Molecular-Recognition-Based DNA Nanodevices for Enhancing the

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Molecular Recognition-Based DNA Nanodevices to Enhance the Direct Visualization and Quantification of SingleVesicles of Tumor Exosomes in Plasma Microsamples Dinggeng He, See-Lok Ho, Hei-Nga Chan, Huizhen Wang, Luo Hai, Xiaoxiao He, Kemin Wang, and Hung-Wing Li Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b04509 • Publication Date (Web): 15 Jan 2019 Downloaded from http://pubs.acs.org on January 16, 2019

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Analytical Chemistry

Molecular Recognition-Based DNA Nanodevices to Enhance the Direct Visualization and Quantification of Single-Vesicles of Tumor Exosomes in Plasma Microsamples Dinggeng He,†, ‡ See-Lok Ho,† Hei-Nga Chan,† Huizhen Wang,‡ Luo Hai,‡ Xiaoxiao He,*, ‡ Kemin Wang*,‡ and Hung-Wing Li*,† †Department

of Chemistry, Hong Kong Baptist University, Kowloon Tong, Hong Kong, China.

‡State

Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha, China. ABSTRACT: Tumor exosomes (Exo) are presumed to expedite both the growth and metastasis of tumor by actively participating in nearly all aspects of cancer development. Tumor-derived Exo is thus proposed as a resource of diagnostic biomarkers in bodily fluids. However, most Exo assays require large samples and are time-consuming, complicated and costly, and thus unsuited for practical applications. Herein, we show an ultrasensitive assay that can directly visualize and quantify tumor Exo in plasma microsamples (1 μL) at the single-vesicle level. The assay uses the specific binding of activatable aptamer probes (AAP) to target Exo captured by Exo-specific antibodies on the surface of flow cell to produce an activated fluorescence. Furthermore, the bound AAP triggers in situ assembly of DNA nanodevice with an enhanced fluorescence that improves the Exo detection sensitivity. By identifying tyrosine-protein kinase-like 7 (PTK7), TIRF assay for PTK7-Exo distinguishes target tumors from control subjects. This assay is also informative in monitoring tumor progression and early responses to therapy. The developed assay can be readily adapted for diagnosis and monitoring of other disease-associated Exo biomarkers.

Recently, circulating tumor-derived exosomes (Exo) have been proposed as emerging non-invasive biomarkers for early cancer diagnosis.1,2 These membrane-enclosed vesicles with diameter of 30-100 nm are naturally secreted by a variety of tumor cells.3-6 They carry key biomolecules (e.g., nucleic acids, proteins, etc.) from parental cells and convey them to the neighboring or even distant cells through circulation.7,8 Emerging evidence demonstrates that tumor-derived Exo facilitates both tumor growth and metastasis by Exo-mediated intercellular communication in whole oncogenic processes, and their vesicular contents are useful for predicting the origin of parent tumors.9-11 Enzyme-linked immunosorbent assay (ELISA) and western blot are the conventional methods to detect Exo, they are capable of differentiating tumor Exo from normal Exo by identifying Exo proteins.12-14 Recently, many other approaches have been developed for specific and sensitive detection of Exo, such as electrochemistry,15 surface enhanced Raman scattering (SERS),16 microfluidic17 and fluorescence,18 nanoplasmonic sensor,19 localized surface plasmon resonance,20 etc.. Nevertheless, most the abovementioned assays are performed under laboratory conditions and their clinical application is limited by tedious, labor intensive and time-consuming pretreatment and purification steps, which scarified the quality and quantity of Exo. In addition, current analysis of Exo focuses mainly in bulk manner, while a single-vesicle

quantitative analysis of tumor-specific Exo abundance and stoichiometry is lacking, thus hindering the understanding on the roles of Exo in cell-cell communication in oncogenesis and tumor heterogeneity. In this context, direct single-vesicle quantitative analysis of tumor Exo in plasma not only can overcome the problem from the conventional methods, but also can gather the stoichiometry measure and composition of Exo to obtain insightful information of Exo-mediated intercellular communications and elevate the early precise tumor diagnosis to the next level.21,22 However, visualization and quantification of Exo at the single-vesicle level has historically been challenging to achieve, because Exo is sub-diffraction limit particle and thus, cannot be directly enumerated by ordinary light microscopy or flow cytometric method.23 Although Exo are readily visualized by electron microscopy or atomic force microscopy, these methods are non-quantitative because of the complex sample preparation procedures. Nanoparticle tracking analysis (NTA) can quantify Exo in solution, but it is lack of selectivity to tumor Exo and powerless to differentiate Exo from protein aggregates. Thus, a simple and mild method for direct and selective quantification of singlevesicles of circulating tumor Exo is highly desired. Total internal reflection fluorescence (TIRF) microscopy as a highly sensitive single-molecule detection tool shows

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a promising potential for visualization and quantification of single-vesicles of nanosized Exo. So far, however, study on single Exo based on the TIRF microscopy is not available mainly due to the lack of desired molecular recognition probes with high sensitivity and selectivity. As an emerging type of immuno-affinity moiety, aptamers have been attractive in the field of single-molecule imaging and detection.24-26 Aptamers are short nucleic acids oligomers that exhibit an excellent binding affinity towards target membrane proteins on the tumor cells and their Exo surfaces.27-32 Compared with antibodies, aptamers are inexpensive, stable to store and can be easily synthesized, modified, and functionalized.33 More importantly, aptamer can be readily combined with a variety of DNA-based reactions, such as hybridization chain reaction (HCR), polymerase chain reaction (PCR), cascade hybridization reaction (CHR) and rolling cycle amplification (RCA), to achieve the amplified detection of target biomolecules.34-37 Thus, aptamer is becoming an attractive alternative for targeted molecular recognition in a wide range of biomedical applications. Here, we describe an ultrasensitive single-vesicle imaging assay with TIRF microscopy for direct visualization and quantification of circulating plasma tumor Exo by in situ building aptamer-based fluorescent DNA nanodevices on target Exo surfaces. We believe this is the first antibody-aptamer sandwich immunoassay for single-vesicle imaging analysis of target tumor Exo. As a proof of concept, the Exo with high expression of PTK7, a protein closely associated with a number of cancers,29 was chosen as a target analyte. The schematic illustration of the detection assay was shown in Scheme 1. In brief, an antibody against CD63, a common Exo membrane biomarker,2,22 was first immobilized on the glass coverslip surface through the EDC/NHS chemistry acting as the capture probe, the plasma samples were then injected into the flow-cell and all Exo are captured by the antibodies. Then target PTK7-containing Exo (PTK7-Exo) was detected by adding activatable aptamer probes (AAP) to sample wells. In our design, the AAP consists of three fragments: a PTK7-targeted aptamer sequence (A-strand), a poly-T linker (L-strand), and a DNA trigger (T-strand) for initiating a hybridization chain reaction (HCR) event (Scheme S1). To show the conformational change of AAP, a fluorophore and a quencher are covalently attached to Astrand and T-strand, respectively. Owing to the hybridization of the T-strand with the A-strand, the AAP forms a stable hairpin, which keeps the fluorophore in close proximity to the quencher, causing the quenched fluorescence in the absence of PTK7. Nevertheless, when the AAP encounters the target PTK7-Exo, it is capable of specific binding with PTK7 on Exo surface, resulting in a conformational reorganization of the AAP and, thus, separating the fluorophore from the quencher. Ultimately, the binding of the AAP to the target PTK7-Exo activates a readable fluorescence signal. Meanwhile, the activated

AAP provides a complete single-stranded T-strand, which further triggers the cross-opening of fluorescent molecular hairpins (H1 and H2), initiating an in-situ self-assembly of aptamer-based DNA nanodevices (ABDNs) that significantly amplify the readout of fluorescence signal. The as-generated ABDNs were then detected under a TIRF imaging system for quantitative analysis.38,39 As the evanescent field generated by the total internal reflection is very shallow (100-300 nm), only PTK7-Exo-ABDNs hybrids that locate at the top of coverslip/water interface are excited by laser, while the bulk solution remains silent. This TIRF assay with the high signal-to-background (S/B) ratio would be used for the direct visualization and quantification of single-vesicles of PTK7-Exo in plasma. EXPERIMENTAL SECTION Chemicals and materials. Carboxylic acid functional glass coverslips (Glass-COOH) were purchased from Nanocs Inc. Anti-PTK7 VioBrightTM FITC was purchased from Miltenyi Biotec. Anti-human CD63 was obtained from R&D Systems. Anti-human CD81 antibody with FITC label was obtained from GeneTex Inc. Dulbecco’s phosphate buffered saline (DPBS), 1-ethyl-3-(3dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysulfosuccinimide sodium salt (Sulfo-NHS) were purchased from Sigma-Aldrich. PTK7 ELISA kit was purchased from Cusabio. All the chemicals were of the highest grade available and used as received without further purification. Nanopure water (18.2 MΩ, Millpore Co.) was used in all experiments and to prepare all buffers. All DNA probes reported in this article were synthesized by Takara Bio Inc. DNA sequences are listed in Table S1. Exo Isolation. Cells were grown in culture media with 10% Exo-depleted FBS for 24, 48 and 72 h. The conditioned media were centrifuged at 3000 rpm for 15 min to remove cell debris, and the supernatant was incubated overnight with ExoQuick-TC (System Biosciences Inc.) solution at 4 °С. The next day, the mixture was centrifuged at 3000 rpm for 30 min. The resulting Exo was carefully collected and re-suspended in 200 μL DPBS (pH 7.0), and stored at 4 °C. The stock solutions of Exo with the concentration of 1010 particles μL-1 were obtained. The total number of Exo was determined by the nanoparticle tracking analysis (NTA) using the NanoSight system. Preparation of Exo Concentration Standards. Plasma samples were centrifuged at 110,000g overnight, and supernatants were collected as Exo-free plasma. Standard Exo samples of known concentration isolated from culture media were dissolved in Exo-free plasma to a final concentration of 1010 particles μL-1, and further diluted to required concentrations (109, 108, 107, 106, 105, 104 and 103 particles μL-1) by 10-fold dilution with Exo-free plasma at time of use.


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Scheme 1. Illustration of the ABDNs-based TIRF assay for single-vesicle imaging and detection of circulating tumor-specific Exo in plasma. Construction of the TIRF Imaging Platform for Plasma Exo Quantification. The flow cell surface was activated with EDC/NHS mixture in MES buffer and functionalized with anti-CD63 antibody. In brief, the flow cell was filled with 10 μL per well of 5 mM EDC/NHS mixture and incubated at room temperature for 2 h in a moist chamber, aspirated and filled with 10 μL per well of 12.5 μg mL-1 anti-human CD63 antibody and incubated for another 2 h. After washing, the flow cell was further filled with 10 μL per well of 5% BSA in PBS (pH 7.0) and incubated at room temperature for 2 h, then PBS (pH 7.0) washed and aspirated three times before filling these flow wells with analysis samples. Sample wells were filled with 10 μL per well of mouse plasma samples (diluted × 10 with pH 7.0 PBS) or CCRF-CEM Exo-spiked plasma samples, incubated at room temperature for 1 h in a moist chamber, washed with PBS three times, and then filled with 10 μL per well of binding buffer solution including AAP1 (30 nM) or the mixture of AAP2 (30 nM), H1 (300 nM) and H2 (300 nM) or anti-PTK7 VioBrightTM FITC (30 nM) and incubated at room temperature for another 1 h, after which the slide was washed with PBS three times, then imaged by TIRF microscopy. To determine the total Exo content in plasma samples, anti-CD81 with FITC label (10 μg mL-1) was also used to bind all Exo, and then the TIRF imaging assays were carried out. Mouse Tumor Model. Male athymic BALB/c (Balb/Cnu) mice were obtained from the Shanghai SLAC Laboratory Animal Co., Ltd. (BALB/c). They were four weeks old at the start of each experiment and weighed 2025 g. All animal procedures were in accord with institutional animal use and care regulations approved by the Laboratory Animal Center of Hunan. The mice were subcutaneously injected with 2 × 106 in vitro-propagated CCRF-CEM cells suspended in 100 µL of DPBS at their backside to establish subcutaneous CCRF-CEM tumor model (n = 3). As a control, Ramos tumor-bearing mice (n = 3) were also established by using the same process. All

mice were retro-orbitally bled using heparin at 0, 10, 20, 30 and 40 days post-injection and the plasma was isolated.11 Plasma (50 μL per mouse) was then diluted with 0.45 mL PBS. Subsequently, the samples were analyzed by the TIRF imaging system for investigating the PTK7-Exo plasma levels. Moreover, tumor size data were also collected at the same time point. A caliper was used to measure tumor length and width. The volume of tumor (V) was estimated by the following equation: V = L × W2 × 0.5, where L and W are the greatest longitudinal diameter and the greatest transverse diameter of tumor, respectively. Treatment Monitoring. Doxorubicin hydrochloride (DOX) was selected as a model chemotherapeutic drug due to its significant therapeutic effect on many tumors clinically. DOX for injection was dissolved in physiological saline to yield the DOX injection solution with a final concentration of 2 mg·mL-1. In most animal experiments of chemotherapy, the dosage was commonly described as the mass of drug used per square meter of body surface area (mg·m-2). Here, the body surface area of nude mice was calculated according to the previously reported literature.40 Subsequently, the CCRF-CEM tumor-bearing mice were treated with DOX. The tumor treatment effect was monitored at indicated time point by investigating the PTK7-Exo levels in plasma samples of mice. Generally, the different dosages (10, 20 and 40 mg·m-2) of DOX were respectively injected into the abdominal cavity of mice (n = 3) with the frequency of every ten days after the subcutaneous injection of mice with CCRF-CEM cells for 15 days. Control mice (n = 3) received the equal volumes of physiological saline without DOX. The PTK7-Exo plasma levels of mice and tumor sizes were respectively measured every five days. Furthermore, to investigate the treatment efficiency for the different stages or sizes of tumor, the treatment for tumor-bearing mice started at 30 days postinjection with CCRF-CEM cells. At this moment, the tumor



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could be seen by naked eye, and tumor volume approached 50-70 mm3. After treatment with different dosages of DOX (40 and 60 mg·m-2), PTK7-Exo levels in plasma samples of tumor-bearing mice (n = 3) were measured once every five days. The changes of relative tumor volume were also studied with the frequency of every two days. Finally, the therapeutic effects for tumor-bearing mice (n = 6) were evaluated by comparing the PTK7-Exo plasma levels before and after serial treatment with 60 mg·m-2 DOX for 40 days. RESULTS AND DISCUSSION Design and Characterization of the ABDNs-Based TIRF Imaging Platform. To start with, target PTK7-Exo derived from CCRF-CEM cells was isolated and purified using the commercial ExoQuick-TC for the preparation of the Exo standards.41,42 Both of the morphology and size distribution of isolated vesicles were studied by TEM and DLS (Figure 1a and Figure 1b). A series of nucleic acid probes were designed and their sequences are listed in Table S1. Fluorescent tests in binding buffer showed that AAP1 emitted a much higher fluorescent signal than control probe upon specific binding with surface proteins of CCRF-CEM Exo (Figure S1). Next, the selectivity of APP1 in buffer was tested by incubating APP1 with control Exo secreted by Ramos, U266 and B95-8 cells, respectively. All control Exo did not trigger significant fluorescence, thus showing a good selectivity. This was also confirmed by the TIRF assays of Exo-spiked plasma with the AAP1 (Figure S2). When the surrounding environment was altered from binding buffer to complex plasma matrix, the activated fluorescence of AAP1 by target CCRF-CEM Exo was not affected and still be detectable by the TIRF imaging system. There was only a weak background signal observed for other control Exo. These results indicated that the TIRF assay based on AAP1 is capable to discriminate target Exo from negative control Exo in plasma samples. Nevertheless, present TIRF signal intensity produced by AAP1-only would be insufficient for ultrasensitive detection of PTK7Exo in plasma. Therefore, a signal amplification technique, hybridization chain reaction (HCR), was used to in-situ build up fluorescent DNA nanodevices on target Exo surfaces to further strengthen the detection sensitivity of this assay. Molecular hairpins were prepared and the successful HCR event in buffer was verified by gel electrophoresis analysis (Figure S3a). The introduction of HCR significantly enhanced the fluorescence signal of target Exo in buffer solution and efficiently improved the S/B ratio (Figure S4).

Figure 1. Characterization of CCRF-CEM Exo and nanoassembly on membrane surface of Exo. (a) TEM image of isolated CCRF-CEM Exo. (b) Hydrodynamic diameters of CCRF-CEM Exo detected by DLS. (c) AFM images of coverslip without (No anti-CD63) and with (anti-CD63-Exo) anti-CD63 after adding CCRF-CEM Exo-spiked plasma, and Exo-ABDNs (white arrows) self-assembled on anti-CD63-modified coverslip surface (anti-CD63-Exo-ABDNs). Nontarget Exo (green arrows) was also observed.

Subsequently, Exo capture on coverslip surface and in situ self-assembly of DNA nanodevices on target Exo surface were intuitively verified by atomic force microscopy (AFM). As shown in Figure 1c, a large number of spherical vesicles were noted on the anti-CD63-modified coverslip surface, while the coverslip without anti-CD63 displayed a negligible nonspecific adsorption of Exo. We found that the height of Exo was about 10 nm, which was inconsistent with the result (50-120 nm) of DLS measurement (Figure 1b). The most probable reason for this is that a collapse of Exo with the vesicle structure occurs after the complex sample preparation process. Thus, the height of Exo in AFM image could not reflect the actual size of Exo. AFM images also showed wire-shaped nanostructures with different lengths (white arrows) on the Exo surface. The heterogeneity in length of observed nanostructures matched roughly with the gel electrophoresis result (ladder shaped bands) of HCR products in solution (Figure S3a). Moreover, the height of wire-shaped nanostructures was in the range of 1.8-2.3 nm, which was almost the same as the width of double helix DNA (Figure S3b). We thus ascertained that the wire-shape nanostructure observed in AFM image was DNA nanodevice self-assembled on target Exo surface by in situ HCR events. The presence of DNA nanodevices could efficiently distinguish target Exo from non-target Exo (green arrows), which strongly supported that the assay possessed a high selectivity towards PTK7-Exo.


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Analytical Chemistry Exo can be simply achieved by counting the bright spots in sample region in the TIRF images.

Figure 2. Characterization of the performance of TIRF assay with CCRF-CEM Exo-spiked plasma. (a) TIRF images of different samples (Exo-free plasma, no anti-CD63, Exo-AAP1 and Exo-ABDNs). Scale bar = 3 µm. (b) Normalized fluorescent signal of different samples in (a). (c) TIRF images of Exo-ABDNs detected in plasma spiked with the indicated Exo concentrations. Scale bar = 3 µm. (d) Correlation of log counts of Exo-ABDNs, Exo-AAP1 and Exo-anti-PTK7 with log Exo concentration in Exo-spiked plasma samples, respectively. Data represent mean ± SEM; n = 3 per group.

The TIRF Imaging Platform for Exo Quantification. The Exo detection performance of the TIRF assay was then characterized. Negative control assay performed with Exofree plasma displayed a negligible signal intensity, and similar result was obtained when the Exo plasma standard (106 particles μL-1) was analyzed on the coverslip without anti-CD63 (Figure 2a and Figure 2b). Assay performed with only AAP1 had a low S/B ratio. Markedly different result was noted, however, when anti-CD63-conjugated coverslip was treated with this concentration standard and the mixture of AAP2, H1 and H2. Owing to the HCR-based signal enhancement effect, the TIRF assay with ExoABDNs exhibited an improved S/B ratio from 5 to ~13. In these TIRF images, single fluorescent Exo was visualized as an individual bright spot. The signal intensity of bright spots is proportional to the PTK7 contents on target Exo. The brightness and spot size for each spot were not uniform (Figure 2a), suggesting that the number of target PTK7 on Exo surface is different. Thus the fluorescence analysis of single bright spot in the TIRF images readily reveals the stoichiometry of PTK7 on each Exo surface (Figure S5). More control assays were demonstrated in Figure S6, and the results further confirmed the signal amplification effect of ABDNs hybrids in the TIRF assay for PTK7-Exo in plasma. Afterward, the obtained TIRF images were used for single-vesicle counting (Figure S6c). This count is directly proportional to the quantity of target Exo in bulk solution.12 Therefore, quantification of target PTK7-

In view of the above, the sensitivity and linearity of the TIRF assay were investigated by single-vesicles counting. We found that the counts of Exo increased with the concentration of Exo (Figure 2c and Figure S7a-c). In the low level of Exo content, the counted Exo number increased greatly with Exo concentration, while in the high level of Exo concentration, the counted Exo number increased slowly. This is because the amount of Exo captured on the coverslip surface tends to the saturation at high concentration. A good linear relationship for the TIRF assay with Exo-ABDNs was obtained in the range from 103 to 108 particles μL-1 (Figure 2d and Figure S7d). The plot was fitted by equation lgY = 0.3674 × lgC - 0.1034 (R2 = 0.9886) (Y was the counted Exo number and C was the known Exo concentration). Limit of detection (LOD) and limit of quantification (LOQ) were respectively calculated to be 103 particles µL-1 (LOD = blank + 3 × standard deviation of mean of blank) and 4 × 103 particles µL-1 (LOQ = blank + 10 × standard deviation of mean of blank). Similarly, the detection limits of TIRF assays with Exo-AAP1 and Exoanti-PTK7 could be also determined to be 3 × 105 and 7 × 105 particles μL-1, respectively (Figure S7d). Notably, the Exo numbers counted by ABDNs labelling were significantly greater than those counted by only AAP1 and anti-PTK7 labelling at all concentrations, and this difference progressively increased with Exo concentration (slopes of 0.1778 for AAP1, 0.2557 for anti-PTK7, and 0.3674 for ABDNs), revealing a signal-amplification effect of DNA device. The TIRF assay with Exo-ABDNs displays a broad linear range (103-107 particles μL-1) with a strong correlation (R2 = 0.9887) between calculated and known Exo concentrations (Figure 3a). An ELISA assay (the standard quantification method for target Exo) was also performed for quantifying target PTK7-Exo. A linear relationship for the ELISA assay was obtained in the range from 106 to 108 particles μL-1 (Figure 3b). The LOD of ELISA was evaluated to be about 5.2 × 103 particles μL-1 based on the 3 times the standard deviations of the background. Compared with ELISA, the developed assay has a boarder dynamic range, lower plasma consumption, shorter analysis time and lower reagent cost (Table S2). Moreover, we compared the developed TIRF assay with other available technologies (Table S3). Among the currently competitive methods, the electrochemical sensors showed high detection sensitivity and short detection time. However, most electrochemical analysis could not avoid the interference of background current and the dependency on the external factor, such as temperature, pH and the surface harshness of electrode. It is challenging to apply electrochemical sensors to analyze real samples. Although the combinations of electrochemical methods and separation enrichment techniques, including magnetic separation and microfluidic technology, can achieve the analysis of target Exo in complicated samples and enhance the detection sensitivity, these methods require longer analysis time and larger sample volume than the TIRF assay. Taken



altogether, our developed assay is superior to most available methods. In addition, the TIRF assay showed the satisfactory recoveries and acceptable standard deviations (Table S4), suggesting that the developed strategy had the potential for reliable and practical detection of circulating tumor Exo in real plasma samples.

ultrasensitive non-invasive assay is readily capable for early tumor diagnosis.

Figure 3. (a) Correlation between Exo concentration calculated by TIRF assay and known Exo concentration. (b) Correlation of ELISA absorbance with log Exo concentration.

TIRF Assays for Identification of Target Exo Markers. Furthermore, the identification of PTK7-Exo and total Exo from CCRF-CEM cells or control Ramos cells was determined at progressive culture time points to study the PTK7-Exo profile with regard to the tumor progression. The content of PTK7-Exo was greater in CCRF-CEM than in Ramos cell culture supernatants at all time points, and it obviously increased with culture duration (Figure 4a), indicating that the TIRF assay for PTK7-Exo in cell culture media is informative for CCRF-CEM tumor progression. The TIRF assay for total Exo was then performed by using an antibody against CD81, which is also an abundant biomarker on most Exo surfaces. Different from the results of PTK7-Exo, the changing trend of total Exo level of both Ramos and CCRF-CEM cells was accordant (Figure 4b). But the number of total Exo and PTK7-Exo counted in the culture media of CCRF-CEM cells is inconsistent. The results suggested that not all Exo derived from CCRF-CEM cells possessed the target PTK7 proteins. Calculations based on NanoSight and TIRF assay data demonstrated that PTK7-Exo represented approximately 0.09% of total Exo in Ramos cells and 8.58% of total Exo in CCRF-CEM cells (Table S5). PTK7-Exo TIRF Assay for Tumor Diagnosis in Mouse Model. To verify the capability of TIRF assay to determine the PTK7-Exo level in plasma during tumor development, athymic nude mice were subcutaneously injected with CCRF-CEM and control Ramos cells, respectively, to establish the mouse tumor models. Then, the PTK7-Exo levels in plasma samples of mice were analyzed by the TIRF assay every 10 days (Figure 4c). Plasma PTK7-Exo levels increased with time in the samples from CCRF-CEM tumor-bearing mice while remained steady in negative control mice (Figure 4d). The TIRF assays significantly distinguish CCRF-CEM tumor-bearing mice from control mice by 10 days post-injection when the tumor was almost invisible to naked eye (Figure S8), thus suggesting that this

Figure 4. Identification of PTK7-Exo as a potential biomarker by the TIRF assay. (a) PTK7-Exo numbers in CCRF-CEM and Ramos culture over time by building PTK7-Exo-ABDNs hybrids. (b) Total Exo numbers in CCRF-CEM and Ramos culture over time by using anti-CD81 with FITC label. (c) TIRF images at indicated time points in plasma samples of mice after injection with CCRF-CEM cells (2 × 106 cells). Scale bar = 3 µm. (d) PTK7-Exo numbers counted by analyzing the TIRF images of plasma samples from the tumor-bearing mice.

PTK7-Exo TIRF Assay for Treatment Monitoring in Mouse Model. Clinical tumor cases are frequently characterized by high rates of therapy resistance, and means for monitoring early responses to treatment are urgently needed to improve therapeutic outcomes. We therefore investigated whether the PTK7-Exo plasma levels reflected the responses of CCRF-CEM tumor to treatment in mice. Plasma samples were collected from tumorbearing mice before and after treatment with doxorubicin (DOX) in different tumor progressions, and analyzed by the TIRF assay every 5 days. The treatment to mice started at 15 and 30 days after tumor implantation, respectively, and frequency of drug administration each group was once every 10 days. PTK7-Exo level in plasma samples of posttreatment mice was found to significantly decrease with the increased dosages of DOX (Figure 5a and Figure 5b). Tumor volume were also monitored in the whole treatment process (Figure 5c and Figure 5d), which matched very well with the PTK7-Exo profile as revealed by TIRF assay. Changes in PTK7-Exo levels are associated with the treatment responses (Figure S9). Therefore, plasma PTK7-Exo level is a useful independent index to detect early responses to treatment, and is highly applicable to the treatment evaluation.


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Analytical Chemistry conventional ELISA. The TIRF assay for plasma PTK7-Exo is also informative in staging tumor progression and in monitoring early responses to treatment. In addition, with the ability to directly determine the quantification and stoichiometry of target Exo in plasma at the single-vesicle level, the developed TIRF imaging platform opens the door to better understand the roles of Exo in cell-cell communication in oncogenesis and tumor heterogeneity, offering the possibility for early diagnosis of diseases including but not restricted to cancers and for designing personalized treatment regimens.

ASSOCIATED CONTENT Supporting Information

Figure 5. (a) PTK7-Exo plasma levels of CCRF-CEM tumorbearing mice that started treatment with DOX (once every 10 days) at 15 days post-injection. (b) PTK7-Exo plasma levels of mice after treatment with DOX (40 and 60 mg m-2 per 10 days) for different times. The treatment to mice started at 30 days post-injection. (c) Changes of tumor sizes after treatment with DOX. The different dosages (10, 20 and 40 mg m-2) of DOX were injected into the abdominal cavity of tumor-bearing mice once every 10 days, respectively. (d) Changes of relative tumor volume (V/V0) upon different treatments after 30 days post-injection. Tumor volumes (V) were normalized to their initial values (V0).

PTK7-Exo TIRF Assay of Clinical Samples. We performed the TIRF imaging analysis of PTK7-Exo in plasma microsamples from five human acute lymphoblastic leukemia patients and five healthy individuals. As shown in Figure S10, plasma samples of cancer patients possessed significantly higher content of PTK7-Exo than that of healthy controls. The results indicated that the developed method is readily applicable to measure the PTK7-Exo level in clinical plasma samples and can distinguish cancer patients from healthy control subjects. The PTK7-positive Exo is suitable for applying to the TIRF imaging analysis, which has the potential to be a convenient tool for the isolation of circulating tumor Exo from clinical plasma samples and preliminary screening in the diagnosis of acute lymphoblastic leukemia in clinical settings. CONCLUSIONS In summary, we have demonstrated an ultrasensitive single-vesicle imaging and detection platform with the TIRF microscopy for quantitative analysis of target tumor Exo by in-situ building molecular recognition-based fluorescent DNA nanodevices on target Exo surfaces. This TIRF assay is able to directly visualize and quantify circulating PTK7-Exo in plasma without any isolation and purification steps, and offers multiple advantages (such as high sensitivity, board dynamic range, high throughput, little sample consumption and low reagent cost) over a

The Supporting Information is available free of charge on the ACS Publications website. DNA sequences, and supplementary tables and figures (PDF).

AUTHOR INFORMATION Corresponding Author * E-mail (H.-W. Li): [email protected]. * E-mail (K. Wang): [email protected]. * E-mail (X. He): [email protected].

Notes Any additional relevant notes should be placed here.

ACKNOWLEDGMENT This work was financially supported by the National Natural Science Foundation of China (21775036, 21675046, 21735002), the Hunan Provincial Natural Science Foundation (2018JJ2033), the Hong Kong Scholars Program, the Research Grant Council of Hong Kong (HKBU12308416 and C2012-15G) and the research committee of Hong Kong Baptist University (FRG2/15-16/028).

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Molecular-Recognition-Based DNA Nanodevices for Enhancing the

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