%), 1.46(RSD=2.20%), 1.02 (RSD=1.12%), respectively, when the concentration of exosomes was 0.4×108, 1.0×108, 2.0×108, 4.0×108, 6.0×108 particles/mL (Fig. S8B). The OD562 nm was linearly with the concentration of exosomes. The relationship between OD562 nm and concentration of exosomes could be derived as OD562 nm = -0.3124×C (particles/mL)+ 2.817 (R2=0.9856), where OD562 nm was the absorbance at 652 nm, and C was the concentration of exosomes. The limit of detection (LOD) of the aptasensor based on the 3σ method was 7.0×106 particles/mL, which was 74 times lower than the existing visible detection method of exosomes32. The improved sensitivity may result from the high recovery efficiency of AE-based isolation and lower impurities. The characterization and quantification of PCa exosomes. PC3 exosomes were spiked into exosomefree plasma to generate simulated PCa plasma samples, and then PCa exosomes were isolated by AE. The purified exosomes were analyzed by TEM and western-blotting, EpCAM and PSA were obviously observed (Fig. S7A, B). PCa exosomes express PSA on their surface. Thus, a PSA aptamer was used to replace EpCAM to specifically detect PCa exosomes. The quantity of PSA aptamer modified on each Fe3O4 NP was

displayed in in Figure S6B. We could see that 357 copies of PSA aptamer were modified on each Fe3O4 NP (Fig.S6B). As displayed in Figure 6, The OD562 nm was 4.68 (RSD=4.23%), 4.48(RSD=2.21%), 4.05(RSD=1.45%), 3.09(RSD=0.17%), 2.17(RSD=0.20%), respectively, when the concentration of PCa exosomes was 0.4×108, 1.0×108, 2.0×108, 4.0×108, 6.0×108 particles/mL. The OD562 nm decreased when the concentrations of PCa exosomes increased from 0.4×108 to 6.0×108 particles/mL (Fig. 6A). OD562 nm scaled linearly with the concentration of PCa exosomes (Fig. 6B). The relationship between OD562 nm and concentration of PCa exosomes could be derived as OD562 nm = -0.4544×C (particles/mL) + 4.915 (R2=0.9989), where C was the concentration of PCa exosomes (Fig. 6B). The LOD of the aptasensor based on the 3σ/S method was 3.58×106 particles/mL. We further tested the selectivity of the aptasensor using HeLa exosomes and BSA to avoid the nonspecific adsorption on the aptamer-Fe3O4 NPs. There was also no significant change in OD562 nm when the concentrations of HeLa exosomes changed from 0.4 ×108 to 6.0×108 particles/mL (Fig. S6C). There was also no significant change in OD562 nm when the concentrations of BSA changed from 0.5 to 5.0×108 mg/mL (Fig. S6D). The OD562 nm due to the PC3 exosomes are significantly lower than those for HeLa exosomes (Fig. S6C), demonstrating excellent selectivity for PCa exosome detection. Therefore, PSA aptamer -capped Fe3O4 NPs could be used for the specific detection of PCa exosomes.

CONCLUSION In this study, an AE-based exosome isolation method was proposed to isolate exosomes directly from plasma and cell culture medium by AE magnetic beads for the first time. Exosomes isolated by AE magnetic beads appear in TEM image as well-defined membrane-bound vesicles (30-120 nm). AE-based isolation



method could effective enrich exosomes and wipe off cell debris and protein purities in plasma. Compared to the conventional UC-based exosome isolation method, AE-based exosome isolation has several advantages: 1) the isolation procedure time was reduced to 30 min, which is significantly shorter than previous studies13,37, 2) the reproducibility of exosomes was higher (recovery efficiency of exosomes > 90%)17,38, and 3) the purity was improved (2.79-fold higher than exosomes isolated by UC, 257.75-fold higher than that of exosomes isolated by AE chromatography). Therefore, the AE-based exosome isolation method displayed better performance than UC. An aptasensor based on aptamer-capped Fe3O4 NPs was also constructed for visual and label-free detection of PCa exosomes from plasma for the first time. This aptasensor could detect PCa exosomes with LOD of 3.58×106 particles/mL. The aptasensor has several advantages: 1) The aptasensor based on aptamercapped Fe3O4 NPs used aptamer to replace antibodies, the stability was better.39 2) the sensitivity was better (the LOD of our method was 74-times lower than the existing visible detection method of exosomes32) 3) the specificity was good, which had excellent selectivity for PCa exosomes, 4) the results could be readout by naked eye, increasing portability than other methods17,40 which improved detection cost, and interfered

the detection accuracy. In the future, we are going to isolate exosomes directly from clinical PCa plasma, then use the aptasensor to detect PCa exosomes in clinical PCa plasma samples for the early diagnosis and staging of PCa. This study provides an efficient and practical approach to the isolation and detection of exosomes, which is promising for the early diagnosis of PCa. Furthermore, the proposed aptasensor holds potential to be used in other diseases by simply changing the aptamer.

ASSOCIATED CONTENT Supporting information The Supporting Information is available free of charge on the ACS Publications website. Eight figures showing characterization of exosomes isolated by AE magnetic beads, optimization of the AE-based exosome isolation method, three tables showing the sequence of EpCAM/PSA aptamer and the quantity of aptamers modified on each Fe3O4 NPs

AUTHOR INFORMATION *Corresponding Author E-mail: [email protected], Phone: (86)-10-62795227 ORCID Junge Chen: 0000-0003-4687-884X Author contributions The manuscript was written through contributions of all authors, who have given approval to the final version of the manuscript. Notes The authors declare no competing financial interest.


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ACKNOWLEDGMENTS This study was supported by the National Key Research and Development Program of China (2016YFC0800703), the National Natural Science Foundation of China (31500691), Beijing Municipal Science & Technology Commission (Z161100000116031) and the Beijing Lab Foundation. The authors thank Xiurui Zhu for advice on the manuscript.

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FIGURES

Figure 1. Schematic of the AE-based isolation of exosomes. (A) Diagram of AE-based isolation of exosomes. (B) TEM characterization of AE magnetic beads. (C) TEM characterization of AE magnetic beads after capture of exosomes. (D) TEM characterization of AE magnetic beads and exosomes after elution.



Figure 2. Characterization of exosomes isolated by AE magnetic beads. (A) TEM characterization of exosomes isolated by AE magnetic beads from simulated plasma samples. (B) NTA analysis of exosomes in simulated plasma samples before AE-based isolation. (C) NTA analysis of exosomes after AE-based isolation. (D) The effect of the concentration of AE magnetic beads on the recovery efficiency of exosomes. (E) The effect of the incubation time on the recovery efficiency of exosomes. (F) The effect of the concentration of NaCl on the elution efficiency of exosomes.


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Figure 3. Characterization of exosomes isolated by AE magnetic beads from plasma samples. (A) TEM characterization of exosomes isolated by AE magnetic beads. (B) NTA analysis of plasma before AE-based isolation. (C) NTA analysis of plasma exosomes after AE-based isolation. (D) The effect of the concentration of AE magnetic beads on the recovery efficiency of exosomes. (E) The effect of pH on the recovery efficiency of exosomes. (F) The effect of the concentration of NaCl on the elution efficiency of exosomes.



Figure 4. Comparison of different exosome isolation methods. (A) NTA analysis of exosomes isolated by UC. (B) NTA analysis of exosomes isolated by AE magnetic beads. (C) Concentrations of exosomes isolated by UC and AE magnetic beads. (D) IG-TEM of exosomes isolated by UC. (E) IG-TEM of exosomes isolated by AE magnetic beads. (F) The purity of exosomes isolated by UC and AE magnetic beads. (G) HPLC/MS characterization of proteins in exosomes isolated by UC (left) and AE (right). (H) Western blotting of exosome biomarkers CD81 and HSP70 in exosomes isolated by UC (left) and AE (right).


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Figure 5. Visible detection of exosomes. (A) Schematic representation of the detection mechanism for visible detection of exosomes. (B) UV–vis absorption spectra of TMB-H2O2 (curve a), TMB-H2O2+ Fe3O4 NPs (curve b), TMB-H2O2+ aptamer-Fe3O4 NPs (curve c), TMB-H2O2+ aptamer-Fe3O4 NPs + exosomes (curve d). (C) Digital images of TMB-H2O2 (image a), TMB-H2O2+ Fe3O4 NPs (image b), TMB-H2O2+ aptamer-Fe3O4 NPs (image c), and TMB-H2O2+ aptamerFe3O4 NPs + exosomes (image d).



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Figure 6. UV–vis absorption spectra of different concentrations of PCa exosomes. UV–vis absorption spectra of different concentrations of PCa exosomes. (A) UV–vis absorption spectra of the solution in the absence and presence of different concentrations of PCa exosomes (from b to f: the concentrations of PCa exosomes were 0.4, 1.0, 2.0, 4.0, and 6.0×108 particles/mL). The inset shows the corresponding photographs. (B) The linear relationship between OD562 concentration of PCa exosomes.


nm

and

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A table of contents graphic


Rapid Isolation and Visible Detection of Tumor-derived Exosomes

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