Single-Exosome-Counting Immunoassays for ... - ACS Publications

Letter Cite This: Nano Lett. 2018, 18, 4226−4232

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Single-Exosome-Counting Immunoassays for Cancer Diagnostics Chunchen Liu,†,‡,§,∥ Xiaonan Xu,§,∥ Bo Li,†,‡ Bo Situ,†,‡ Weilun Pan,†,‡ Yu Hu,§ Taixue An,†,‡ Shuhuai Yao,*,§ and Lei Zheng*,†,‡ †

Department of Laboratory Medicine and ‡Guangdong Engineering and Technology Research Center for Rapid Diagnostic Biosensors, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China § Department of Mechanical and Aerospace Engineering, The Hong Kong University of Science and Technology, Hong Kong, China

Nano Lett. 2018.18:4226-4232. Downloaded from pubs.acs.org by UNIV OF SUNDERLAND on 10/13/18. For personal use only.

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ABSTRACT: Exosomes shed by tumor cells have been recognized as promising biomarkers for cancer diagnostics due to their unique composition and functions. Quantification of low concentrations of specific exosomes present in very small volumes of clinical samples may be used for noninvasive cancer diagnosis and prognosis. We developed an immunosorbent assay for digital qualification of target exosomes using droplet microfluidics. The exosomes were immobilized on magnetic microbeads through sandwich ELISA complexes tagged with an enzymatic reporter that produces a fluorescent signal. The constructed beads were further isolated and encapsulated into a sufficient number of droplets to ensure only a single bead was encapsulated in a droplet. Our droplet-based single-exosome-counting enzyme-linked immunoassay (droplet digital ExoELISA) approach enables absolute counting of cancer-specific exosomes to achieve unprecedented accuracy. We were able to achieve a limit of detection (LOD) down to 10 enzyme-labeled exosome complexes per microliter (∼10−17 M). We demonstrated the application of the droplet digital ExoELISA platform in quantitative detection of exosomes in plasma samples directly from breast cancer patients. We believe our approach may have the potential for early diagnosis of cancer and accelerate the discovery of cancer exosomal biomarkers for clinical diagnosis. KEYWORDS: Droplet microfluidics, exosomes, droplet digital ExoELISA, cancer diagnostics

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measurement.16−18 However, NTA only provides an estimated number of exosomes at a high concentration level (1 × 107− 109 particles/mL) and lacks specificity.19 Western blot, ELISA, and flow cytometry all require large amounts of sample input and have limited sensitivity.20,21 Unfortunately, in the early stage of cancer limited tumor-derived exosomes in peripheral blood circulation can hardly be detected with these conventional quantification methods. Many efforts have been made by researchers to improve the sensitivity of the detection methods, including miniaturized microfluidic platforms,22−25 aptamer-based electrochemical sensors,26−29 surface plasmon resonance (SPR).30,31 and Raman scattering.32,33 However, these detection methods are performed in a bulk solution, which hardly enables absolute quantification or classification. As the cancer biomarkers that present in the early stage in liquid biopsy are at low concentrations in the range of 10−12− 10−16 M,34 to quantitate such low abundance markers the required sensitivity for detection needs to be at the single molecule level.35−37 Recently, single extracellular vesicle analysis (SEA), based on photon-counting techniques, has been applied for multiplexed profiling of single extracellular

xosomes are extracellular vesicles of 30−150 nm in size that are derived from eukaryotic cells that circulate in body fluids.1,2 They carry numerous molecular information like proteins and nucleic acids from the parent cells and therefore play a vital role for intercellular communication.3,4 In the past decade, accumulated evidence has indicated that the exosome molecular cargo shed from tumor tissues can be identified as potential noninvasive biomarkers for cancer diagnosis because it reflects the genetic or signaling alterations of the parent tumors.5−9 For instance, Glypican-1 (GPC-1), an exosomal membrane protein, was discovered to have much higher expression on the cancerous exosomes than the noncancerous by immunoblotting analysis,10 revealing its clinical value as an exosomal biomarker for the early diagnosis of pancreatic, breast, and colorectal cancer.11−13 Exosomes secreted by nucleated cells are widely present in human biofluids14,15 and there exists various exosome subpopulations. Recently, the subpopulation of tumor-derived exosomes is found to be valuable for clinical diagnostics. To accurately quantify and classify the tumor-derived exosomes from biofluids is potentially significant for cancer diagnostics, prognosis, and monitoring the response of therapy. Conventional methods such as nanoparticle tracking analysis (NTA), Western blot, ELISA, and flow cytometry have been widely adopted in research laboratories for exosome quantity © 2018 American Chemical Society

Received: March 23, 2018 Revised: May 28, 2018 Published: June 11, 2018 4226

DOI: 10.1021/acs.nanolett.8b01184 Nano Lett. 2018, 18, 4226−4232

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Figure 1. Schematic showing the droplet digital ExoELISA for exosome quantification. (a) Single exosome immunocomplex constructed on a magnetic bead. (b) Substrate and beads are coencapsulated into microdroplets. (c) Droplet digital ExoELISA chip. (d) Fluorescent readout for counting the positive droplets with the target exosomes.

Figure 2. Characterization of exosomes. (a) TEM shows exosomes with double-wall lipid membrane layers ranging approximately 30−150 nm in diameter. (b) size distribution of MDA-MB-231 exosomes by NTA analysis. Red band depicts three repetitive experiments. (c) The expression of CD63 (the exosomal marker) and GPC-1 (the diagnostic marker) in MDA-MB-231 exosomes and parent cells by Western blot analysis. Equal amounts of proteins (20 μg) in exosomes and cells were loaded.

vesicles using ELISA.38 Careful buffer washing and complex imaging procedures are required to differentiate single vesicles from protein complexes or other clusters due to their low signal-to-noise ratios, and the detection limit is still quite high (e.g., with an intensity cutoff of 102 counts).38 Nevertheless, these methods are still impractical for wide adoption due to the throughput and cost. Reliable platforms for quantification of exosomes with high sensitivity and specificity are still lacking. In recent years, digital PCR and digital ELISA platforms have revolutionized detection technologies for absolute quantification of nucleic acids and proteins.39−41 In contrast

to the conventional biological and chemical assays conducted in large volumes in pipettes, beakers, tubes, or flasks, the basic principle of digital quantification of molecules is to divide the sample uniformly into a large quantity of small compartments (either in microwells or in droplets).42,43 By doing so, an individual molecule is confined in a small volume where the signal can be amplified and concentrated for detection.44 Compartmentalization technology that ensures the isolation of molecules in each compartment to follow the Poisson distribution is the core to the success of digital quantification.45,46 Droplet microfluidics that generates uniform droplets 4227


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Figure 3. (a) Prepared beads and FDG substrate are coencapsulated into 40 μm diameter droplets which spread in one layer in the device for detection. (b) Droplet digital ExoELISA calibration results showing the dynamic range of the captured exosomes spans 5 orders of magnitude. Dashed line is the background plus 3 times the standard deviation indicating the LOD (∼10 exosomes/μL). (c) Negative control without target exosomes. (d−h) Gradient of the fluorescence readout by serial dilution of the exosome sample isolated from MDA-MB-231. NanoSight was used as a benchmark measurement for the exosome number concentration.

at the pico- to nanoliter scale in high throughput (in kHz) has enabled numerous single-molecule assays to be performed in parallel.47,48 In recent years, there has been tremendous progress in the development of droplet-based platforms for the formation and manipulation of monodispersed droplets and the associated use of a range of fluorescence-based techniques for high-throughput and highly sensitive analysis of droplet content.49 In this Letter, we develop a droplet-based single-exosomecounting immunoassay approach for digital quantification of exosomes. Exosome enzyme-linked immunosorbent assay (ExoELISA) is adopted to identify the exosomes with target membrane protein biomarkers. We named this method as droplet digital ExoELISA, the procedure of which is illustrated in Figure 1; magnetic beads serve as a medium for capture and separation of the target exosomes. First, the exosome suspension is mixed with a sufficient number of magnetic beads conjugated with capture antibodies that can selectively bind a specific protein on the exosome membrane. After effective magnetic separation and washing, one target exosome is immobilized and captured onto a magnetic bead. A detection antibody tagged with an enzymatic reporter further recognizes the antigen on the captured exosome, forming a single enzymelinked immunocomplex on the bead (Figure 1a). Second, the prepared beads and the enzymatic substrate are coencapsulated into a sufficient number of microdroplets to ensure that a majority of droplets contain no more than one bead, using a microfluidic chip (Figure 1b,c). Third, for those droplets that contain the beads with exosome immunocomplex, the substrate is catalyzed by the enzyme to emit fluorescein within the droplets (Figure 1d). On the basis of the statistics of the fluorescent droplets, the target exosome concentration can be calculated. We have demonstrated that the droplet digital ExoELISA approach is able to detect as few as ∼5 exosomes per microliter. Other than high sensitivity, the droplet digital ExoELISA offers high specificity and absolute quantification for

targeting exosomes with specific protein biomarkers. For clinical demonstration, we quantified the GPC-1(+) exosomes from breast cancer patients and the results yielded distinct GPC-1(+) expression level before and after surgery, suggesting the great potential of the droplet digital ExoELISA platform for cancer diagnostics. Exosomes were purified and isolated from a breast tumor cell line (MDA-MB-231) by multiple steps of ultracentrifugation following our previous work.50 Standard characterization of exosomes was performed using transmission electron microscopy (TEM), NTA ,and Western blot. As shown in Figure 2a, the TEM image revealed that the lipid bilayer structure remained intact on the purified exosomes after ultracentrifugation and the size of the exosomes ranged from 50 to 150 nm in diameter. With NTA analysis, we obtained the size distribution and concentration of the exosomes (Figure 2b). The prepared exosomes had an average size of 104.2 ± 3.9 nm in diameter and the corresponding concentration was 6.39 × 108 ± 4.90 × 106 particles per milliliter. In our experiments, CD63 protein, a member of the transmembrane 4 superfamily, was selected as the protein biomarker for capturing exosomes because CD63 is the exosome-enriched protein located on the membrane and, according to the literature, is commonly used for exosome capture.51,52 We performed the Western blot analysis, which showed the exosomal marker CD63 on the exosomes isolated from the MDA-MB-231 culture media was consistent with the CD63 protein extracted from the same cell line as a positive control, clearly indicating the existence of CD63 on these samples (Figure 2c, top row). Also, we used a dual-color super resolution microscopy to confirm the localization of CD63 on the exosome membrane (Figure S1a−c). We selected GPC-1 protein as the breast cancer reporter in our experiments as it has already been reported for breast cancer detection.12 The high expression of GPC-1 on exosomes from the MDA-MB-231 cell line and the location of GPC-1 on exosome membranes was confirmed by western bolt 4228


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Figure 4. Specificity of the assay. (a) Western blot analysis showing different expressions of GPC-1 in MDA-MB-231 cells (positive control) and exosomes isolated from MDA-MB-231, HL-7702, RAW264.7, and hES cell culture media. Each lane was loaded with 20 μg of proteins. (b) The specificity of the droplet digital ExoELISA with exosomes isolated from MDA-MB-231, HL-7702, RAW264.7, and hES cell culture media. Cases of the magnetic beads without CD63 Ab and detection sample solution without exosomes served as the negative controls. Each sample solution contained 6.39 × 104 exosome particles per microliter.

number of beads per droplet at 99.53%) capture at most one target exosome.54 Therefore, in our experiments we input 10× more beads than the expected exosomes to ensure single-exosome capture. To prove the successful capture of exosomes via CD63 antibody−antigen binding on beads, we carried out the TEM experiments for validation. The magnetic beads coated with CD63 capture antibody were exposed to two samples, one with MDA-MB-231 exosomes and the other without exosomes as the control group. Figure S2a shows a bare bead without exosomes on the surface while Figure S2b clearly shows that one exosome was constructed on a magnetic bead. These results demonstrated that the functionalized magnetic beads were able to bind the exosomes specifically in a single complex through ExoELISA. After single exosomes were captured on beads, we used anti-GPC-1, previously biotinylated with a biotin tag, as the detection antibody to bind GPC1 protein marker on the membranes of the target exosomes. After forming immunocomplex on the beads, the detection antibody was further conjugated with an enzymatic reporter, βgalactosidase, which catalyzes the fluorescein-di-β-D-galactopyranoside (FDG) substrate to produce a fluorescent signal for detection in the droplet microfluidic system. A flow-focusing droplet generation device with two sample inlets for the prepared bead sample and FDG substrate solutions respectively40 was used to generate droplets of 40 μm diameter in mineral oil (Figure 3a). Likewise, the encapsulation of beads in microdroplets is also based on the Poisson distribution. In our experimental protocol, we set the mean 4229


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detection of GPC-1(+) exosomes using clinical samples from serum of 5 healthy sample (HS), 5 patients with benign breast disease (BBD), 12 patients with breast cancer (BC), and 2 patients with breast cancer after surgery (BC-AS) (Figure 5).

methods for detection of exosomes (Table S1), our method achieved the lowest LOD. Because of in sample discretization, we input a sufficient quantity of beads to mix with exosomes and compartmentalize the beads into a sufficient number of droplets; in most measurements, we were able to achieve one fluorescent droplet representing one target exosome with over 99% confidence. Figure 3c shows the background of the assay, possibly caused by nonspecific binding to the surface of the beads or carry-through of free reporter enzymes into the encapsulated droplets, as suggested by previous reports.40,54 Therefore, special surface treatment and more effective washing steps may be needed to further improve the accuracy of our approach.55 Figure 3d−h shows the images of the fluorescent droplets in the chamber by taking the 10-fold serial dilution. It is noted that among the fluorescent droplets some droplets emitted stronger fluorescence signals than others. The variations could be due to various expressions of GPC-1 on a single exosome or the heterogeneous nature of single-enzyme catalysis.56 In our experiments, one million droplets were generated and the dynamic range allowed us to reach the range of 5 log of the linear regime. The dynamic range can be further extended by employing the two dependent Poisson statistics.41,54 The variety of exosome subpopulation protein biomarkers significantly complicate exosome counting. The differentiation of exosome subpopulations in our approach is based on immunoassay, which possesses excellent specificity. To check the specificity of GPC-1(+) exosome detection in breast cancer exosomes (MDA-MB-231 exo), we performed control experiments using three kinds of noncancerous exosomes including human normal liver exosomes (HL-7702 exo), mouse normal macrophage exosomes (RAW264.7 exo), and human embryonic stem exosomes (hES exo). We first used Western blot analysis to identify the expression levels of GPC-1 in MDAMB-231 exo, HL-7702 exo, RAW264.7 exo, and hES exo and found that the expression of GPC-1 in MDA-MB-231 exo was slightly higher than the other three groups (Figure 4a). Because of the limited detection capacity of Western blot, if the sample contains a small amount of GPC-1(+) exosomes, other proteins on the exosomes in the sample may interfere with the GPC-1(+) in Western blot analysis. Moreover, the Western blot analysis can only qualitatively indicate whether GPC-1 is expressed in the sample as it cannot measure the specific number of GPC-1(+) exosomes. Next, we investigated the specificity of our droplet digital ExoELISA for GPC-1(+) exosome detection among the four chosen exosomes and two negative controls: a sample using magnetic beads without CD63 Ab and a sample with no exosomes (Figure 4b). Before the experiments, NTA analysis was used to estimate the exosome number concentrations. The measured values were 4.22 × 108, 2.86 × 108, and 2.85 × 108 particles per milliliter for HL-7702 exo, RAW264.7 exo, and hES exo, respectively (Figure S5a−c). After proper dilution, each sample contained 6.39 × 104 exosomes per microliter. Among these samples, only MDA-MB-231 exo showed a significantly high number of GPC-1(+) exosomes (40141 exosomes per microliter). For the negative control cases, we consistently observed very few fluorescent droplets per experiment (∼5 detectable copies per microliter), confirming the background of our assay is mainly due to the low enzyme nonspecific binding to the magnetic beads.40,54 To demonstrate a clinically relevant application of our approach, we performed the droplet digital ExoELISA for

Figure 5. Clinical analyses of GPC-1(+) exosomes by droplet digital ExoELISA. (a) Quantification of GPC-1(+) exosomes from serum samples of 5 healthy samples (HS), 5 patients with benign breast disease (BBD), and 12 patients with breast cancer (BC). (b) Scattered dot plots show significant overexpression of GPC-1(+) exosomes of BC patients compared to HS and BBD (****, p < 0.0001). (c) Quantification of GPC-1(+) exosomes in two patients with breast cancer (BC) and breast cancer after surgery (BC-AS). Error bars represent the standard deviation of three independent experiments.

Serum samples obtained from HS were used as the control for this study. According to previous reports,10 there are about 0.3%−4.7% (average of 2.3%) GPC-1(+) exosomes even in healthy human serum samples. Figure 5a shows that there was an average of 5448 GPC-1(+) exosomes per microliter in HS and similar GPC-1(+) exosomes (∼6914 exosomes/μL) in BBD, whereas the average GPC-1(+) exosomes in the BC group increased by five- to seven-fold. Our data were in great concordance with the previously published data,12 revealing that the expression of GPC-1 significantly increased on tumorderived exosomes as compared to the normal and benign breast disease samples. The increase may be a result of a higher level of GPC-1(+) exosomes shed by tumor cells than normal cells. Figure 5b shows that the BC patients overexpressed GPC-1(+) exosomes and can be well discriminated from the HS and BBD groups (p < 0.0001). Notably, for BC1-AS and BC2-AS two samples of patients BC1 and BC2 after surgery, 4230


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the measured values of GPC-1(+) exosomes in BC1-AS and BC2-AS were significantly lower than BC1 and BC2 (Figure 5c), respectively, but relatively higher than HS and BBD (Figure 5a). Therefore, these data not only verified the GPC-1 can be regarded as an exosomal biomarker to distinguish nonBC subjects from patients with breast cancer but also suggested that our method may be suitable for detection of GPC-1(+) exosomes for pre- and postsurgical monitoring. Our droplet digital ExoELISA has been demonstrated as a reliable method for quantifying target exosomes from HS, BBD, and BC-AC from BC clinical samples, however, in current studies we have not maximized the superior sensitivity of our approach because of the relatively high baseline value of GPC-1(+) exosomes (∼5448 exosomes/μL) in HS. It is worth mentioning that in the early stage of the diseases (especially cancer), where some exosome subpopulations only secreted by tumor cells are extremely small, our approach can be more suitable for detecting the extremely low abundance exosomes than other reported methods (Table S1). Therefore, our approach is promising for early cancer diagnostics and postsurgical monitoring in clinical research. In this study, to leverage the droplet microfluidics for single molecule/copy detection, we extended the standard ExoELISA techniques to detection of ultralow ambulance exosomes with specific target proteins. Our droplet digital ExoELISA method is able to achieve unprecedented accuracy and high specificity for exosome quantification and has the potential to distinguish the target protein expression level on single exosomes through the fluorescence signal level in droplets. We demonstrated that our system detected the target exosomes in a dynamic range of 5 log and the detection limit can be as few as 10 exosomes per microliter. The high specificity was also demonstrated by quantifying the exosomes with target GPC-1 biomarker from a variety of exosome subpopulation protein biomarkers. We successfully used this method for absolute quantification of exosomes in serum samples from breast cancer patients, manifesting the prospective clinical value of the droplet digital ExoELISA method that may propel the discovery of cancer exosomal biomarkers.

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C.L. and X.X contributed equally. The manuscript was written through contributions of all authors. All authors 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 Innovation and Technology Fund (ITS/224/16), the National Natural Science Foundation of China (81702100), the Science and Technology Program of Guangzhou (201510010097), and the Major Program of Health Care and Innovation of Guangzhou Project (201704020213, 201604020015).

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The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.nanolett.8b01184.

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Details of methods. Figures S1−S5: dual-color superresolution images of fluorescence labeled exosomes; TEM images of an immunomagnetic captured single exosome; bright-field images of beads in droplets; incubation time optimization; NTA plots of exosomes isolated from HL-7702, RAW264.7, and hES cell culture media. Table S1: Comparison of current assays for detection of exosomes (PDF)

AUTHOR INFORMATION

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Shuhuai Yao: 0000-0001-7059-4092 4231


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