Advances in biosensing technologies for analysis of cancer-derived exosomes

Trends in Analytical Chemistry 123 (2020) 115773

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Advances in biosensing technologies for analysis of cancer-derived exosomes Huiying Xu a, Bang-Ce Ye a, b, c, * a Lab of Biosystem and Microanalysis, State Key Laboratory of Bioreactor Engineering, East China University of Science & Technology, Shanghai, 200237, China b Institute of Engineering Biology and Health, Collaborative Innovation Center of Yangtze River Delta Region Green Pharmaceuticals, College of Pharmaceutical Sciences, Zhejiang University of Technology, Hangzhou, 310014, Zhejiang, China c School of Chemistry and Chemical Engineering, Shihezi University, Xinjiang, 832000, China

a r t i c l e i n f o

a b s t r a c t

Article history: Available online 13 December 2019

Exosomes play an important role in intercellular communication that contribute to the interaction of cancer cells with the tumor microenvironment. The original features endow tumor-derived exosomes with distinctive opportunities to serve as sensitive biomarkers for identifying early-stage tumors and monitoring of therapeutic response non-invasively. Conventional detection techniques face a number of drawbacks including sensitivity and reproducibility, therefore considerable efforts have been made to establish reliable biosensors for exosomes analysis. This review summarizes recent progress in exosomes quantification techniques, mainly focused on novel molecular recognition methods and signal transduction strategies. Furthermore, we offer a perspective of exosomes research towards the development innovative concepts on future applications. © 2019 Elsevier B.V. All rights reserved.

Keywords: Cancer-derived exosomes Biosensing technologies Molecular recognition methods Signal transduction strategies Cancer diagnostics

1. Introduction The tumorigenesis and progression is a stepwise and complex process which highly depend on the ability of primary tumor to adapt and colonize the surrounding microenvironment [1]. To develop a comfortable microenvironment for tumor invasion and metastasis, cancer cells can secrete numerous growth factors and matrix metalloproteinase to promote the growth of tumor and surrounding stromal compartments, such as cancer-associated fibroblasts, vascular endothelial cells and immune cells. These surrounding cells can also continuously release different types of cytokines and growth factors that directly or indirectly remould the tumor microenvironment [2,3]. The symbiotic relationship between tumor cells and different stromal cells brings about uncontrollably differentiation which alters tissue homeostasis to provide support for the survival of tumors and facilitates of tumor progression, invasion and drug resistance [4]. Bidirectional communication between cancer cells and their surroundings drive the

* Corresponding author. Lab of Biosystem and Microanalysis, State Key Laboratory of Bioreactor Engineering, East China University of Science & Technology, Shanghai, 200237, China. E-mail address: [email protected] (B.-C. Ye). https://doi.org/10.1016/j.trac.2019.115773 0165-9936/© 2019 Elsevier B.V. All rights reserved.

development of the tumor microenvironment and understanding the communication pattern is critical for the prevention, diagnosis and therapy of malignant tumors [2]. Recently, emerging evidence indicated that exosomes play an important role in intercellular communication and molecular exchange that contribute to the interaction of cancer cells with the tumor microenvironment [5,6]. Exosomes, a branch of extracellular vesicles (EVs), are membrane-enclosed phospholipid nanovesicles with a diameter of 30e150 nm and cup-shape appearance, which are actively secreted by all eukaryotic cells, including various tumor cells and normal cells through an endolysosomal pathway [7,8]. These nanovesicles carry enriched proteins and nucleic acids originated from parents’ cells, including tetraspanin family (CD9, CD63 and CD81), adhesion molecules (integrins, lactadherin), heatshock proteins (Hsp 60, Hsp 70 and Hsp 90), messenger RNA and microRNA [3,9]. Exosomes exist in a variety of bodily fluids and can serve as transmission medium in cell-to-cell signaling through transferring their cargo to remote sites to regulate the receptor cells [6]. Tumor-derived exosomes are potential contributors to tumorigenesis and angiogenic process via releasing relevant signal molecules which results in enhanced proliferation and migration of endothelial cells [10]. The immune system can also be affected by tumor-derived exosomes which block tumor recognition and

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counter the anti-tumor immunity systemically [11]. Furthermore, the number of tumor-associated vesicles and their molecular profiles have correlated with tumor heterogeneity, temporal evolution and treatment response [12e14]. Compared to circulating tumor cells, exosomes are actively secreted in large quantities and provide a rich genetic profile of their original cells [15]. These original features endow tumor-derived exosomes with distinctive opportunities to serve as stable and sensitive biomarkers for identifying early-stage tumors, performing personalized medicine and realtime monitoring of therapeutic response non-invasively [16e18]. Unfortunately, the small size and low buoyant density of exosomes pose significant challenges to their isolation and detection in the complex biofluids [19]. Although being considered as the gold standard, ultracentrifugation isolation requires expensive instruments and tedious steps, which may suffer from low recovery with high expenditure [17,20]. To quantify the isolated/concentrated exosomes, conventional detection techniques mainly includes nanoparticle tracking analysis (NTA), dynamic light scattering (DLS), flow cytometry, transmission electron microscope (TEM), and enzyme-linked immunosorbent assay (ELISA) [21]. NTA is the most popular technology to characterize exosomes by tracking their Brownian motion in a suspension and calculate their concentration and size information [22]. However, it is still difficult to achieve high reproducibility by different users because of the higher request to the operation skills. As the instrumental and software settings require manual adjustments to eliminate deviations, it is time-consuming accompanied by the risk of individual error [23]. DLS is widely used to characterize the particle-size distribution of a variety of particles such as nanoparticles and proteins by quantifying fluctuations in scattered light intensities [24]. DLS is user-friendly and does not need a large sample volume, but it is not amenable to molecular labeling when required to profile specific molecular information on exosomes [21]. Although Flow cytometry allows to measure multiple surface markers of exosomes, it faces the challenge to recover such weak signals from the smaller size of exosomes [25]. Besides, TEM imaging mainly provides the morphology and heterogeneity of exosomes and ELISA is limited by large sample demand and low sensitivity. To improve the sensitivity, speed and efficiency, numerous researchers attempt to establish novel biosensors for the detection of exosomes with the addition of several detectors, such as ultravioletevis spectroscopy, fluorescence, surface-enhanced raman scattering (SERS), and electrochemical detector. The instrumental simplicity and popularization of biosensors could provide sufficient selectivity, diversity of analysis, high sensitivity and rapid response time. The rapid development of exosomes analysis platforms based on newly identified biomarkers, highpurity capture, newly developed signal transduction pathway and signal amplification strategies holds promise for the future production of high-throughput detection methods and advanced diagnostic tools for cancer patients. This review presents an overview of recent developments in innovative exosomes sensors to more reliably and accurately quantify tumor-derived exosomes. 2. New development in exosomes quantification techniques When establishing convenient and reliable sensing strategies, the characteristic lipid bilayer and abundant proteins expressed on exosomes provide unique binding sites to transduce the recognition event into a measurable signal. Taking advantages of the highly enriched cancer-associated antigens on the surface, tumor-derived exosomes can be detected by utilizing the differences of their specific protein composition (such as CD24, EpCAM, MUC1, PSMA, PTK7, HER2, and CA125), which are used as diagnostic targets for a

variety of cancers [26]. In addition to the classic antigen-antibody interaction, many aptamers and biocompatible anchor molecules are also introduced to act as the recognition elements because of their binding affinity on exosomes. During the past few years, various types of signal conversion methods have been used to develop biosensors for exosomes analysis with the help of several detectors. According to the different signal generator mechanism, we divides the previously reported biosensors for the detection of exosomes into five parts: ELISA based sensing protocol, directly exosomes quantification, aptamer based signal amplification, interaction between aptamer and nano-materials and correlated with other aptamer or materials. 2.1. ELISA based sensing protocol Some ELISA-based sensing protocol have been developed to improve the sensitivity of traditional ELISA. Doldan et al. described an electrochemical exosomes biosensor by immobilizing rabbit aCD9 antibodies on gold electrodes and using another monoclonal a-CD9 antibody against CD9 for detection of captured exosomes (Fig. 1A) [27]. Signal amplification can be obtained from the multiple detector antibodies bind to different copies of the CD9 protein exposed on the surface of each captured vesicle. With sample volumes as low as 1.5 mL, this electrochemical sensor achieved sensitive detection of exosomes, with a limit of detection of 2
102 particles/mL. Chen et al. designed a ZnO nanowires coated three-dimensional scaffold chip device for effective immunocapture and colorimetric detection of exosomes (Fig. 1C) [28]. The hierarchical nanointerface of the modified 3D porous scaffold not only provides more binding site to immobilize more anti-CD63 antibody, but also benefits for the contact frequency between exosome and ZnO nanowires because of size exclusion-like effect and chaotic mixing. The high capture efficiency greatly facilitates the following colorimetric assay to achieve good reproducibility and enough sensitivity with a minimal detectable concentration of 2.2
104 particles/mL. Boriachek et al. introduced CD9 or CD63 antibody functionalized AueNPFe2O3NC to direct isolation and electrochemical quantification of placenta alkaline phosphatase (PLAP) specific exosomes (Fig. 1C) [29]. The intrinsic horseradish peroxidase (HRP)mimicking activity of AueNPFe2O3NC was used to catalyze the oxidation of TMB and the electroactive yellow-colored product further generated electrochemical signal which was also convenient for naked-eye visualization. This nanozyme-based biosensor achieved high sensitivity with a LOD of 103 particles/mL which is 104-fold better in comparison with ELISA. ELISA-based sensing protocol are easy to be integrated in multiplexer channel analysis. Jeong et al. reported an integrated magneto-electrochemical approach for rapid, on-site exosome analyses [30]. Parallel measurements were achieved in the miniaturized and expanded sensors after exosomes enrichment by immunomagnetic beads. Unlike the binding reaction of primary antibodies and secondary antibodies in ELISA, HRP was brought into the sensing system through the interaction between sulfoeNHSebiotin labeled antibodies and streptavidin-HRP. They applied this platform to profile multiple protein markers of exosomes though altering different antibodies. The results validated the protein expression of CD63-positive exosomes have a high correlation with their parent cells and revealed the expression levels of CD24 and EpCAM in exosomes were much higher in ovarian cancer patients than in healthy samples. Chiu et al. presented an exosome secretion assay which accommodates different cellular types and efficient single-cell culturing [31]. A PDMS mesh with an array of through holes was fabricated and adhered to a glass substrate to form microwells for

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Fig. 1. ELISA-based sensing protocol for exosomes analysis. (A) Schematic representation of the amperometric immunosensor for determination of exosomes. Reprinted with permission from Ref. [27], Copyright 2016 American Chemical Society. (B) The assay for direct exosome magnetic isolation and AueNPFe2O3NC based detection from cell culture media. Reprinted with permission from Ref. [29], Copyright 2019 American Chemical Society. (C) The immunocapture, colorimetric detection and controlled release of exosomes by ZnO nanowires coated three-dimensional scaffold chip. Reprinted with permission from Ref. [28], Copyright 2018 Elsevier.

single cells. Each antibody-treated glass slide can capture secreted exosomes which were subsequently labeled with another antibody coupled with Quantum Dots (Qdots). The exosomes secretion can be investigated by counting quantum dots. The noninvasive collection and surveying of single-cell secretions makes it suitable for evaluating environmental effects in combination with drug screening. To detect low levels of cancer-derived exosomes, Zhang et al. designed a microfluidic chip with self-assembled three-dimensional nanostructured herringbone (nano-HB) [32]. This 3D nanostructure promotes microscale mass transfer of bioparticles and has unique drainage to reduce near-surface hydrodynamic resistance, which can enhance surface binding efficiency of particles resulted from particles enrichment on the increased surface area. Combined with an ELISA method, the nano-HB chip can achieve a low limit of detection of 10 exosomes/mL. To verify the feasibility for biomarker profiling of cancer, they investigated alteration of CD24, EpCAM and folate receptor alpha (FRa) protein expression in exosomes derived from ovarian cancer plasmas. Only 2 mL plasma sample was tested every time and the results suggested exosomal FRa as a potential biomarker for monitoring of ovarian cancer.

2.2. Directly exosomes quantification With the help of different sensing interface, directly exosomes quantification can be implemented without any other signal molecules. The innovation of sensing interface can bring opportunities to shorten response time and improve sensitivity. Field effect transistors (FET) can transform and amplify microelectrical signals into readable electrical signals when interaction occurs between biomolecules and sensing interface [33]. Recently, a CD63 antibody-functionalized reduced graphene oxide FET biosensor was developed to directly detect exosomes in a sensitive and labelfree manner (Fig. 2A) [34]. After the capture process, the negative charges of exosomes will change the net carrier density on the substrate surface, leading to the migration of the Dirac point. A good linear relationship between changes in the Dirac point and exosome concentration was obtained and nonspecific proteins such as APF, CEA and BSA didn't disturb the Dirac point changes. By quantifying exosomes directly, the RGO FET biosensor can achieve a relatively high sensitivity with a LOD of 33 particles/mL. Electrochemical biosensors have intrinsic advantages of rapid response, low cost, sensitive recognition, and excellent portability.

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Fig. 2. Directly exosomes quantification strategies. (A) Schematic diagram of a CD63 antibody functionalized RGO FET sensing device for detection of exosomes. Reprinted with permission from Ref. [34], Copyright 2019 American Chemical Society. (B) Schematic illustration of the NTH-assisted electrochemical aptasensor. Reprinted with permission from Ref. [35], Copyright 2017 American Chemical Society. (C) Schematic illustration of TiN functionalized by anti-CD63 antibody for the detection of U251 GMs-derived exosomes. Reprinted with permission from Ref. [37], Copyright 2018 Wiley-VCH.

Electrochemical-based exosomes analysis have unique superiority in implementing miniaturized and flexible design. Wang et al. reported a nanotetrahedron (NTH)-assisted aptasensor for capture hepatocarcinoma (HepG2) exosomes as well as direct electrochemical detection (Fig. 2B) [35]. They introduced a modified ATGCZP library to screen out the aptamer LZH8 which exhibited superior binding selectivity for HepG2 exosomes. A DNA NTH supportor was combined on gold electrodes to maintain spatial orientation of LZH8 and decrease hindrance effect which was beneficial for biomolecular recognition between LZH8 and exosomes. The electric signals were generated when captured exosomes decreased electrode surface area and suppressed signals of ferricyanideferrocyanide redox couple. Compared to the single modification of LZH8 aptamer, the NTH supportor significantly improves sensitivity of the electrochemical sensor of HepG2 exosomes with the LOD of 2.09
104 particles/mL and a wide linear range from 105 to 1012 particles/mL.

Surface plasmon resonance (SPR) can reflect the interaction information between positive and negative permittivity materials at the interface through the resonant oscillation of stimulated electrons [36]. SPR biosensing is not only a label-free and real-time molecular sensing technique which can transmit fast analysis, but also a non-destructive method with no need of tedious sample preparation steps [22]. Qiu et al. innovatively took advantage of Titanium nitride (TiN) film in SPR biosensing to devise a biotinylated antibody-functionalized TiN (BAF-TiN) biosensor for the detection of malignant U251 glioma cells (GMs)-derived exosomes (Fig. 2C) [37]. To capture GMs-derived exosomes, the synthesized TiN film was functionalized by biotinylated-anti-CD63 and biotinylated anti-EGFRvIII antibody. This method showed better sensitivity than that of the conventional gold-film-based SPR biosensors because of the much closer spatial extent between GMsderived exosome and the TiN thin film. Furthermore, BAF-TiN biosensors exhibited good selectivity toward U251GMs-derived

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exosomes and serum-derived exosomes, which demonstrated their potential to quantify exosomes in real biological fluids. Im et al. reported a SPR-based assay for high-throughput exosome protein analysis [12]. The chip comprises arrays of plasmonic nanoholes functionalized with different antibody for specific exosomes binding and the following spectral shifts or intensity changes are proportional to protein levels of exosomes. The LOD of the nPLEX chip was calculated to be 3000 particles/mL when functionalized with an anti-CD63 antibody. By analyzing ascites samples from cancer patients, they found that the expression of CD24 and EpCAM can be used to identify ovarian cancer exosomes. 2.3. Aptamer based signal amplification As an emerging alternative to antibodies, aptamers are singlestranded oligonucleotides that fold into three-dimensional structures and exhibit high affinity for a specific target, showing great promising for analysis of exosomes surface proteins [38e40]. Compared with antibodies, aptamers not only have better stability with low immunogenicity, but also can be modified with functional groups facilely and synthesized with little batch-to-batch variation [41,42]. More importantly, aptamers can be readily incorporated into diversified DNA-based reactions, such as rolling-cycle amplification (RCA), polymerase chain reaction (PCR), hybridization chain reaction (HCR) and cascade hybridization reaction (CHR), to obtain amplified signals for targets detection [43e46]. In recent years, a variety of aptamer-mediated approach have been reported to quantify exosomes and analysis of exosomal functional proteins. Zhou et al. first reported a CD63 aptamer-based electrochemical biosensor for exosomes detection [47]. CD63 aptamers were immobilized onto electrode surface and hybridized with probing strands which were pre-labeled with redox moieties. CD63positive exosomes can displace probing strands resulted in decreased redox signal. This aptasensor exhibited 100 times lower detection limit (1
106 particles/mL) than commercial immunoassay based on CD63 antibody. After that, an increasing number of reports utilized CD63 aptamer as the recognition unit to capture or detect exosomes because of the simple sequences of CD63 aptamer and the abundant CD63 on the surfaces of exosomes [48e55]. Xu et al. presented a two-stage microfluidic platform which integrates on-chip isolation and a new signal transduction strategy for exosomes analysis (Fig. 3A) [56]. After efficient capture through an array of Y-shaped micropillars, a label-free electrochemical aptasensor was introduced to quantify exosomes. CD63-positive exosomes can open the original single-stranded DNA hairpin and form a hemin/G-quadruplex complex which was employed as the HRPmimicking DNAzyme and NADH oxidase for signal enhancement. This platform enables sensitive detection for CD63 positive exosomes as low as 4.39
103 particles/mL without expensive nucleic acid modification and complicated signal amplification. Most importantly, clinical sample analysis showed satisfactory results to differentiate liver cancer patients from healthy serums. Zhang et al. found mucin 1 protein (MUC1) was also highly expressed on the surface of MCF-7 cells-derived exosomes compared to normal breast cells-derived exosomes [57]. They designed an aptasensor based on MUC1 aptamer which was labeled with a luminophore and quenching group. MCF-7 exosomes can open the hairpin-like MUC1 aptasensor and separate the luminophore from the quenching group, which was resulted in strong fluorescence. The simple and rapid method had a LOD of 4.2
104 particles/mL. With the help of MUC1 aptamer, Huang et al. presented a label-free electrochemical aptasensor for detection of gastric cancer exosomes by combining RCA and hemin/G-

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quadruplex system [58]. By linking the MUC1 aptamer to a primer sequence, RCA can be triggered by gastric cancer exosomes to produce multiple G-quadruplex units which can efficiently catalyze the H2O2-mediated oxidation of TMB to achieve amplified electrochemical signal. The aptasensor exhibits high specificity and sensitivity toward gastric cancer exosomes with the detection limit of 9.54
102 particles/mL. Taking advantage of naturally anchoring of cholesterol on lipid membranes of exosomes, He et al. developed a versatile method for direct exosome quantification [59]. After immunomagnetic capture, exosomes were anchored by a bivalent-cholesterol-labeled DNA probe which was used as a trigger for HCR for signal amplification. HRP-tagged DNA probe was used as a signal transducer and the enzyme-linked HCR amplification was proved to enhance detection efficiency for exosomes significantly. The LOD was calculated to be 2.2
103 exosomes/mL. In addition, they presented a SERS based method by immobilizing the bivalent cholesterol (B-Chol)-labeled DNA anchor with as-prepared gold nanostar@4-mercaptobenzoic acid@nanoshell structures (AuNS@4-MBA@Au) to form the SERS nanoprobes [60]. The monitored SERS signals can indicate the corresponding exosome concentration. The LOD of the B-Chol anchor based biosensor was reduced to be 27 particles/mL. To achieve precise and digital detection at a single-particle level, Tian et al. synthesized a biocompatible anchor molecule (BAM)-DNA to insert into exosomes which were distributed into chambers with less than one exosome per chamber on the microchip, and quantitative analysis was applied by digital PCR to amplify the nanoscale signals to microscale digital fluorescence points (Fig. 3B) [61]. After BAMDNA-labeled total exosomes analysis, antibodyDNA was specific binding on exosomes to generate a dual signal for selective exosomes detection. In another work, a tyrosine-protein-kinase-like 7 (PTK7)-contained aptamer was designed to identify PTK7 positiveexosomes and initiate assembly of HCR in the presence of two fluorescent molecular hairpins (Fig. 3C) [43]. The enhanced fluorescence can be detected by a total-internal-reflection-fluorescence (TIRF) assay for directly visualizing and quantification PTK7positive exosomes at the single-vesicle level without any purification steps. The TIRF imaging platform offers an intuitive method for monitoring of disease-associated exosomes and provides a new way to research the roles of exosomes in oncogenesis and tumor heterogeneity. Liu et al. developed a l-DNA-mediated size-selective separation and subsequent aptamer-mediated detection of extracellular vesicles (EV) subpopulations (Fig. 3D) [62]. A viscoelastic microfluidic system and Newtonian sheath fluid was designed for separation of exosomes, microvesicles (MVs), and apoptotic bodies (ABs). Human epidermal growth factor receptor 2 (HER2) and epithelial cell adhesion molecule (EpCAM) aptamers were pre-labeled with different fluorophore for detection of EVs subpopulations by fluorescence microscopy. They revealed that the isolated MVs have the highest discrimination ability than exosomes and ABs in classifying breast cell lines and breast cancer patients with varied HER2 expression. Besides, they reported a thermophoretic aptasensor for the enrichment of EVs and profiled of surface proteins (PTK7, LZH8, HER2, PSA, CA125, EpCAM and CD63) via a panel of seven fluorescent aptamers [63]. When using DiI-labeled HepG2 EVs to evaluate the performance, the thermophoretic aptasensor showed a low LOD of 3.3
103 particles/mL. By profiling of EVs in hundreds of serum samples and applying a linear discriminant analysis algorithm, this system can detect stage I cancers with high sensitivity and classify six cancer type with an overall accuracy of 68%. The low sample volumes (<1 mL) and rapid analysis make the assay convenient for early cancer screening and classification.

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Fig. 3. Aptamer based signal amplification for exosomes detection. (A) Schematic diagram of the ExoPCD-chip and the electrochemical sensor on the surface of ITO electrode. Reprinted with permission from Ref. [56], Copyright 2018 American Chemical Society. (B) Schematic of digital exosomes analysis at the single-nanoparticle level through nucleic acid-based amplification. Reprinted with permission from Ref. [61], Copyright 2018 American Chemical Society. (C) Illustration of the single-vesicle imaging based on TIRF assay and HCR for detection of circulating tumor specific exosomes. Reprinted with permission from Ref. [43], Copyright 2019 American Chemical Society. (D) Schematic of l-DNA-mediated sorting of EV subpopulations and individual EVs analysis based on multiple aptamers. Reprinted with permission from Ref. [62], Copyright 2019 American Chemical Society.

2.4. Interaction between aptamer and nano-materials Nanomaterials-based biosensors are revolutionizing in molecular diagnostics and biomarkers analysis through simple reaction and readout strategy [64]. The combination of nanomaterials and aptamers offers them not only the unique photoelectric properties and catalytic activity of nanomaterials but also the target-specific of aptamers, which is ideal to develop various nanoprobes with desired properties [65]. The intrinsic peroxidase-like activity of graphitic carbon nitride nanosheets (g-C3N4 NSs) endows this graphene-like carbon-based nanomaterial great potential in biomedical applications [66]. Wang et al. found that single stranded DNA (ssDNA) adsorbed g-C3N4 NSs have improved catalytic activity than unmodified NSs (Fig. 4A) [54]. Inspired by this capability of

ssDNA, they introduced CD63 aptamer to bind onto exosomes which can generate variable catalytic ability under different concentrations of exosomes. Absolute quantification of exosomes was measured based on changes of the colored product. They selected TMB as peroxidase substrate by evaluating the molecular structure of TMB and ABTS, and the LOD was calculated as 13.52
105 particles/mL. Xia et al. also reported an exosomes quantification method based on the interaction between CD63 aptamer and single-walled carbon nanotubes (s-SWCNTs) which possess intrinsic peroxidase-like activity [55]. The DNA-capped s-SWCNTs exhibited enhanced peroxidase activity which can be decreased after adding exosomes and accompanied by color changes from deep to moderate. In addition, Chen et al. first developed an anionexchange-based isolation method and then they detected

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Fig. 4. Detection exosomes based on interaction between aptamer and nano-materials (A) Illustration of the nanozyme sensor combined with DNA aptamer and g-C3N4 NSs for the detection of exosomes. Reprinted with permission from Ref. [54], Copyright 2017 American Chemical Society. (B) Schematic of the Cy3-CD63 aptasensor coupled with MXenes for exosomes detection. Reprinted with permission from Ref. [50], Copyright 2018 American Chemical Society. (C) Schematic of aptamer/GO nanoprobe mediated molecular recognition with the aid of DNase I that can recycle the specific binding events for signal amplification. Reprinted with permission from Ref. [64], Copyright 2018 American Chemical Society. (D) Synthetic route of ASP and sensing mechanism of ASPNC. Reprinted with permission from Ref. [70], Copyright 2019 Wiley-VCH.

prostate cancer exosomes by means of the interaction between EpCAM aptamer and Fe3O4 NPs which also possess peroxidase activity [67]. After an anion-exchange-based exosome isolation process, the aptasensor could detect exosomes as few as 3.58
106 mL1. Due to the complete metal atomic layers and abundant of hydroxyl or oxygen terminated surface, MXenes have unique nanobiointerface units and exhibit intrinsic fluorescence quenching ability [68]. Zhang et al. constructed a self-standard FRET biosensing platform based on the Cy3 labeled CD63 aptamer/Ti3C2 MXenes nanocomplex as nanoprobe for detection of exosomes (Fig. 4B) [50]. The selectively adsorption of CD63 aptamer on Ti3C2 MXenes could quench the fluorescence of the labeled Cy3 dye. The addition of exosomes turned on the fluorescence quickly attributed to the release of aptamer from the surface of MXenes which resulted from the relative strong specific recognition between exosomes and CD63 aptamer. This fluorescence sensor showed a limit of detection exosomes of 1.4
103 particles/mL with a wide dynamic range from 104 to 109 particles/mL. Taking advantages of the similar absorption between graphene oxide (GO) and singlestranded fluorescent aptamer, Jin et al. developed an aptamer/ GO-based nanoprobe to quantify prostate cancer exosomes and phenotype surface proteins (Fig. 4C) [64]. When the aptamer binds with specific exosomal marker and moved away from GO protection, the exposed aptamer sequence can be digested by

deoxyribonuclease I (DNase I). The recognition between aptamer and exosomal surface protein recycles to amplify fluorescence signal. To profile exosomal surface proteins, they used seven protein aptamers (CD63, EpCAM, PSMA, PTK-7, CEA and PDGF) to measure their expression level on exosomes derived from five cell lines. With the help of DNase I-mediated signal amplification, the ExoAPP assay achieved a detection limit of 1.6
105 particles/mL. Moreover, this sensitive strategy allows for monitoring epithelialmesenchymal transition (EMT) through exosomes. They verified the increased expression of EpCAM and PSMA on exosomes from serum samples of prostate cancer patients compared to healthy individuals. Jiang et al. reported a multiplexed exosomes sensor platform consists of a gold nanoparticle (AuNP) coupled with a panel of five aptamers [69]. Because of the stronger binding between the aptamer and exosomal surface protein, exosomes can break the non-specific binding equilibrium between AuNP and aptamers. Without aptamers protection, AuNPs aggregates in a high salt solution and the color of the solution changes from red to blue. Taking this simple approach, they investigated the differential expression of CD63, EpCAM, PDGF, PSMA, and PTK7 on exosomes secreted from HeLa, human prostate cancer (PC3), human acute lymphoblastic leukemia (Ramos) and human acute lymphoblastic leukemia (CEM) cells, respectively. Lyu et al. synthesized an afterglow semiconducting polyelectrolyte nanocomplex (ASPNC) to construct a

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nanoprobe electrostatic attracted to the quencher BHQ-2-tagged aptamer for the detection of exosomes (Fig. 4D) [70]. The electron transfer between ASPNC and BHQ-2 can quench the initial fluorescence and afterglow signals of ASPNC. When aptamer encounters exosomes, their specific binding increases the distance of the quencher, and recovers both fluorescence and afterglow signals. To validate the recognition ability, they employed five kinds of exosomes to examine expression levels of CD63, EpCAM, HER2, and MUC1 and the results were consistent with previous reports [71]. 2.5. Correlated with other aptamer or materials In order to obtain significant signal amplification, some researchers tried to build some relationship between exosomes and other materials or DNA probes [72e75]. Then the exosomes quantification can be transferred to the detection of the additional probes. These strategies bring more diversified chooses for exosomes analysis with smaller limits on the characteristic of exosomes. Rational utilizing the intrinsic of supplementary along with smart design may accomplish multiple signal amplification without complicated modification. Wang et al. proposed an exosomeszirconium (Zr4þ) -liposomes structure to detect exosomes (Fig. 5A) [76]. Because of the strong binding ability to phospholipid membranes, Zr4þ ion could adsorb on the surface of exosomes and mediate the cross-linking of liposomes which encapsulated lots of calcein. After removing the unbound liposomes by magnetic separation and the following treatment with 1% Triton X-100, quantity of exosomes can be measured by analysis the concentration of

calcein released from liposomes. The resulting fluorescence intensity is positively correlated to the quantity of exosomes, and the LOD of the assay is calculated to be 7.6
103 particles/mL. He et al. presented a copper-mediated signal amplification strategy for rapid exosomes quantification (Fig. 5B) [77]. Exosomes were first captured by cholesterol-modified magnetic beads. Subsequently, the bead-binding exosomes and CD63 aptamer-modified copper oxide nanoparticles (CuO NPs) can form sandwich complexes through the special recognition of CD63 aptamer. After magnetic separation, the acidolysis transforms CuO NP into Cu2þ, which can be further reduced to fluorescent copper nanoparticles (CuNPs). Finally, the fluorescence intensity increases with the increased Cu2þ concentration, which is direct correlation with the concentration of exosomes. This strategy allows quantitative analysis of exosomes with a LOD of 4.8
104 particles/mL in biological sample. In view of the better understanding of nucleic acid, Dong et al. reported an electrochemical method which converted exosome detection to the cyclic enzymatic amplification for nucleic acid detection through PSMA aptamer recognition induced multi-DNA release (Fig. 5C) [78]. They first prepared a PSMA aptamermagnetic bead bioconjugate and hybridized three kinds of messenger DNAs (mDNAs) with the PSMA aptamers. When LNCaP cells derived exosomes binded to PSMA aptamer, triple the amount of mDNA released and hybridized with probe DNAs to amplify the electrochemical signal generated from electroactive Ru(NH3)63þ. Subsequent Exo III cyclic digestion can decrease the peak current of Ru(NH3)63þ which is correlated with the mDNA concentration. The LOD of the assay was experimentally estimated to be 70 particles/

Fig. 5. Exosomes quantification transferred to the detection of the additional probes. (A) Working principle of the signal amplification assay based on phosphate-Zr4þ-liposome coordination interaction. Reprinted with permission from Ref. [76], Copyright 2019 The Royal Society of Chemistry. (B) Working principle of the exosome detection strategy based on a copper-mediated signal amplification strategy. Reprinted with permission from Ref. [77], Copyright 2018 American Chemical Society. (C) Schematic of the electrochemical method which converted exosome detection to the cyclic enzymatic amplification for nucleic acid detection through PSMA aptamer recognition induced multi-DNA release. Reprinted with permission from Ref. [78], Copyright 2018 American Chemical Society. (D) Schematic illustration of the exosome detection method based on DNA assembly caused dual signal amplification. Reprinted with permission from Ref. [49], Copyright 2019 The Royal Society of Chemistry.

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Table 1 Summary of the exosomes quantification strategies. Methods

Detection limit

Detection time

Target biomarker

Advantages

Disadvantages

Ref.

Colorimetric PLA-RPA-TMA assay

102 particles/mL

2.5 h

[79]

13.52
105 particles/mL

30 min

High specificity and high sensitivity Simple, rapid and low-cost

Complicated operation

ssDNA-NSs hybrid

Surface protein (LPM1 and EGFR) Exosomal CD63

[54]

High selectivity, visibledetection Simple and multiplexed protein analysis Label-free, rapid and visible

Lack specificity and signal-off signal-off strategy

6

Aptamer-capped Fe3O4 NPs

3.58
10 particles/mL

70 min

EpCAM and PSA

Aptamer/AuNP complex

e

e

DNA-capped s-SWCNTs

5.2
105 particles/mL

40 min

CD63, EpCAM, PDGF, PSMA, and PTK7 Exosomal CD63

ZnO-chip device

2.2
10 particles/mL

e

nano-HB chip

10 particles/mL

e

Exosomal CD9 and CD63 CD24, EpCAM and FRa

B-Chol anchor assay based HCR

2.2
103 particles/mL

~16.5 h

Exosomal CD9

2.5 h

LMP1 and EGFR

Effective capture and direct visualization Programmable 3D assembly and increased binding area by the 3D nanostructure Cost-efficient and easy to operate High sensitivity and specificity

1.4
10 particles/mL

1h

Exosomal CD63

4

2

[67]

Lack quantitative information Lack specificity and signal-off Lack specificity

[69]

[28]

e

[32]

Lack specificity

[59]

Complicated operation

[79]

Simple and high sensitivity

Lack specificity

[50]

Low-background and profiling exosomal surface proteins

Rely on fluorescent labeling

[64]

Require expensive instruments Unable to be used in clinical samples Rely on fluorescent labeling Complicated operation

[43]

[61]

e

[62]

Profiling one marker per run

[63]

Lack specificity

[76]

Lack specificity

[77]

Complicated operation and lack specificity Lack clinical samples

[49] [80]

Lack specificity

[27]

Lack clinical samples

[29]

Poor linearity

[30]

Lack clinical samples

[35]

Lack specificity

[47]

Lack specificity

[56]

Narrow linear range

[58]

[55]

PLA-RPA-TMA assay Fluorescence Cy3-CD63 aptamer/Ti3C2 MXenes nanocomplex ExoAPP platform

10 particles/mL

1.6
105 particles/mL

e

ABDN-Based TIRF Assay

103 particles/mL

e

CD63, AFP, CEA, EpCAM, PTK-7, PSMA, and PDGF PTK7

Single-cell assay

e

e

CD63, CD9 and CD81

“on-off”-type aptasensor

4.2
104 particles/mL

32 min

MUC1

BAM-DNA based analysis

e

e

GPC-1

l-DNA -mediated approach

e

e

HER2 and EpCAM

thermophoretic aptasensor

3.3
103 particles/mL

3h

Zr4þ -liposomes based signal amplification copper-mediated signal amplification strategy HDCR/DNA dendrimer assembly strategy polymer-based, fluorescencesensing platform Electrochemical Gold electrodes functionalized with a-CD9 antibodies AueNPFe2O3NC

7.6
103 particles/mL

e

PTK7, LZH8, HER2, PSA, CA125, EpCAM and CD63 Exosomal CD63

4.8
10 particles/mL

2h

Exosomal CD63

Direct quantification at the single-vesicle Quantify exosome secretion by single cells Rapid, high specificity and lowbackground Single exosome digital detection Enable deciphering of heterogeneity of single EVs Small serum volumes and profiling exosomal surface proteins Label-free and relatively simple measuring procedure Ease of operation and low cost

1.16
103 particles/mL

e

Exosomal CD63

Low experimental cost

6 pg/mL

10 min

CD9 and GGT1

Rapid, high specificity and pretreatment-free

2
102 particles/mL

~1.5 h

Exosomal CD9

103 particles/mL

4h

iMEX

3
104 particles/mL

1h

Placenta alkaline phosphatase EpCAM, CD24, CA125, HER2, MUC18 and EGFR

Low sample volumes and wide linear range Simple, visible and portable

NTH-assisted aptasensor

2.09
104 particles/mL

30 min

e

CD63 aptamer immobilized gold electrode ExoPCD-chip

1
106 particles/mL

e

Exosomal CD63

4.39
103 particles/mL

3.5 h

Exosomal CD63

9.54
10 particles/mL

e

MUC1

Integrate chip, label-free and immobilization-free probe High specificity and sensitivity

70 particles/mL

~6 h

PSMA

High specificity and selectivity

Time-consuming and signal-off

[78]

e

CD63 and EGFRvIII

Real-time and good selectivity

e

[37]

nPLEX assay

4.29
103 and 2.75
103 mg/mL 3
103 particles/mL

60 min

CD63, CD24 and EpCAM

e

[12]

B-Chol based SERS RGO FET chip ASPNC detection

27 particles/mL 33 particles/mL 0.24 mg/mL

50 min 30 min e

Exosomal CD9 Exosomal CD63 CD63, EpCAM, HER2, and MUC1

Portable platform, highthroughput and multiplexed protein analysis Easily fabricated Direct and label-free. Multiplex protein profiling and minimized signal background

Lack specificity Lack specificity e

[60] [34] [70]

RCA based hemin/Gquadruplex system Exo III assisted target recycling strategy Others BAF-TiN biosensor

3

4

2

Direct profiling surface proteins and portable for parallel measurements Rapid and direct method, wide linear range Easy readout without labeling

[31] [57]

10

H. Xu, B.-C. Ye / Trends in Analytical Chemistry 123 (2020) 115773

mL. Liu et al. employed a proximity ligation assay (PLA) to generate a unique surrogate DNA signal for exosomes analysis [79]. This DNA signal can be synchronously amplified twice by recombinase polymerase amplification coupled with transcription-mediated amplification. The final RNA products are proportional to the initial concentration of exosomes, which were quantified by a gold nanoparticle (AuNP)-based colorimetric assay. This strategy showed high sensitivity with a low detection limit of 102 particles/ mL and was applied to quantify Epstein-Barr virus latent membrane protein 1 (LPM1)-positive exosomes and epidermal growth factor receptor (EGFR)-positive exosomes from nasopharyngeal carcinoma patients plasma. Gao et al. designed a dual signal amplification method through converting the exosome capture event to nucleic acid detection [49]. Exosomes can be captured by CD63 aptamers which were linked to the magnetic beads and blocked by DNA probes. The binding exosomes could further switch the conformation of aptamer to release the DNA probes. Then the released DNA probes initiated a catalytic hairpin DNA cascade reaction (HDCR) and DNA dendrimer self-assembly process, in which FAM-labeled Y shaped DNAs were assembled on the AuNPs. The final fluorescence signal is correlated with the concentration of exosomes. After two signal amplification processes, this method achieved a detection limit of 1.16
103 particles/mL, and it provides the advantages of convenient operation, high sensitivity, and low experimental cost. Unlike most reports, Mori et al. designed exosome-binding cavities through molecular imprinting to create a fluorescencebased sensing platform (Fig. 5D) [80]. Firstly, they immobilized PC3 cell exosomes on a substrate by anti-CD9 antibodies and introduced methacryloyl disulfide groups onto the exosomes surface. After building the cavity around intact exosomes, they removed the template exosomes and transformed the disulfide linker into a free thiol. With the help of the thiol groups exist inside the cavities, post-imprinting in-cavity modifications were employed to site-specifically introduce fluorescent reporter molecules into the cavities. Without direct exosomes quantification, the resulting fluorescence generated by the reporter molecules modified thiol groups can indicate different concentrations of exosomes. 3. Conclusion and outlook Tumor-derived exosomes have been proved to provide great information in identification early-stage tumors and real-time monitoring of therapeutic response non-invasively. Although the extremely small size increases difficulty of exosomes detection, numerous researchers have established sensitive biosensors based on novel signal transduction and amplification strategies. A general overview of the LOD, detection time, recognition site, advantages and disadvantages of the various biosensing strategies for exosomes detection have been summarized in Table 1. With the aid of adding nanoprobes, such as aptamers and nano-materials, most of the advancements here have demonstrated effective exosome quantification or profiling of protein markers, and the LOD can be reduced to 102 particles/mL. After significant signal amplification via self-assembly of DNA nanodevices, direct visualization and quantification of exosomes at the single-vesicle level can be accomplished by TIRF imaging system. Moreover, microfluidic technologies have extended their capabilities to achieve integrated isolation and high throughput detection. Combining these two techniques may offer a promising strategy for selective separation and ultrasensitive analysis tumor-derived exosomes in complex samples. It is notable that a part of reports employed CD63 antibody or CD63 aptamer to capture or detect tumor-derived exosomes because of the abundant CD63 on the surfaces of exosomes and the

simple sequences of CD63 aptamer. Although CD63 also exist on the non-tumorigenic cells-derived exosomes, tumor-derived exosomes carry more plentiful CD63 because of the more active secretion of exosomes in tumor cells and higher expression level of CD63 on the surface of exosomes secreted by tumor cells than normal cells. With the in-depth research of exosomes, an increasing number of tumorassociated protein biomarkers have been identified in exosomes such as PTK7, HER2, PSA, CA125, EpCAM and MUC1. By using one or combination of these exosomal surface protein as the recognition sites, exosomes based biosensors can obtain better tumor specificity directed against particular kinds of cancer. In addition, most of the current platforms evaluated their diagnostic potential by comparison the samples of cancer patients with healthy controls only. The patients and controls can be well discriminated by the specific biomarkers, however, clinical translation still need rigorous validation with much larger cohorts. Despite great improvements have been made in exosomes detection and present a promising prospect of exosomes in diagnosis of cancer, some challenges still exist which will hinder their clinical application. Standardization procedure for isolation and detection of exosomes needs to be developed to reduce inconsistencies caused by different research groups and operation protocols. Many reported platforms focused on exosomes quantification and have obtained high sensitivity towards specific tumorderived exosomes. However, thorough research is also required to fully understand the biological mechanism of exosomes in mediating intercellular communication and therapeutic response. New tracer techniques and surface modification strategies are also imperative to explore the internalization pathways and final destination of exosomes. Meanwhile, the number, surface protein and molecular content of exosomes vary between different individuals and cell origins, it is necessary to build definite correlation between the expression levels of multiplex exosome markers and other physiological indicators, which will contribute to clinical diagnosis and therapeutic values. Acknowledgement This work was supported by the National Natural Science Foundation of China (Grant No. 21705047, and 31730004), the Fundamental Research Funds for the Central Universities (Grant No. 222201814030). References [1] [2] [3] [4] [5] [6] [7] [8] [9]

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