Aptamer‐based extracellular vesicle isolation, analysis and therapeutics

Extracellular vesicles (EVs) play an important role in many physiological processes. Thus, EV analysis has a great value for the understanding of mechanisms underlying disease progress or diagnosis, prognosis and therapy. The overlapped physical and immune properties between EVs and events in body fluids, as well as the phenotypic heterogeneity of EVs, require efficient isolation and analysis methods. The unique properties of aptamers, such as facile modification and programmability, make them easily assembled as powerful platforms for EV isolation and analysis. EVs can also be used as vehicles for drug delivery, benefiting from the properties of homing ability, hypo‐immunogenicity, and strong tolerance. The affinity recognition ability to targets and the feature of single stranded DNA of aptamers make them useful in promoting the targetability of EVs and delivery of nucleic acid drugs. This review summarizes recent progress in aptamer‐based EV isolation, analysis, and aptamer‐functionalized EVs for therapeutics.


METHODS FOR EVs
Current isolation and purification methods for EVs mainly rely on differential and buoyant density centrifugation or ultrafiltration, which are time-consuming, labor-intensive and have low EV recovery rate. Leveraging the properties of precise fluid control and convenient integration of microfluidics, a series of rapid and efficient EV isolation methods have been developed, such as DLD-based microfluidics, 100 acoustofluidics, 101 and the viscoelastic fluid microfluidic chip. 102 However, these label-free methods cannot differentiate exosomes from other vesicles nor isolate-specific EVs. Utilizing the abundant biomarkers on the membranes of EVs, affinitybased EV isolation/capture approaches have been proposed using antibodies or aptamers as recognition ligands with high specificity and efficiency. The ease of chemical modification of aptamers enables them to be modified on different interfaces for EV isolation, including Au electrodes, 11,79 MoS 2 -Fe 3 O 4 nanostructures (MOFE), 13 graphene oxide (GO) decorated Fe 3 O 4 @SiO 2 magnetic nanoparticles (NPs), 14 Fe 3 O 4 @TiO 2 NPs, 18 and magnetic beads. 78,103 Instead of modifying aptamers directly on magnetic beads for EV capture, Zhang's group marked EVs with biotin-labeled CD63 aptamers and separated EVs using Streptavidin-modified magnetic beads via Streptavidinbiotin interaction (Figure 2A). 17 Considering the heterogeneity of exosome surface biomarkers, Li's group modified two types of aptamers targeting CD63 or MUC1 on Fe 3 O 4 @SiO 2 NPs to improve the capture efficiency of exosomes. 16 Free aptamers are easily digested in clinical samples, causing loss of effective concentration of aptamers and low capture efficiency of targets. 106 To increase the effective concentration of aptamers, Wan's group employed rolling circle amplification (RCA) to prepare a biotin-labeled multivalent, long single-stranded DNA aptamer with a PTK7-targeting aptamer sequence as repeat units for EV enrichment ( Figure 2B). 90 EVs were isolated with 45% recovery rate and elimination of 84.7% albumin contamination in 40 min. Ye's group designed a satellite network tethered with long (tens to hundreds of microns) DNA molecules containing multiple CD63 aptamers, spacers, and linkers by RCA to isolate exosomes. 15 When CD63 aptamers were combined with CD63 on exosomes, the DNA linkers were released and paired with the probe on the surface of AuNPs, thus forming AuNP-exosome satellite structures, which enabled isolation of exosomes by low-speed centrifugation.
However, conventional interface recognition partially only overcomes the fundamental limits in the interfacial molecular interaction, leading to compromised performance. To improve the efficiency of interfacial affinity reaction, Yang's group proposed a microfluidic chip decorated by supported lipid bilayers with fluid aptamers on nanoporous herringbone (Pore-HB) to increase mass transfer, interface contact, and the binding affinity between aptamers and EV surface proteins simultaneously for enhanced EV detection ( Figure 2C). 104 Nevertheless, T A B L E 1 Summary of aptamers used for isolation (in blue), analysis (in orange) of EVs, and therapeutics (in green).  EVs 12,24,33,41,75,80,98,99 Abbreviation: EVs, extracellular vesicles. most isolation methods cannot handle the heterogeneity of EVs, which are critical for understanding the biological mechanism of EVs. Song's group developed a modular platform for distinguishing PD-L1 EVs from tumor and non-tumor origins ( Figure 2D). 105 Tumor cell-derived PD-L1 EVs co-bind with extended EpCAM and PD-L1 aptamers and induce the AND logic operation, while immune cell-derived PD-L1 EVs express only PD-L1 and induce the NOT logic operation. Two independent outputs were used for tandem microfluidic separations for tumor cell-and immune cell-derived PD-L1 EVs. Generally, the capture and isolation of EVs are critical preconditions for the detection, bio-functional study, and biomedical application of EVs. But the development of EV enrichment and isolation methods with high efficiency, high specificity, and high throughout is still needed. Moreover, the isolation of subtypes of EVs is critical for exploring their biological functions and transforming their molecular signatures to clinic applications.

EVs
The quantification and biological analysis of EVs will promote the exploration of their physiological and F I G U R E 2 Aptamer-based isolation methods for EVs. (A) Isolation of exosomes by immunomagnetic beads. Reproduced with permission. 17 Copyright 2019, American Chemical Society. (B) RCA-induced multivalent aptamers for EV isolation. Reproduced with permission. 90 Copyright 2021, The Royal Society of Chemistry. (C) Fluid nanoporous microinterface (FluidporeFace) enhanced affinity interaction for EV detection. Reproduced with permission. 104 Copyright 2022, Elsevier. (D) DNA computation-mediated microfluidic tandem separation for PD-L1 EV subpopulation isolation. Reproduced with permission. 105 Copyright 2022, American Chemical Society. EVs, extracellular vesicles; RCA, rolling circle amplification. ZHU ET AL. pathological regulation mechanisms and the transformation of EV-related biomarkers into clinical diagnostic indicators. The programmability of aptamers makes them easily adapted to the manufacture of various signal sensors, which converts the recognition signal features of aptamers into physical and chemical signals. Based on the signal transduction mode, a variety of EV analysis strategies, including fluorescence, colorimetry, electrochemistry, and surface-enhanced Raman scattering (SERS), have been proposed and used for EV quantification and analysis.

| Fluorescence detection
Fluorescence spectroscopy is widely used in the biological analysis because of its high sensitivity and good selectivity, affording multi-dimensional biological information. Due to their flexible programmability, aptamers can be designed as a series of responsive probes, and the binding information of aptamer-target protein can be read out by fluorescence quenching 43,107 or recovery 29,66,108,109 of the aptasensor. The combination of aptamers with biocompatible nanomaterials can also be adopted to improve the detection sensitivity of EVs. 29,107,110 For example, Wang et al. used GO through the destruction of the stable π-π stacking interaction by displacement of aptamers on GO by exosome binding to achieve efficient fluorescence quenching of fluorescently labeled aptamers and a detection limit of exosomes with 2.1 � 10 4 particles/μL. 29 Wu et al. used in situ growth of AuNP on EVs to form a gold nano-shell, leading to local quenching of the fluorescent probe targeting the same vesicle and resulting in decrease of the amount of sample needed to 1 μL. 107 The size of EVs is near to the detection limit of commercial fluorescence instruments. Therefore, with the continuing progress of molecular machines, DNA amplification techniques, such as hybridization chain reaction (HCR), 41,66,108,111 RCA, 39 and toeholdmediated strand displacement reaction (TSDR), 40,109 have been developed to amplify the fluorescence signal. 110,112,113 For example, Zhao et al. proposed a dualprobe strategy, which combined CD63 and cholesterol dual-recognition-induced proximity ligation assay (PLA) for accurate exosomal protein analysis and RCA for signal amplification ( Figure 3A). 39 Thermophoresis is a phenomenon that solvated molecules deplete from the heated spot of non-uniform temperature field, which can be quantified by the Soret coefficient that is related to the molecule charge, size, and solvation energy. As a homogeneous, label-free reaction system with advantages of efficient reaction rate, high detection sensitivity, and separation-free qualities, thermophoresis is widely used in the rapid analysis of specific proteins on EVs. 33,41,57,114 For example, Huang et al. developed an aptamer-based thermophoresis method for exosomal PD-L1 quantification ( Figure 3B). 57 Based on the different diffusion rates of free aptamer and aptamer-exosome complexes in the infrared laser beam, sensitive detection of exosomal PD-L1 was successfully achieved with a detection limit as low as 17.6 pg/mL. Since using one marker cannot distinguish tumor-derived EVs from a mixture of heterogeneous EVs, Deng et al. proposed a thermophoresis and a gated operation analysis method for tumor-derived EV detection by labeling EVs with aptamers targeting PSMA or EpCAM proteins, followed by a connector probe to induce aptamer connection and fluorescence resonance energy transfer (FRET) ( Figure 3C). 114 The method can be used to distinguish patients with prostate cancer (PCa) and benign prostatic hyperplasia with 91% accuracy within 15 min. Liu et al. employed a set of fluorescent aptamers to analyze the expression levels of seven types of surface proteins on serum EVs ( Figure 3D). 33 This method can distinguish patients with stage I cancer from healthy F I G U R E 4 Aptamer-based electrochemical detection of EVs. (A) NTH-assisted electrochemical aptasensor for exosome quantification. Reproduced with permission. 50 Copyright 2017, American Chemical Society. (B) Protein subpopulation analysis of exosomes. Reproduced with permission. 24 Copyright 2020, American Chemical Society. (C) Two-stage microfluidic platform (ExoPCD-chip). Reproduced with permission. 23 Copyright 2018, American Chemical Society. (D) Self-calibrating aptasensor. Reproduced with permission. 20 Copyright 2020, American Chemical Society. EVs, extracellular vesicles; NTH, nanotetrahedron. ZHU ET AL.
-7 of 21 donors with 95% sensitivity and 100% specificity and can be used for differentiation of different tumor types. In general, fluorescence detection for EVs is flexible and universal. With the development of new fluorescent labels, sensing strategies and detection equipment, high sensitivity, and low background can be achieved for EV quantification and molecular fingerprint analysis.

| Electrochemical detection
Aptamer-based electrochemical detection transduces the recognition events of EV-bonded aptamers into changes in electric potential or current to achieve rapid, sensitive, and selective detection of EVs. 115 Thiolated aptamers can be efficiently immobilized on gold electrode surfaces, forming a high-density self-assembled monolayer without further complicated modifications. By introducing electroactive redox labels or probes, the number of EVs can be determined by the electrochemical response caused by EV recognition by aptamers modified on the electrode surface. 79 To guarantee the vertical orientation of aptamers, mercaptohexanol was used to displace the nonspecifically adsorbed parts of the aptamers on Au electrodes. 11,116 In addition, Szunerits's group modified commercial Au electrodes with polyethyleneimine/ reduced GO films 117,118 and immobilized azide aptamers on the electrode surface via click chemistry for EV analysis. 117,118 However, aptamers coated on electrodes are disordered and intertwined, preventing the accessibility of aptamers to EVs. To overcome this barrier, Tan's group proposed a nanotetrahedron (NTH)-assisted aptasensor to enhance the accessibility of aptamers to suspended exosomes ( Figure 4A). 50 Compared to the singlestranded aptamer-functionalized aptasensor, the NTHassisted aptasensor detects exosomes with 100-fold higher sensitivity.
The recognition between EVs suspended in the solution and aptamers coated on the electrode interface can be compromised because of the limited mass transfer between the solid and liquid phases. To surmount this limitation, free immobilization strategies were developed, commonly with a sandwich-like assay "capture probe-EV-response probe." Zhang's group developed a magneto-mediated electrochemical sensor with the "MB/ CD63 aptamer-exosome-SiO 2 NPs" sandwich strategy for simultaneous analysis of protein subpopulations of breast cancer exosomes, using an electrochemical indicator molecule N-(2-((2-aminoethyl)disulfanyl)ethyl) ferrocene carboxamide (FcNHSSNH 2 ) ( Figure 4B). 24 Huang et al. 23 proposed a two-stage microfluidic platform (ExoPCDchip) to form an "MB/Tim4-exosome-aptasensor" sandwich ( Figure 4C). The designed aptasensor contains a CD63 aptamer and a mimicking DNAzyme sequence. On binding to CD63-positive exosomes, a G-quadruplex is generated as an electrochemical signal reporter with the aid of hemin. To avoid inaccurate detection results caused by a single-signal output, Sun et al. developed a self-calibration aptasensor with dual redox-signal responses of MB (labeled on aptamer) and Fc (doped into ZIF-67) ( Figure 4D). 20 The limit of exosome detection was as low as 100 particles mL −1 . Due to the high sensitivity and user-friendliness, aptamer-based electrochemical sensors are emerging as effective analytical tools for clinical markers, but more research is needed to expand their applicability in complicated samples.

| Colorimetric detection
Colorimetric detection converts the signals of EVs to the change of color, which can be observed by the naked eye. Moreover, the detection results can be read out with easily accessible equipment, thus enabling practical point-of-care testing.
As one of the most developed colorimetric methods, the enzyme-linked affinity assay employs enzymes to catalyze chromogenic substrates, such as 2,2 0 -azinobis[3ethylbenzothiazoline-6-sulfonic acid]-diammonium salt and 3,3 0 ,5,5 0 -tetramethylbenzidine (TMB), for a color change. Aptamer-based enzyme-linked affinity assays integrate aptamer-specific recognition and enzymecatalyzed visual signal amplification for rapid, facile, and sensitive EV detection. Lee's group developed an aptamerbased enzyme-linked affinity assay by constructing a sandwich of "capture antibody-EV-biotin-SQ 2 aptamer" followed by SA-HRP, which catalyzes the oxidation of TMB to a blue color for quantitative detection of alkaline phosphatase placental-like 2 on EVs. 56 Although aptamer-based enzyme-linked affinity assays provide sensitive and rapid detection of EVs, the storage conditions of natural enzymes are stringent. Moreover, the assay requires multiple operations and is time-consuming.
To counteract the deficiencies of natural enzymes, NPs having a characteristic peroxidase-like catalytic activity (termed as nanozymes) have been used for EV analysis. Wang et al. adsorbed CD63 aptamers on graphitic carbon nitride nanosheets (g-C 3 N 4 NSs) to improve the intrinsic peroxidase-like mimic catalytic activity of the nanosheets ( Figure 5A). 30 The quantification of exosomes was determined by the reduced reaction rate of H 2 O 2 -mediated TMB oxidation caused by the capture of exosomes by CD63 aptamers adsorbed on g-C3N4 NSs, which reduced the number of aptamers adsorbed on g-C 3 N 4 NSs. In addition, aptamers can improve the peroxidase-like activity of single-walled carbon nanotubes ( Figure 5B), 32 Fe 3 O 4 NPs, 52 and Gold nanospheres 119 and were integrated with the above nanomaterials for EV quantification and EV-related marker analysis. Aptamers can also prevent AuNPs from salt-induced aggregation. The recognition between aptamers and EV surface proteins breaks the non-specific and weaker binding equilibrium between AuNPs with aptamers, thus resulting in the aggregation of AuNPs and a red-to-blue color change. Using this principle, Tan's group profiled five types of exosome surface proteins in a simple manner ( Figure 5C). 28 Compared with the monochromic intensity change, multicolor-based colorimetric methods are more distinguishable by naked eyes, thus affording more reliable signal read-out. Zhang et al. introduced alkaline phosphatase (ALP)-labeled DNA probes for HCR amplification after capturing exosomes by CD63 aptamer-modified beads. The reaction of Ag + and ascorbic acid by ALPcatalyzed dephosphorylation of L-Ascorbic acid 2phosphate (AAO) leads to the deposition of Ag shells on Au NRs. As a result, the localized surface plasmon resonance peak of Au NRs is shifted, producing a distinctive multicolor change ( Figure 5D). 31 The detection limit was 9 � 10 3 particles/μL by naked eyes. Overall, the user-friendly colorimetric methods are emerging as potential strategies for EV analysis especially in resource-limited areas. However, the sensitivity of colorimetric methods remains to be improved.  advantages of single molecule sensitivity, rapid response, and resistance to photobleaching, SERS has been widely applied in liquid biopsy.
Two types of aptamer-based SERS detection models have been developed for EVs. The first type involves the release of SERS reporters from a metal-SERS reporter complex via the combination of EVs and aptamers, allowing the quantification of aptamers by the reduction of the SERS signal. For example, Ye's group 80 generated SERS detection probes composed of gold-silver-silver core-shell-shell nanotrepangs (GSSNTs) with three kinds of SERS reporter molecules and linker DNAs, which were complementarily paired with CD63 aptamermodified MBs ( Figure 6A). GSSNTs were competed for release into the supernatant after exosomes were captured by the CD63 aptamer, thus decreasing the strength of SERS signals on GSSNTs. Another type of aptamer-based SERS detection model involves the detection of SERS signals on "MB/aptamer-EV-SERS probe" sandwiches. Wang et al. formed a sandwich-type apta-immunocomplex with CD63 aptamer-modified MB@SiO 2 @Au as a capture substrate with the HER2/ PSMA/CEA aptamer and Raman reporter dual-labeled gold NPs as SERS probes for multiple detection of cancer exosomes ( Figure 6B). 12 The hot spots at the junctions between metal nanostructures result in significantly enhanced SERS signals. However, the limited number of hot spots (0.1%) compared to the total SERS active sites and the irregular distribution of hot spots caused by random aggregation of metal NPs led to poor and irreproducible analytical performance. 121 To circumvent this limitation, Zhang's group assembled Raman molecules and the recognition unit on triangular pyramid DNA to form strong and definite electromagnetic hot spots at junctions between AuNPs ( Figure 6C). 120 The LOD of exosomes was 1.1 � 10 2 particles/μL by an aptamer/MB-exosome-SERS probe sandwich for exosome capture and quantification. Overall, aptamer-based SERS detection for EVs is a rapid and sensitive method. Further research should be attempted to improve the accuracy of SERS in EV quantification.
Generally, high specificity and sensitivity are the basic requirements of EV detection. Benefiting from the modifiability and programmability of aptamers and the advances in engineering, the ideal EV detection performance can be easily achieved. Innovation of fluorescent labeling strategy and abundant signal output devices enable fluorescence to be one of the most widely used techniques for EV detection in multiple physiological samples. Electrochemistry and SERS are fast, sensitive, and user-friendly methods for EV detection. However, their performance in complicated clinical samples is compromised. Usually, the above methods need cumbersome equipment for signal readout, which are inappropriate for resource-limited areas. Colorimetry converts the recognition events of EVs to the change of color, which can be observed by the naked eye, making it suitable for point-of-care (POC) diagnostics.

| Signal amplification
To reduce the interference of "biological noise" and track the subtle changes of EVs in clinical practice, signal amplification is required to improve the detection sensitivity of EVs. Due to the flexible modifiability and assembly of aptamers, it is convenient to couple aptamers with signal amplification strategies for enhanced EV detection sensitivity. There are two types of signal amplification approaches, including direct amplification and competitive amplification.

| Direct amplification
In the direct amplification methods, aptamer probes have been designed with functional sequences. Moieties of functional sequences can be triggered for nucleic acidbased signal amplification, such as RCA and HCR, to improve the detection sensitivity of EVs.
RCA is an enzyme-catalyzed thermostatic singlestrand DNA amplification technology, which has been widely used in biosensing. 122 Aptamer-based RCA  amplification can be easily achieved by customizing the templates and repetitive sequences. For example, Huang et al. 65 designed a DNA probe with a gastric cancer exosome-specific aptamer and a primer complementary to a G-quadruplex circular template so that the probe can trigger RCA followed by multiple G-quadruplex units. The G-quadruplex mimiced a DNAzyme to catalyze the reduction of H 2 O 2 for electrochemical signal output with an exosome detection limit of 9.54 � 10 2 particles mL −1 ( Figure 7A). He's group 125 proposed a branched rolling circle amplification (BRCA) strategy by cyclizing a padlock probe after aptamer incubation and triggering BRCA from a second primer with SYBR Green I as the fluorescent dye. Similarly, Zhang's group introduced nicking endonuclease (Nb·BbvCI) to release FAM from gold NP (GNP)-DNA-FAM complexes followed by hybridization with the repeat RCA sequences from the primer padlock ( Figure 7B). 123 HCR is an enzyme-free amplification approach with rigid double DNA strands as products. 111,126 A simple HCR assay can be implemented by designing a DNA probe with an aptamer targeting an EV-specific protein and the trigger sequence for HCR reaction. 127 For example, He et al. combined a DNA probe containing a PTK7 aptamer, a poly-T linker, and a trigger DNA-activated HCR strategy with total internal reflection fluorescence microscopy for single EV visualization and quantification. 128 Shen et al. designed a conformation-switchable probe composed of CD63 aptamer and a trigger domain for initiation of HCR amplification ( Figure 7C). 124 The HCR products bond to multiple fluorophores to amplify EV signals, thus enlarging the overall size of EVs to over 500 nm, to enable detection of a signal EV by conventional flow cytometry. Zhang's group reported an enzyme-assisted signal amplification assay (EXOAPP) for exosome quantification and phenotype profiling. 36 Aptamers are adsorbed on GO with quenched fluorescence. On binding to exosomes, the fluorescence of the nano-confined aptamers is recovered. DNase I cleaves exosome-captured aptamers and allows the exposure of exosomes for the next round of aptamer-binding, thus amplifying the fluorescence signals ( Figure 7D). A similar strategy was used for quantification of colorectal cancer exosomes by adsorbing CD63 and EpCAM aptamers on GO. 29

| Competitive amplification
In a direct amplification assay, nucleic acid amplification products are generated on the EV surface, which is spatially confined for adequate amplification and may lead to entanglement of nucleic acid amplification products. In a competitive amplification assay, aptamers competitively bound on EVs can release trigger DNA sequences upon interaction between aptamers and proteins on EVs for subsequent amplification. Compared with direct amplification, competitive amplification converts EV recognition to the detection of ssDNA, which is conducive to more flexible amplification.
In the cyclic enzymatic digestion competitive amplification strategy, trigger DNA sequences initially coupled with aptamers are released for hybridization with reporter DNA sequences to form a double strand DNA complex. Nuclease is used to cleave nucleotides and trigger DNA sequences are released for cyclic digestion. For example, Xu's group proposed an endonuclease Nb. BbvCl-assisted aptamer-binding DNA walking machine for exosome ECL ( Figure 8A). 129 Li's group combined aptamer-induced release of trigger DNA probes and Exonuclease III-assisted recycling amplification for electrochemical detection of exosomes using doxorubicin (DOX) as a redox-active indicator. 19 Similarly, Shen's group combined aptamer recognition-induced release of messenger DNAs and cyclic enzymatic amplification with electroactive Ru (NH 3 ) 6 3+ as a signal reporter for exosome quantification ( Figure 8B). 78 The detection limit was as low as 70 particles/μL. A cascade toehold-triggered strand displacement reaction (TSDR) can be initiated by releasing the trigger DNA strands via aptamer-target interaction and involving them in multiple cycles of DNA hybridization in an enzyme-free manner. For example, Li et al. proposed a platform with superparamagnetic conjunction and molecular beacon, which transferred molecule signals on exosomes to signal strand DNA detection ( Figure 8C). 81 After release via exosome-PSMA aptamer interaction, the complementary trigger DNAs are hybridized with hairpin DNA probe HP1 followed by replacement by hairpin DNA probe HP2 with FRETbased fluorescence quenching to generate an HP1 −HP2 complex for fluorescence activation via TSDR. The free trigger DNAs can be used for the next signal amplification round. The LOD of exosomes was around 100 particles μL −1 in urine samples. To simplify the competitive amplification procedure, Wu et al. developed a one-step quantification of salivary exosomes based on quantum dot loaded self-assembled DNA concatamers tethered to aptamers and the direct release of DNA concatamers by CD63 aptamer recognition on exosomes ( Figure 8D)

| Aptamer-based EV protein and nucleic acid analysis
Exosomes carry abundant genetic information from their parental cells, such as proteins and nucleic acids. Accurate analysis of the amount and status of heterogenous biomacromolecules provide indications for the diagnosis of diseases and the evaluation of curative effects. For EV protein analysis, methods have been developed for multiple protein analysis 33,130,131 or for tracing the source of EV proteins. 61 Recently, post-translation modification of exosomal proteins, such as glycosylation, has been found to be critical in the implementation of biological functions of exosomes. In addition, EV nucleic acids, such as microRNA (miRNA), play an important role in disease progression. The development of EV nucleic acid detection methods has revealed the potential of EV nucleic acids in clinical use. This section will discuss methods for aptamer-based EV protein-specific glycosylation and nucleic acid analysis.

| EV protein-specific glycosylation analysis
Recently, glycoproteins on EVs have drawn widespread attention due to their roles in the regulation of various physiological and pathological processes, such as tumor progress and metastasis. 132,133 The current proteinspecific glycosylation characterization methods, such as lectin assays and mass spectrometry, are not suitable for in situ analysis. Benefiting from the tiny size and scalability of aptamers, several facile and noninvasive aptamer-based protein-specific glycosylation analysis methods have been developed, promoting the discovery of new biomarkers for cancer diagnosis. For example, Ju's group developed a quantitative localized analysis method using localized chemical remodeling for electrochemical quantification of exosomal MUC1-specific galactose/Nacetylgalactosamine (Gal/GalNAc) ( Figure 9A). 45 Metabolic glycan labeling (MGL) is a highly biocompatible and uncoded glycan labeling method for remodeling glycan with functional chemical modifications. Yang's group visualized exosomal PD-L1 (exoPD-L1) glycosylation via FRET between MGL and subsequent click chemistry-induced Cy5-and Cy3-PD-L1 aptamer tagging ( Figure 9B). 59 With the advantage of the nondestructive method, the biofunction of exosomal PD-L1 glycosylation in recognition between PD-1 and the inhibition of CD8 + T cells were verified for the first time. Further work attempted to promote the visualized sensitivity of exosomal protein-specific glycosylation by a proximity dual-tagging-induced HCR from non-functional epitope. 58 For accurate quantification of exosomal protein-specific glycosylation, Song's group developed a lectin-and PD-L1 aptamer-induced PLA for specific screening and quantification of glycosylated exosomal PD-L1 combined with quantitative real-time PCR for amplification ( Figure 9C). 60 The detection limit of glycosylated exosomal PD-L1 was 1.09 pg/mL. For the first time, glycosylated exosomal PD-L1 was verified to be a more accurate marker than total exosomal PD-L1 for cancer diagnosis.
Generally, the aptamer-based non-destructive EV protein-specific glycosylation analysis provides a magnifier for post-translational modification of EV proteins and serves as a powerful tool for precision medicine. Current approaches can only characterize glycosylation of onespecific protein and mainly focus on EV PD-L1 protein.
More research studies on the existence and biological functions of EV protein-specific glycoprotein will be conducted for the precise description of the roles of EVs in normal physiology.

| EV nucleic acid analysis
EVs encapsulate specific microRNAs (miRNAs) for regulating disease progression, such as cancer, 134 neurodegenerative disease, 135 and diabetes. 136 Thus, EV miRNAs have been confirmed to be potential biomarkers for noninvasive disease diagnosis. Several methods have been proposed for EV miRNA detection, such as nucleaseassisted signal amplification, 137 next-generation sequencing, 138 and nanomaterials-based methods. 139,140 The programmability of aptamers enables the design of aptamer-based capture and detection probes for EV nucleic acid analysis. For example, Huang's group 141 developed an aptamer-functionalized SERS sandwich assay with aptamers targeting miR-122 coupled MB or Au shell NP as capture elements or SERS tags for miR-122 detection ( Figure 10A). However, EVs need to be lysed in the above methods, which are a time-consuming and labor-intensive process. Yang's group developed a one-step in situ detection method for tumor-derived EV miRNAs by employing aptamer-mediated selective fusion without EV lysis ( Figure 10B). 142 The surfaces of liposomes hold aptamers for fuse of EVs expressing aptamer-targeting proteins by aptamer-target protein recognition. The encapsulated MBs are hybridized to the target miRNA so that the hairpin structure is opened with recovered fluorescence. Moreover, the increased size caused by fusion enables detection of exosomes by commercial flow cytometry. Using this strategy, the level of miR-21 in PD-L1 positive EVs was quantified and found to be an effective biomarker for cancer diagnosis.

IN DRUG DELIVERY AND TREATMENT OF DISEASES
With the advantages of small size, homing ability, hypoimmunogenicity, and strong tolerance of physiological barriers, EVs have been designed as vehicles for delivering diagnostic or therapeutic agents, such as small molecule drugs, 142 proteins, 143 and RNAs. 94,[144][145][146][147] To improve the selectivity of drug delivery, aptamers targeting specific proteins on receptor cells can be functionalized on EVs loading therapeutic agents, such as DOX, 73,96 miRNA, 71 and siRNA. 147 For example, Zou et al. assembled the diacyl lipid-sgc8 aptamer on exosomes loading DOX by hydrophobic interaction to form Apt-Exos particles ( Figure 11A). 92 The selective recognition of Apt-Exos by cells overexpressing PTK7 enhanced the therapeutic effect compared to free DOX. To enhance the cell-targeting ability of aptamer functionalized exosomes, Liu et al. generated a DNA scaffold with multivalent structureswitchable aptamer-sensing probes on exosomes. 149 The structural switching upon protein targeting produces a fluorescence signal for dynamic monitoring of exosomecell interactions. Guo's group constructed a row-shaped fluorescent PSMA aptemer -3WJ/cholesterol RNA ligand on EVs loaded with siRNA, altering the orientation of the arrow-shaped RNA, to regulate the intracellular trafficking of siRNA ( Figure 11B). 82 Using this strategy, PCa xenograft was inhibited. In addition, aptamers can be used as carriers to transport specific mRNA into EVs. Zhang et al. designed a DNA aptamer with a single-strand section to recognize the target mRNA and a double-strand section, which could be recognized by a zinc finger (ZF) motif ( Figure 11C). 148 The DNA aptamer promoted the capacity of mRNA of interest into CD9-ZF-engineered EVs for treatment of obesity and inflammatory bowel disease. In addition, Wang's group provided a light-inducible vehicle to load and deliver long endogenous RNA with MS2 RNA aptamer as the connecter for miR-21 sponge and AS1411 aptamer-modified on exosomes as the ligand for targeted delivery of miR-21 to leukemia cells ( Figure 11D). 72 In general, EVs might serve as superior drug vehicles for drug delivery than lipidic NPs and synthetic polymeric, which may cause toxicity and immune response. The targetability of aptamers improves the selectivity of EVs as drug carriers to lesions, thus reducing side effects. In addition, the feature of single-strand of aptamers makes them carriers to transport therapeutic mRNA into EVs and the target cells. Aptamer-functionalized EVs have great potential in the development of highly efficient and selective drug delivery systems for treatment of diseases. Insights into the in vivo fate of aptamerfunctionalized EVs should be attempted to enhance their clinical translation.

| CONCLUSION AND OUTLOOK
EVs offer significant clinical application values because of their convenient and non-invasive sampling and abundant molecular information for disease diagnosis and treatment. Aptamers hold numerous advantages, such as small size, good selectivity, facile modifiability, and multifunctional programming, which contribute to the development of EV-related liquid biopsy and disease treatment. In this review, we mainly discussed the use of aptamers in EV isolation and analysis and aptamerfunctionalized EVs for drug delivery and treatment of diseases.
Although emerging approaches and devices have been developed for EV quantification and signature characterization and preliminary correlations have been made between EVs and disease progression, there is still a gap between the basic research of EVs and its clinical application. Several bottlenecks remain to be overcome. First, the phenotypic heterogeneity of EVs provides ambiguous information of mixed EV subtypes. Subtypes of EVs share similar physical characteristics in terms of size, density, and membrane orientation. Therefore, current label-free EV isolation methods, such as ultracentrifugation, cannot efficiently separate specific EV subtypes. Combining the specific affinity of aptamers against targets on EVs with special signal output modes, numerous methods have been developed for EV isolation. However, vesiclefree biomarkers like free proteins coexist in complex biological systems, obstructing the recognition between aptamers and proteins on EVs, and giving rise to inaccurate EV analysis. Besides, current EV isolation methods based on EV capture by one ligand and detection by another ligand cannot differentiate the origin of EVs, which defines the biological function of EVs and is critical for revealing the physiological significance of EVs. Therefore, future efforts are needed for precise EV profiling and for tracing the origin of EVs. Second, the current methods mainly focus on EVs in a certain point of time or sample and ignore the spatiotemporal heterogeneity of EVs, which are important to understand the dynamic conversion of EVs and the regulatory molecules EVs released in cell-to-cell communication. New mouse models with the ability to trace the phenotype and homing of EVs will be possible for characterizing spatiotemporal heterogeneity of EVs. In summary, EVs highlight their application potential in disease diagnosis and prediction. The renovation of advances in EV research will speed up EV-based liquid biopsy for individual treatment.

ACKNOWLEDGMENTS
We thank the National Natural Science Foundation of China Grants (22022409) and the Program for Changjiang Scholars and Innovative Research Team in University (Grant IRT13036) for their financial support.

CONFLICT OF INTEREST STATEMENT
Prof. Chaoyong Yang is the member of Interdisciplinary Medicine editorial board. The author declares no other conflicts of interest.