Dual Rolling Circle Amplification-Assisted Single-Particle Fluorescence Profiling of Exosome Heterogeneity for Discriminating Lung Adenocarcinoma from Pulmonary Nodules

Abstract

Open AccessCCS ChemistryRESEARCH ARTICLES25 May 2022Dual Rolling Circle Amplification-Assisted Single-Particle Fluorescence Profiling of Exosome Heterogeneity for Discriminating Lung Adenocarcinoma from Pulmonary Nodules Yan Zhou†, Haoxiang Li†, Min Hou, Jianjun He and Jian-Hui Jiang Yan Zhou† Hunan Key Laboratory of Organ Fibrosis, Department of Pulmonary and Critical Care Medicine, The Third Xiangya Hospital, Central South University, Changsha 410013 †Y. Zhou and H. Li contributed equally to this work.Google Scholar More articles by this author , Haoxiang Li† State Key Laboratory of Chemo/BioSensing and Chemometrics, College of Biomedical Sciences, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082 †Y. Zhou and H. Li contributed equally to this work.Google Scholar More articles by this author , Min Hou State Key Laboratory of Chemo/BioSensing and Chemometrics, College of Biomedical Sciences, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082 Google Scholar More articles by this author , Jianjun He *Corresponding authors: E-mail Address: [email protected] E-mail Address: [email protected] State Key Laboratory of Chemo/BioSensing and Chemometrics, College of Biomedical Sciences, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082 Google Scholar More articles by this author and Jian-Hui Jiang *Corresponding authors: E-mail Address: [email protected] E-mail Address: [email protected] State Key Laboratory of Chemo/BioSensing and Chemometrics, College of Biomedical Sciences, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082 Google Scholar More articles by this author https://doi.org/10.31635/ccschem.022.202202028 SectionsSupplemental MaterialAboutAbstractPDF ToolsAdd to favoritesDownload CitationsTrack Citations ShareFacebookTwitterLinked InEmail Exosomes secreted by tumor cells carry abundant molecular biomarkers that reflect the status of their originating cells. These tumor-derived exosomes (TDEs) have emerged as attractive diagnostic targets. However, the identification and characterization of highly heterogeneous TDEs remain practically challenging. Here, we report a dual rolling circle amplification (DRCA)-assisted approach for the selective encapsulation of single TDEs for fluorescence microscopic and flow cytometric analysis. TDEs have been targeted by aptamers that recognized their surface tumor marker and exosomal marker CD63, following DRCA that produced entangling polymeric DNA chains, resulting in facile particle enlargement that allows single-particle fluorescence profiling of exosome heterogeneity. We have demonstrated the use of a dual-marker positive ratio for exosome differentiation and applied division and multiplication operations for normalized and magnified marker heterogeneity analysis. We further applied this assay to distinguish lung adenocarcinoma and pulmonary nodule patients and found an accuracy of 90%. We anticipate promising transformations of this straightforward assay into clinically implantable diagnostic methods. Download figure Download PowerPoint Introduction Exosomes (40–160 nm) are a kind of highly heterogeneous extracellular vesicle shed by different cell types.1 Exosomes can shuttle their cargo, such as proteins, nucleic acids, and lipids, from parental cells to recipient cells and play essential roles in tumor development and progression.2 Especially, tumor-derived exosomes (TDEs) that share a variety of tumor biomarkers have become an attractive category for tumor diagnosis.3–5 So far, numerous researchers have developed various technologies for the isolation and characterization of TDEs and demonstrated their theranostic applicability in tumor diagnostics, prognosis, and treatment response assessment.6–8 Despite being a promising diagnostic target in liquid biopsy, the use of TDEs as clinical indicators is limited by complicated exosome composition and cumbersome detection processes.9,10 Moreover, their nanoscale size makes it very hard to directly analyze single integrated TDEs by conventional analytical methods such as flow cytometry and fluorescence microscopy, resulting in exosome heterogeneity deciphering difficulty.11–13 Furthermore, the sheer volume of non-TDEs places great challenges on identifying and isolating TDEs.2,14 The recently developed methods for TDE detection usually require procedures for exosome immobilization, such as microfluidic chips or magnetic beads, in which further downstream analysis of specific TDEs is often limited.10,14–20 Lung cancer is the leading cause of cancer-related deaths worldwide. Although clinical treatment has improved significantly, the overall 5-year survival rate for lung cancer is <20%, mainly because a large number of patients (>70%) have already developed advanced or metastatic lung cancers at the time of diagnosis.21,22 Therefore, early detection of lung cancers is the critical component of improving lung cancer survival rates. Low-dose computed tomography (LDCT) is an effective early screening modality recommended in the United States.23 Despite being sensitive to finding early lung cancer, LDCT screening is expensive and involves irradiation exposure. Moreover, LDCT suffers from high false-positive rates; many patients with benign nodules are misdiagnosed with tumors and undergo unnecessary tests and treatments.24 Therefore, a simple, cost-effective, and accurate assay is of great importance for lung cancer diagnosis in clinical practice. Thus, we searched to develop a TDE-based strategy for lung cancer diagnosis. Aptamers are molecular affinity probes with high specificity and versatile functionality, which have been widely applied to target surface exosomal markers for isolation and identification.25 And the incorporation of isothermal nucleic acid amplification methods, such as rolling circle amplification (RCA), can facilitate the downstream characterization of exosomes.26 Standing on the shoulders of these achievements, we designed two aptamer-guided RCA-tailed probes (ARPs) consisting of an aptamer and an RCA primer to trigger a dual RCA (DRCA)-assisted enlargement of TDEs for single-particle fluorescence analysis. The probes can recognize a specific tumor marker and a general exosomal marker that is expressed on the surface of target TDEs. DRCA is triggered after simultaneously binding to the target TDE, generating ultralong DNA chains designed to cross-hybridize.27 The entangled TDE would be heavier and larger and can therefore be enriched by standard centrifugation. By labeling TDEs with supplementary single-strand DNA (ssDNA)-conjugated fluorophores, marker expression and heterogeneity of single TDEs can be fluorescently profiled with microscopy and flow cytometry. Moreover, we have applied this method to detect clinical plasma samples to differentiate lung adenocarcinoma (LUAD) patients from pulmonary nodule (PN) patients. Experimental Methods Chemicals and materials All oligonucleotides used in this work ( Supporting Information Table S1) were synthesized by Qingke Biotechnology Co., Ltd. (Beijing, China). T4 DNA ligase and ligase buffer were purchased from Shanghai Sangon Biotechnology Co., Ltd. (Shanghai, China). Phi29 DNA polymerase (10,000 U/mL) and Phi29 buffer were obtained from Beyotime Biotechnology (Shanghai, China). Anti-CD63 antibody was purchased from Abcam (Cambridge, United Kingdom), and anti-PTK7 (PTK7 = protein tyrosine kinase 7) antibody was purchased from Zhengneng Biotechnology (Chengdu, China). Horseradish peroxidase (HRP)-conjugated goat antimouse IgG secondary antibody was purchased from Beyotime Biotechnology (Shanghai, China). DNase was purchased from Thermo Fisher (Waltham, MA, United States). All other chemicals were of analytical grade and purchased from Aladdin Bio-Chem (Shanghai, China). Gel electrophoretic analysis was performed using a ChemiDoc MP system (Bio-Rad, Inc., Hercules, CA, United States). Cell culture and exosome isolation CCRF-CEM (CEM) and Ramos cells were cultured in RPMI 1640 medium under a 100% humidified atmosphere containing 5% CO2 at 37 °C. MCF-7 and HEK-293T cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) under the same conditions. When 70% confluency was reached, the supernatant from the cell culture was collected and centrifuged according to previous reports in the literature.28 In brief, the cell culture supernatant was centrifuged at 4200 rpm for 10 min to remove detached cells, and the supernatant was collected and filtered through 0.22 μm filters (Merck Millipore, Burlington, VT, United States). Then the collected supernatant was centrifuged using a Beckman Coulter Optima TM L-100XP Ultracentrifuge at 110,000 gavg at 4 °C for 70 min with a Type 32 Ti rotor. The supernatant was carefully removed to obtain exosomes. The total protein concentration of exosomes was determined by bicinchoninic acid assay. The exosome pellet was resuspended in 200 μL 1× phosphate-buffered saline (PBS) and stored at −80 °C for later use. Preparation of plasma samples Patient blood samples were obtained from the Third Xiangya Hospital of Central South University with permission. Plasma samples were collected by centrifuging blood at 3000 × g for 10 min at 4 °C. Then, the collected supernatant was centrifuged at 4200 × g for 10 min at 4 °C. Plasma was collected and stored at −80 °C before use. Crude exosomes from 400 μL plasma were separated by ultracentrifugation using a Beckman Coulter Optima TM L-100XP Ultracentrifuge at 120,000 gavg at 4 °C for 70 min with an NVT 100 rotor. The supernatant was carefully removed to obtain exosome pellets that were then resuspended in 100 μL 1× PBS for DRCA-assisted fluorescence analysis. Preparation of ARPs ARPs (100 μL) were prepared by mixing 25 μL of the primer probes (10 μM), 25 μL of template probe (10 μM), and 50 μL ddH2O. The mixture was first heated at 95 °C for 5 min and slowly cooled to room temperature. Then, 10 μL of 10× T4 DNA ligase reaction buffer [400 mM Tris–HCl, 100 mM MgCl2, 100 mM dithiothreitol (DTT), 5 mM adenosine 5′-triphosphate (ATP)], 38.5 μL of dd H2O, and 2.5 μL of T4 DNA ligase (1000 U/μL) were added to the mixture. The mixture was incubated at 16 °C overnight and then heated at 65 °C for 10 min to terminate the reaction. The ARPs were saved at 4 °C. RCA reaction The RCA reaction was performed in a volume of 20 μL containing 2 μL circular ligation template (5 μM), 2 μL of 10 × phi29 DNA polymerase reaction buffer (330 mM Tris-acetate, 100 mM Mg-acetate, 660 mM K-acetate, 1% (v/v) Tween 20, 10 mM DTT), 1 μL dNTPs (25 mM), 14.5 μL of H2O, and 0.5 μL phi29 DNA polymerase (10 U/μL). The RCA reaction was performed at 37 °C for 8 h and inactivated by heating it at 65 °C for 10 min. Then the products were detected by 2% agarose gel electrophoresis. Electrophoresis analysis Gel electrophoresis was used to characterize the preparation of ARPs and RCA reactions. 16 μL products mixed with 4 μL of 5× loading buffer were loaded onto the gels. 2% agarose gel electrophoresis was carried out in 1× TAE (TAE = Tris-acetate-EDTA buffer) at 80 V for 60 min, and 12% nondenaturing polyacrylamide gel electrophoresis was performed in 1× TBE (TBE = Tris-borate-EDTA buffer) at 120 V for 90 min. Subsequently, the gel was analyzed using a gel imaging system. DRCA-assisted encapsulation of target exosomes Exosomes were suspended in 100 μL PBS at various concentrations. 100 nM ARPs were added to bind target exosomes at 4 °C for 40 min. Unbound ARPs were removed by ultracentrifugation. Then, 2.5 μL phi 29 DNA polymerase (10 U/μL), 10 μL 10× phi 29 DNA polymerase reaction buffer, and 1 μL dNTPs (25 mM), 100 μL were added and diluted to 100 μL by ddH2O. This allowed the in situ DRCA to take place at 37 °C for 8 h. The DRCA-enveloped exosomes were centrifuged at 12,000 rpm for 30 min to remove the supernatant- and isolate-enveloped exosomes for further analysis. Transmission electron microscopy Exosomes were adsorbed on carbon-coated copper grids for 10 min, washed twice with PBS and water, stained with 1% phosphotungstic acid for 10 min, and washed again with PBS. After being dried under a white woven lamp overnight, the samples were observed on a JEOL-3010 transmission electron microscope (JEOL USA, Inc., Peabody, MA, United States). Dynamic light scattering The size (n = 3 for each sample) of exosomes and DRCA-enveloped exosomes were measured using a Zetasizer (Malvern Nano 3000 HS, Malvern Instruments Ltd., Malvern, England, United Kingdom). Fluorescence measurements MCF-7 and HEK-293T cells were cultured in confocal dishes for 24 h in a humidified incubator. Then, the cells were washed with PBS buffer three times. Subsequently, the cells were incubated with 10 μL of 5 μM EpCAM ARPs and 10 μL of 5 μM CD63 ARPs for 2 h at 37 °C. The cells were washed with PBS three times before being incubated with 5 μL phi 29 DNA polymerase (10 U/μL), 20 μL 10× phi 29 DNA polymerase reaction buffer, and 2.5 μL dNTPs (25 mM) and DMEM for 8 h at 37°C. Then, 2 μL 100 nM ssDNA-Cy5 (complement to CD63 ARPs) and 2 μL 100 nM ssDNA-FAM (FAM = 6-Carboxyfluorescein) (complement to EpCAM ARPs) fluorescent probes were added and incubated at 37°C for 1 h. Finally, cells were washed with PBS three times and kept in 200 μL DMEM. The cells were imaged under a 60× objective by a Nikon Ti-E+A1 SI microscope. All images were processed with ImageJ, the multiplication and division operations were performed by clicking Process → Image Calculator → Select Images and Operations. The multiplied images were further analyzed with 3D Surface Plot. DRCA-enveloped exosomes were incubated with 100 nM ssDNA-Cy5 and 100 nM ssDNA-FAM (complement to tumor-specific PTK7/EpCAM ARPs) fluorescent probes at 37 °C for 30 min. TDEs were separated by centrifugation at 12,000 rpm for 30 min. For confocal fluorescence imaging, the enveloped exosomes were imaged under a 60× objective by an OLYMPUS FV1200 microscope. Acquired images were analyzed with ImageJ. The flow cytometric analysis was carried out in a Gallios flow cytometer (Beckman Coulter, Brea, CA, United States). All data were acquired and analyzed by the FlowJo software. TDE measurements were performed in parallel with negative controls to eliminate background noise. Western blot Western blotting was employed to confirm the presence of typical protein markers on exosomes. Exosomes were incubated with loading sample buffer at 95 °C for 10 min. Then, the protein samples were analyzed by 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (PAGE) and blotted to the nitrocellulose membrane. After blocking with 5% nonfat milk for 8 h, the blots were incubated overnight at 4 °C with anti-CD63 antibody or anti-PTK7 antibody (1:1000 dilution). After washing, the blots were incubated with HRP-conjugated goat antimouse IgG antibody (1:5000 dilution) for 3 h. The blots were imaged by a gel image system using chemiluminescence. Statistical analysis Significance analysis, receiver operating characteristic (ROC) analysis, and area under the curve (AUC) were performed using GraphPad Prism 9 (United States). Results and Discussion Design and characterization of DRCA To achieve the selective enveloping of targeted TDEs, we designed two ARPs. One contains an aptamer that recognizes the specific surface marker of target TDEs and is extended with a primer that complemented a circle template. The other has the same structure but contains an aptamer that binds to CD63, a general surface marker of exosomes (Scheme 1). The two probes can simultaneously bind to the target TDEs, allowing in situ DRCA-assisted chain prolongation. The two RCA-amplified polymeric DNA chains were designed to hybridize with each other, resulting in a cross-linked DNA network that encapsulated the targeted exosome. Therefore, the enlarged DNA-cloaked submicron particle can be separated via normal centrifugation and is compatible with single-particle fluorescence analyses (fluorescence imaging and flow cytometry). While for the nontarget exosomes, only the CD63 probe will bind and trigger its RCA, resulting in a lower degree of cross-linking, which greatly differs from TDEs regarding size and fluorescence identification. The number of TDEs can be quantitatively detected by fluorescence microscopy or flow cytometry using two short fluorophore-labeled ssDNA chains that specifically bind to the repeated RCA-amplified sequences, allowing direct TDE counting and marker heterogeneity profiling. Moreover, the enveloped TDEs can be released by DNase-induced breakage of DNA networks, which can potentially be reused in further applications. Scheme 1 | Schematic illustration of DRCA-assisted single-particle fluorescence profiling of TDE heterogeneity. TDEs are targeted by ARPs that weave cross-hybridized DNA networks by DRCA-assisted chain elongation. The encapsulated TDEs can be separated via normal centrifugation for further single-particle fluorescence analysis. Download figure Download PowerPoint We first evaluated the assembling of aptamers and circle templates and their ability to trigger RCA reactions. As the agarose gel electrophoresis has shown, the aptamers can hybridize with corresponding circle templates and produce ultralong DNA products after the RCA reaction (Figure 1a and Supporting Information Figure S1). We then chose CEM and Ramos cell lines as the cell model for cancer and normal cells. PTK7 is a surface marker highly expressed only in CEM cells.29 Thus, we have utilized the Sgc8 aptamer that specifically targets PTK7 to design a CEM-specific PTK7 ARP and used it for the following validations.6,30 Before usage, exosomes were first isolated from CEM and Ramos cell lines and characterized with transmission electron microscopy (TEM), dynamic light scattering (DLS), and western blot (WB). As confirmed by WB (Figure 1b), the CEM exosomes overexpressed PTK7 while Ramos exosomes did not. These exosomes were then individually mixed with CD63 and PTK7 ARPs and triggered the DRCA reaction in situ. The captured exosomes were expected to be packaged in DNA networks. We employed DLS and TEM to evaluate the morphology change of the encapsulated exosomes. As the DLS analyses have shown, the sizes of CEM exosomes were enlarged to 571 (Figure 1c) and 475 nm for Ramos exosomes ( Supporting Information Figure S2a), increased to a detectable range for fluorescence microscopy and flow cytometry. Especially, the CEM exosomes were enlarged 7.7-fold, significantly larger than the DRCA-enlarged Ramos exosomes (Figure 1d), suggesting the DRCA-amplified cross-linked DNA network contributes more to the particle enlargement than the single RCA reaction. Figure 1 | Characterization of DRCA-enveloped TDEs. (a) Gel electrophoresis analysis of ARPs. L1: DNA mass marker, L2: CD63 template, L3: PTK7 template, L4: CD63 primer, L5: PTK7 primer, L6: mixed CD63 and PTK7 templates, L7: assembled CD63 ARPs, L8: assembled PTK7 ARPs. (b) WB analysis of PTK7 and CD63 protein expression of CEM and Ramos exosomes. (c) DLS analysis of CEM exosomes and DRCA-enveloped CEM exosomes. (d) Analysis of exosome particle size before and after DRCA reaction. Unpaired, one-tailed t test, *P < 0.05. (e) TEM images of CEM and DRCA-enveloped CEM exosomes, scale bar: 100 nm. Download figure Download PowerPoint Moreover, the TEM image showed that DRCA-enveloped CEM exosomes were intensely stained with enlarged particle sizes (Figure 1e), completely masking the typical exosome morphology. And the TEM image of the captured Ramos exosomes was lightly stained as to CEM ( Supporting Information Figure S2b). Overall, these results suggested a larger and higher degree of cross-linked DNA network for DRCA-assisted TDE encapsulation. It is worth noting that normal centrifugation (i.e., 12,000 rpm using an Eppendorf 5424 R centrifuge equipping an FA-45-24-11 rotor) can easily enrich the DRCA-enveloped exosomes, greatly simplifying the isolation process. We further optimized the DRCA reaction time. As shown in Supporting Information Figure S3, the particle size of DRCA-enveloped TDEs increased with reaction time. As the larger size is more favorable for the subsequent isolation and characterization, we choose 8 h as the standard DRCA reaction time in the following experiments. Fluorescence imaging of DRCA-enveloped exosomes After the DRCA reaction, TDEs were individually packaged by a cross-linked DNA network. To visualize and count the single-particle DRCA-enveloped TDEs, ssDNA-linked Cy5, and FAM fluorescent probes that target the RCA-amplified regions of the CD63 and PTK7 ARPs were used, respectively. The target TDEs are expected to exhibit fluorescence in both channels, while only Cy5 fluorescence should be observed for non-TDEs. As imaged by a confocal fluorescence microscope, DRCA-enveloped CEM exosomes displayed good FAM and Cy5 colocalization (Figure 2a and Supporting Information Figure S4a), confirming the formation of DRCA networks. We then searched to evaluate the marker heterogeneity across exosomes. First, we applied the divide operation between the PTK7 image and the CD63 image for normalization (Figure 2b and Supporting Information Figure S4b). While this offered a normalized comparison in PTK7 marker expression, the nonuniform background made it challenging for data interpretation. And we found that the multiplication operation offered magnified expression differences for heterogeneity profiling. We then counted the PTK7 and CD63 dual-positive (PTK7+CD63+) exosome ratios across 10 image frames in both groups (Figure 2c). While Ramos exosomes displayed low levels of PTK7 expression, various levels of PTK7 expression were observed for CEM exosomes, suggesting high molecular heterogeneity across TDEs. Figure 2 | Confocal fluorescence microscopic analysis of DRCA-enveloped exosomes. (a) Fluorescence images of DRCA-enveloped CEM and Ramos exosomes, scale bar: 10 μm. (b) Divided and multiplied images between CD63 and PTK7 channels. scale bar: 10 μm. (c) PTK7+CD63+ exosome ratios. Each spot represents the ratio of PTK7+ counts to CD63+ counts per fluorescence image taken. **P < 0.01. (d) Fluorescence images of DRCA-assisted encapsulation of different CEM (C) and Ramos (R) exosome mixtures, scale bar: 10 μm. (e) Fluorescence microscopic PTK7+CD63+ counts of serially diluted CEM exosomes. For each condition, 10 frames were counted per replicate, n = 3. Download figure Download PowerPoint To further validate the DNA network formation, we applied DNase to digest DNA and release DRCA-enveloped TDEs. As shown in Supporting Information Figure S5a, few fluorescently visible particles were observed in DNase-treated DRCA-TDEs. Moreover, the TEM image of DNase-treated DRCA-TDEs showed a clear exosome structure ( Supporting Information Figure S5b). These results confirmed the DRCA-assisted fluorescence observation of target TDEs. To evaluate the specificity of DRCA-based TDE encapsulation, we mixed CEM and Ramos exosomes at different ratios. As shown in Figure 2d, more CEM exosomes resulted in more spots getting fluoresced in both channels while more Ramos exosomes resulted in more spots with only CD63 fluorescence. These results demonstrated the specificity of DRCA. Next, we prepared TDEs at different concentrations to test the DRCA-based fluorescence microscopic analysis in quantifying TDEs. As shown in Figure 2e and Supporting Information Figure S6, the PTK7+CD63+ counts are positively correlated with the TDE concentrations. Flow cytometric analysis of DRCA-enveloped TDEs Flow cytometry is widely regarded as a high-throughput, rapid, and quantitative fluorescence analysis method.31 Thus, it is advantageous for fluorescence imaging regarding large-number TDE counting. However, conventional flow cytometry cannot detect the nanosized exosomes. Therefore, we first evaluated whether DRCA-enlarged exosomes can be detected by flow cytometry. As shown in Figure 3a and Supporting Information Figure S7, plenty of fluorescent particles was recorded for DRCA-enveloped CEM exosomes while fewer were detected for DRCA-enveloped Ramos exosomes and little for exosomes directly labeled with fluorescent aptamer probes. Moreover, the fluorescence intensities (FIs) of DRCA-enveloped CEM exosomes were much higher than those of Ramos in both channels ( Supporting Information Figure S8a). The forward scatter (FSC) parameter of flow cytometry can directly reflect the difference in particle sizes. As shown in Figure 3b and Supporting Information Figure S8b, the FSC of DRCA-enveloped CEM exosomes is higher than that of Ramos, consistent with our previous experimental findings that the DRCA-enveloped TDEs exhibit a larger particle size than those nontarget exosomes. As larger particle sizes led to higher sensitivity of flow cytometry, the statistical analysis of DRCA-enveloped CEM exosomes showed a much smaller standard deviation compared to Ramos exosomes ( Supporting Information Figure S8a). Figure 3 | Flow cytometric analysis of DRCA-enveloped exosomes. Histogram analysis of the captured exosomes in CD63 and PTK7 (a), and FSC (b) channels. (c) Exosome’s FI in PTK7 and CD63 channels was multiplied to reflect marker expression heterogeneity. Error bars represent median ± interquartile range, ****P < 0.0001. Statistical analysis of PTK7+CD63+ ratios (d) and counts (e) of the DRCA-enveloped exosomes. Error bars represent mean ± S.D., n = 5, ***P< 0.001, ****P < 0.0001. (f) The linear relationship between particle counts and CEM exosome weights. Error bars represent mean ± S.D., n = 3. Download figure Download PowerPoint We further compared the PTK7+CD63+ counts (gated as shown in Supporting Information Figure S7a) between DRCA-enveloped CEM and Ramos exosomes. First, we analyzed the PTK7+CD63+ counts with the division and the multiplication operations to profile the single exosome heterogeneity. As shown in Figure 3c and Supporting Information Figure S8c, both operations reflected the large differences and broad distribution of marker expressions. Furthermore, as shown in Figures 3d and 3e, significantly higher PTK7+CD63+ particle ratios and counts were detected for CEM exosomes compared to Ramos, providing a straightforward differentiation approach for marker-specific TDEs. We then investigated the relationship between particle counts and exosome concentrations. As shown in Figure 3f and Supporting Information Figure S9, the counts were linearly proportional to the input exosome mass within the range of 5–30 μg, proving the capability of this DRCA approach in exosome quantification. Overall, we have demonstrated that this DRCA-based approach can enlarge the target TDEs into submicron DNA-encapsulated particles for subsequent single-particle fluorescence imaging and flow cytometric analysis. DRCA-assisted analysis of clinical exosome samples Having shown the effectiveness of the DRCA-assisted fluorescence counting and profiling of TDEs, we next applied the developed assay for the diagnosis of clinical plasma samples. Plasma samples from 10 LUAD patients and 10 PN patients (common false-positive cases in LDCT screening) were collected. EpCAM is a widely acknowledged lung cancer marker that has been previously used for LUAD exosome capture and characterization.32 Therefore, we designed an EpCAM ARP for the DRCA-based fluorescence analysis of LUAD exosomes. We first evaluated the success of the assembly of the EpCAM ARP and RCA reaction, as well as its specific binding to EpCAM overexpressed MCF-7 cells ( Supporting Information Figure S10). Then, this EpCAM-based DRCA-assisted fluorescence analysis was performed with MCF-7 exosomes, and obvious differences were observed with both fluorescence imaging and flow cytometric analysis ( Supporting Information Figure S11), consistent with previous PTK7-based DRCA results, which demonstrated the general applicability of DRCA-based single-particle profiling of TDEs. Then, we evaluated the feasibility of DRCA-based approach for targeting and enveloping patient-derived exosomes. As shown in Figure 4a, EpCAM+CD63+ particle