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Back to Journal »International Journal of Nanomedicine» Volume 16

Advances in optical and electrochemical methods for the detection of exosomes based on nanomaterials

Authors: Ma Xin, Hao Yan, Liu Li

Published on November 13, 2021, the 2021 volume: 16 pages 7575-7608

DOI https://doi.org/10.2147/IJN.S333969

Single anonymous peer review

Editor approved for publication: Dr. Ebrahim Mostafavi

Ma Xiaohua, 1 Hao Yuanqiang, 1 Liu Lin1,2 1 Henan Provincial Key Laboratory of Biomolecular Recognition and Sensing, Shangqiu Normal University, Shangqiu, Henan, 476000; 2 School of Chemistry and Chemical Engineering, Anyang Normal University, Anyang, Henan, 455000 Corresponding Authors: Hao Yuanqiang; Lin Liu Email [email protected]; [Email protection] Abstract: Exosomes with a diameter of 30-150 nm are membrane-bound small vesicles secreted by various cells. They play an important role in many biological processes, such as tumor-related immune response and intercellular signal transduction. Exosomes have been considered as emerging and non-invasive biomarkers for cancer diagnosis. Recently, a large number of optical and electrochemical biosensors have been proposed for sensitive detection of exosomes. To meet the growing demand for ultra-sensitive detection, nanomaterials have been combined with various technologies as powerful components. Due to its inherent biocompatibility, excellent physical and chemical properties and unique catalytic ability, nanomaterials have significantly improved the analytical performance of exosomal biosensors. In this review, we summarize the latest advances in nanomaterial-based biosensors for detecting cancer exosomes, including fluorescence, colorimetry, surface plasmon resonance spectroscopy, surface-enhanced Raman scattering spectroscopy, electrochemistry, and electricity. Chemiluminescence, etc. Keywords: exosomes, nanomaterials, circulating tumor biomarkers, electrochemical biosensors, optical biosensors

Cancer is the main cause of death, and its occurrence and development is a gradual and complex process. Early diagnosis and treatment of cancer can greatly improve the survival chances of cancer patients. Extracellular vesicles (EV) are secreted by various cell types and circulate in different body fluids. They were first discovered as "cell junk" about 40 years ago. 1,2 Due to the lack of specific and reliable markers, the clear assignment of EV subtypes has become extremely difficult. According to the operating terms of EV subtypes proposed by the International Association of Extracellular Vesicles (ISEV), EVs can be divided into three groups according to their size: exosomes, small EVs (sEV) (< 200 nm), medium EVs (mEV), And large EV (lEV). 3 Exosomes are nano-sized extracellular vesicles (EV) (30-200 nm). For the convenience of the reader, this review uses a relatively broad term "exosomes" to refer to heterogeneous mixtures of sEVs smaller than 200 nm, as it is increasingly used in bioassays. 4 Compared with circulating tumor cells and circulating DNA, exosomes are present in body fluids (such as serum, urine, and ascites) with higher abundance and stability. 5 Emerging evidence shows that most types of cancer secrete a large number of exosomes, which carry abundant molecular information from maternal tumor cells, including nucleic acids, proteins, bioactive lipids, and metabolites. 6,7 Exosomes can act as cell messengers, transmitting information between cells through endocytosis. 8,9 They also play a key role in cancer metastasis and progression in the tumor microenvironment. 10,11 In addition, various unique cargo exosomes represent meaningful physiological and pathological states of diseases. 12,13 Therefore, exosomes have been recognized as the most reliable promoters of early diagnosis and treatment of cancer in non-invasive organisms. 14-17

Various complex biological fluids containing exosomes contain a large number of non-vesicular macromolecules, such as proteins, proteases, nucleases and RNA complexes, which may interfere with the analysis of exosomes. For example, proteases and nucleases can digest biological recognition elements such as antibodies and aptamers. Therefore, the growing interest in exosomes and their potential applications in cancer detection has prompted researchers to develop various isolation and detection techniques. Generally, traditional separation techniques, including ultracentrifugation, density gradient centrifugation, and ultrafiltration, are mainly based on the physical properties of exosomes (such as size and density). 5,18 However, these technologies face some problems, such as low purity, cumbersome procedures, and the need for expensive equipment. Traditional detection techniques used to quantify isolated exosomes include nanoparticle tracking analysis (NTA), flow cytometry, western blotting, dynamic light scattering, scanning electron microscopy (SEM), and transmission electron microscopy (TEM). 2,19–22 Although these methods have been widely used, their low reproducibility, time-consuming, large sample volume, and low sensitivity limit their further application.

In the past few years, many types of proteins have been discovered on membranes, such as four transmembrane proteins (such as CD9, CD63, CD81, and CD82), adhesion molecules (such as integrins and lactadherin), and lipid rafts (such as Cholesterol, phosphatidylserine and ceramide) 23,24 These biomolecules can be used as targets for the detection of total exosomes. However, exosomes from various tumor cells exhibit different cancer-associated antigens on the surface. These different antigens have been used as biomarkers to identify certain cancer-derived exosomes. 25 For example, exosomes secreted by MCF-7 have highly expressed MUC1 on the surface. 26 Protein tyrosine kinase 7 and CD147 are overexpressed on the surface. They are the surface of human leukemia lymphoblasts (CCRF-CEM) and colorectal cancer exosomes. 27,28 These proteins are promising markers for the separation and detection of exosomes with the aid of biorecognition elements. 29,30 By modifying magnetic beads (MBs), chips or microfluidics with antibodies against exosomal membrane markers, a technology based on immunoaffinity capture is proposed for separation and enrichment with high selectivity and specificity And complete biologically active exosomes. 23,31-44 However, the high cost and instability of antibodies limit their practical applications. With low cost, small size and relatively excellent stability, aptamers and peptides are screened as biorecognition elements for the isolation and detection of exosomes. 45-47 In addition, exosomal glycans provide valuable ways and glycans to label exosomes through lectin interactions. 48,49 Lipophilic cholesterol anchors can penetrate into the lipid bilayer for membrane modification, which is considered a promising candidate for labeling exosomes. 50,51 Compared with the separation technology based on physical properties, the separation technology based on affinity shows higher enrichment efficiency, which is conducive to the sensitive detection of exosomes.

In recent years, many new biosensors with high sensitivity and efficiency have been established for the determination of exosomes through specific identification, including colorimetry, fluorescence, surface plasmon resonance (SPR), and surface enhanced Raman scattering ( SERS) spectroscopy, mass spectrometry and electrochemistry, electrochemiluminescence (ECL) and photoelectrochemical (PEC) determination. 4,52-59 In addition, many works based on microfluidic devices implemented using optical or electrochemical techniques have been reported for the comprehensive determination of exosomes. 60-64

With the continuous development of nanotechnology, countless nanomaterials and nanostructures have had a huge impact on biosensing. Significant progress has made it possible to controllably prepare nanomaterials with various chemical compositions, morphologies, and physical and chemical properties. For example, nanomaterials with excellent luminescence characteristics have become an important substitute for traditional dyes in optical measurement due to their excellent advantages such as adjustable emission wavelength, high luminescence quantum yield and good light stability. Due to the interesting size and shape-dependent local surface plasmon resonance phenomenon, noble metal nanoparticles (NP), especially gold and silver, have been widely used to enhance the signal intensity in SPR and SERS measurements. Carbon-based nanomaterials with high surface-to-volume ratio and high electrical conductivity (for example, carbon nanotubes and graphene oxide) are often used for electrode modification to accelerate electron transfer and increase electrode surface area. In addition, recently, the photothermal and enzyme mimic properties of nanomaterials have aroused great interest in the development of portable bioassays. Various nanomaterial-based signal amplification strategies combined with different detection technologies have been developed for ultra-sensitive detection of biomolecules, including DNA/RNA, proteins, exosomes and cells. In the detection of exosomes, there are two main goals, including improving the capture rate of exosomes and improving the performance of the detection method. For the former, magnetic beads (MB) as a classic material are increasingly used to selectively capture exosomes from clinical samples when modified with antibodies or aptamers.

In this review, a comprehensive summary of the latest advances in the use of nanomaterial-based biosensors for the detection of exosomes. Detection techniques mainly include optical and electrochemical analysis (Tables 1 and 2). Nanomaterials involve semiconductor quantum dots (QD), metal nanoparticles, metal oxides and sulfides, and carbon-based nanomaterials. The purpose of this review is to gain an in-depth understanding of the multifunctional role of nanomaterials in assays and to illustrate their potential advantages in further applications. Table 1 Overview of the optical methods for the detection of exosomes based on nanomaterials Table 2 Overview of the electrochemical methods for the detection of exosomes based on nanomaterials

Table 1 Overview of optical methods for exosomes detection based on nanomaterials

Table 2 Overview of electrochemical methods for the detection of exosomes based on nanomaterials

Fluorescence biosensors have the advantages of simple operation, comparable sensitivity, and strong ability to detect multiple targets. Exosomes can be labeled with dyes or dye-modified biorecognition elements through various targeting strategies, and then determined by fluorescence imaging or spectroscopy. 50,65-72 However, the low fluorescence intensity of the dye always limits the sensitivity of exosome detection. 73 Therefore, some have proposed signal amplification strategies to improve detection sensitivity. 74,75 For example, β-galactosidase-labeled antibodies are used to label captured exosomes, where β-galactosidase acts as a reporter enzyme to catalyze the breakdown of luciferin disaccharides. β-D-galactopyranoside produces a strong fluorescent signal. 41,76 Combined with a microfluidic platform based on a nano-interface, exosomes are effectively enriched and sensitively detected.

For homogeneous fluorescence detection, converting exosome detection to DNA detection or other detection is one of the most effective strategies, which usually produces a "one-to-many" amplification effect. 77-81 Due to the flexible structure, the previously reported DNA-based signal amplification methods, such as terminal deoxynucleotidyl transferase (TdT) mediated polymerization and hybridization chain reaction (HCR), can be detected ultra-sensitively and uniformly DNA. 82-89 For example, Gao et al. reported a double-signal amplification method for indirect detection of exosomes based on the catalytic hairpin DNA cascade reaction (HDCR) and self-assembly of DNA dendrimers on the surface of gold nanoparticles (Au NPs) . 90 In this work, streptavidin (SA) modified MB was labeled with a biotin-modified CD63 aptamer, and then combined with probe S by hybridization. After adding exosomes, the aptamer binds to CD63 on the exosomes, and the probe S is released to trigger HDCR on the surface of the nanoparticles. Then, the opened metastable hairpin (HP) DNA probe captures the fluorescently labeled DNA dendrimer. After several rounds of Y-type DNA assembly, the complexes of AuNPs, HP, and DNA dendrimers are separated. With the help of β-mercaptoethanol, the fluorescently labeled dendrimer is released and the fluorescent signal is recorded for the determination of exosomes. This method shows a higher signal-to-noise ratio, with a linear detection range of 1.75×103-7.0×106 particles/μL. Pan et al. reported a steric hindrance-controlled signal amplification fluorescence strategy for exosome detection. 91 In the absence of exosomes, cholesterol-bound DNA 1 can hybridize with SA-modified DNA 2 to form blunt-ended dsDNA. Due to the lack of a single-stranded initiator (more than three deoxynucleotide residues), the formed dsDNA cannot be recognized and extended by the TdT enzyme. However, when exosomes are added, DNA 1 is inserted into the lipid membrane through the hydrophobic interaction between cholesterol and the lipid bilayer. The huge steric hindrance of exosomes strongly inhibited the hybridization of DNA 1 and SA-modified DNA 2. Therefore, SA modified DNA 2 can be extended by TdT enzyme to generate abundant G-quadruplex structure and increase fluorescence intensity. The "signal on" method maintains high sensitivity and excellent selectivity when analyzing complex samples. Li et al. developed a reversible nanoplatform for fluorescence detection of urinary exosomes by using superparamagnetic binding and molecular beacons. 92 As shown in Figure 1, the prostate-specific membrane antigen (PSMA) aptamer is modified on the surface of superparamagnetic NPs and then has reduced hybridization energy with two ssDNA strands. Exosomes that bind to the aptamer with high affinity result in the release of a double amount of ssDNA to initiate the amplification cycle of the two hairpin DNA strands. Turn on the molecular beacon HP2 to restore the fluorescence of the probe. The two released low-concentration ssDNA sequences start the amplification cycle with two hairpin DNA strands. Many molecular beacons HP2 are turned on and the fluorescence of the probe is restored. The detection limit of this method for urine samples is 100 particles/μL. Figure 1 Schematic diagram of SMC-MB platform. (A) Construction of SMC-MB platform. (B) The procedure of the SMC-MB platform in the analysis of exosomes. (C) Principle of non-enzymatic amplification cycle. (D) Purification of exosomes by restriction enzymes. Reprinted with permission from Li Ping, Yu X, Han Wei, etc. Ultra-sensitive and reversible nano-platform of urinary exosomes for prostate cancer diagnosis. ACS Sensor 2019; 4:1433-1441. Copyright 2019 American Chemical Society. 92

Figure 1 Schematic diagram of SMC-MB platform. (A) Construction of SMC-MB platform. (B) The procedure of the SMC-MB platform in the analysis of exosomes. (C) Principle of non-enzymatic amplification cycle. (D) Purification of exosomes by restriction enzymes. Reprinted with permission from Li Ping, Yu X, Han Wei, etc. Ultra-sensitive and reversible nano-platform of urinary exosomes for prostate cancer diagnosis. ACS Sensor 2019; 4:1433-1441. Copyright 2019 American Chemical Society. 92

Compared with traditional fluorescent dyes, QD has adjustable size/component luminescence and excellent photobleaching resistance, and has been widely used in bioimaging, luminescent biomarkers and light-emitting devices. 93,94 Wu et al. proposed a "one-step" strategy to detect exosomes using aptamers as biorecognition elements and QD as signal amplification reporter genes (Figure 2A). The 95 CD63 aptamer is anchored on the surface of the magnetic microsphere (MM) and connected to the self-assembled DNA concatemer. Then, streptavidin (SA)-coupled quantum dots are used to label the biotin-modified DNA concatemers. Exosomes preferentially bind to aptamers and induce the release of QDs-labeled DNA concatemers. After magnetic separation, the fluorescence signal in the supernatant was monitored for exosomes detection. Zhang et al. prepared a diagnostic biochip based on bionic periodic nanostructures for exosomal detection using QD. 96 As shown in Figure 2B, QD modified with glypican-1 (GPC1) antibody is used to label exosomes. When the solution is dropped on a biochip coated with photonic crystals, the fluorescence is significantly amplified. In addition, QDs-embedded silica NPs are also used for extracellular vesicles in lateral flow assays (LFA) through membrane biotinylation strategies. 97 Figure 2 (A) Schematic diagram of the construction process of magnetic and fluorescent biological probe (MFBP) and the principle of MFBP sensing-based on exosomal quantification. Reprinted with permission from Wu Min, Chen Zhong, Xie Q, etc. One-step quantification of salivary exosomes based on junction aptamer recognition and quantum dot signal amplification. Biosens Bioelectronics. 2021; 171: 112733–112742. Copyright 2021 Elsevier BV95 (B) Photonic crystal assisted signal amplification schematic for measuring tumor-derived exosomes. Reprinted with permission from Zhang Jie, Zhu Yan, Shi Jie and others. Amplify the sensitive signals of diagnostic biochips based on bionic periodic nanostructures for the detection of cancer exosomes. ACS application program interface. 2020; 12: 33473–33482. Copyright 2020 American Chemical Society. 96 (C) Schematic diagram of exosomes detection method based on copper-mediated signal amplification strategy. Reprinted with permission from He Fei, Wang Jie, Yin BC, Ye BC. Quantification of exosomes based on a copper-mediated signal amplification strategy. Anal Chemistry 2018; 90: 8072-8079. Copyright 2018 American Chemical Society. 98 (D) Schematic diagram of ASPNC's design and sensing mechanism. (a) The synthetic route of ASP. Reagents and conditions: i) Tris(dibenzylideneacetone)dipalladium(0)[Pd2(dba)3], tris(p-tolyl)phosphine (TPTP), chlorobenzene, 100℃, 24 hours; ii) Trimethyl Amine, tetrahydrofuran (THF), methanol, 24 hours. (b) Description of the formation of ASPNC and the detection of exosomal afterglow. Reprinted with permission from Lyu Y, Cui D, Huang J, Fan W, Miao Y, Pu K. Near-infrared afterglow semiconductor nano-multiplexes are used for the multiple differentiation of cancer exosomes. Angew Chem Int Ed. 2019;58:4983–4987. Copyright 2019 Wiley-VCH.69

Figure 2 (A) Schematic diagram of the construction process and sensing principle of the MFBP-based quantitative magnetic and fluorescent biological probe (MFBP) for exosomes. Reprinted with permission from Wu Min, Chen Zhong, Xie Q, etc. One-step quantification of salivary exosomes based on junction aptamer recognition and quantum dot signal amplification. Biosens Bioelectronics. 2021; 171: 112733–112742. Copyright 2021 Elsevier BV95 (B) Photonic crystal assisted signal amplification schematic for measuring tumor-derived exosomes. Reprinted with permission from Zhang Jie, Zhu Yan, Shi Jie and others. Amplify the sensitive signals of diagnostic biochips based on bionic periodic nanostructures for the detection of cancer exosomes. ACS application program interface. 2020; 12: 33473–33482. Copyright 2020 American Chemical Society. 96 (C) Schematic diagram of exosomes detection method based on copper-mediated signal amplification strategy. Reprinted with permission from He Fei, Wang Jie, Yin BC, Ye BC. Quantification of exosomes based on a copper-mediated signal amplification strategy. Anal Chemistry 2018; 90: 8072-8079. Copyright 2018 American Chemical Society. 98 (D) Schematic diagram of ASPNC's design and sensing mechanism. (a) The synthetic route of ASP. Reagents and conditions: i) Tris(dibenzylideneacetone)dipalladium(0)[Pd2(dba)3], tris(p-tolyl)phosphine (TPTP), chlorobenzene, 100℃, 24 hours; ii) Trimethyl Amine, tetrahydrofuran (THF), methanol, 24 hours. (b) Description of the formation of ASPNC and the detection of exosomal afterglow. Reprinted with permission from Lyu Y, Cui D, Huang J, Fan W, Miao Y, Pu K. Near-infrared afterglow semiconductor nano-multiplexes are used for the multiple differentiation of cancer exosomes. Angew Chem Int Ed. 2019;58:4983–4987. Copyright 2019 Wiley-VCH.69

Ultra-small metal nanoclusters (such as gold, silver and copper) have excellent fluorescence properties. Ye's team proposed a copper-mediated signal amplification method to quantify exosomes (Figure 2C). 98 In this work, cholesterol-modified MB was used to capture exosomes through the hydrophobic interaction between cholesterol groups and lipid membranes. The CuO NPs modified with CD63 aptamer are used to label exosomes and release Cu2 ions under acid hydrolysis after magnetic separation. Using poly(thymine) as a template, a large amount of released Cu2 ions can be reduced to fluorescent Cu nanoclusters. In addition, Lyu et al. used the afterglow semiconductor polyelectrolyte nanocomposite (ASPNC) to construct a luminescent nanosensor for exosome detection. 69 As shown in Figure 2D, the main chain of polyphenylene vinylene (PPV) modified with cationic quaternary ammonium groups is combined with tetraphenylporphyrin (TPP) for emission red shift and afterglow signal enhancement. The positively charged ASPNC can adsorb the aptamer labeled with the quencher black hole quencher 2 (BHQ-2) through electrostatic interaction, and the afterglow and fluorescence signals are quenched by the effective electron transfer between the PPV framework and BHQ-2. In the presence of exosomes, the specificity and strong affinity between the exosomal protein and the BHQ-2 labeled aptamer leads to the desorption of the aptamer from ASPNC. As a result, afterglow and fluorescence signals are restored.

Nanomaterials with excellent fluorescence quenching ability are very attractive for the development of "switch" biosensors. Zhang reported a dual-signal amplification platform based on rolling circle amplification (RCA) and nick endonuclease-assisted target recovery for the analysis of leukemia-derived exosomes. 88 In addition, Yu et al. proposed a 3D DNA motor-based detection platform 99 As shown in Figure 3A, AuNP is modified by a fluorescein-labeled substrate chain and a motor chain locked by an aptamer. In the presence of exosomes, the aptamer binds to the target protein, and the kinematic chain is unlocked to trigger the DNA movement process. Driven by restriction endonucleases, the kinematic chain walks along the track autonomously, leading to the release of many fluorescent molecules. Gold nanorods (AuNR) with adjustable aspect ratio-related plasma extinction bands can also be used to quench luminescence. For example, Chen's team reported a simple paper-supported biosensor with AuNRs that quenched the luminescence of upconversion nanoparticles (UCNPs) through luminescence resonance energy transfer (LRET). 100 As shown in Figure 3B, the aptamer sequence of the CD63 protein is divided into two flexible ssDNA fragments with different sequences (CP and DP). The branched polyethyleneimine (PEI) modified UCNPs and CP are fixed on the surface of the filter paper by forming a Schiff base. When exosomes were added, CD63 on the surface of exosomes promoted the combination of DP and CP into a complete tertiary aptamer, resulting in the closure of AuNRs and UCNPs. The distance between AuNRs and UCNPs was shortened to allow LRET to occur. However, for CD63, there is no interaction between the two fragments, and LRET does not occur. Figure 3 (A) A schematic diagram of an enzyme-driven DNA motor triggered by exosomes for exosome detection. Reprinted with permission from Yu Y, Zhang WS, Guo Y, Peng H, Zhu M, Miao D, Su G. The engineering of DNA motors driven by enzymes triggered by exosomes is used for high-sensitivity fluorescence detection of tumor-derived exosomes. Biosens Bioelectronics. 2020; 167: 112482–112490. Copyright 2020 Elsevier BV99 (B) Schematic diagram of a paper-supported aptamer sensor based on LRET between UCNP and AuNR for the determination of exosomes. Reprinted with permission from Chen X, Lan Jie, Liu Yan, etc. A paper-supported aptamer sensor based on up-conversion luminescence resonance energy transfer is used for the accessible determination of exosomes. Biosens Bioelectronics. 2018; 102: 582-588. Copyright 2018 Elsevier BV100

Figure 3 (A) A schematic diagram of an enzyme-driven DNA motor triggered by exosomes for exosome detection. Reprinted with permission from Yu Y, Zhang WS, Guo Y, Peng H, Zhu M, Miao D, Su G. The engineering of DNA motors driven by enzymes triggered by exosomes is used for high-sensitivity fluorescence detection of tumor-derived exosomes. Biosens Bioelectronics. 2020; 167: 112482–112490. Copyright 2020 Elsevier BV99 (B) Schematic diagram of a paper-supported aptamer sensor based on LRET between UCNP and AuNR for the determination of exosomes. Reprinted with permission from Chen X, Lan Jie, Liu Yan, etc. A paper-supported aptamer sensor based on up-conversion luminescence resonance energy transfer is used for the accessible determination of exosomes. Biosens Bioelectronics. 2018; 102: 582-588. Copyright 2018 Elsevier BV100

Graphene oxide (GO) can interact with DNA or RNA through π-π stacking interactions, thereby quenching the fluorescence of dye-labeled DNA/RNA probes through FRET. The DNA-based "signal on/off" fluorescent biosensor developed for exosome detection is a fascinating nanomaterial. 101 Wang et al. designed a DNase I enzyme-assisted signal amplification strategy for the fluorescence analysis of the difference between GO and aptamer based on the interaction of colorectal cancer (CRC) exosomes. 102 As shown in Figure 4A, the fluorescence of two aptamers (CD63 and Epithelial Cell Adhesion Molecule or EpCAM) labeled with different fluorophores was quenched by GO. In the presence of exosomes, the two aptamers bind to the CD63 and EpCAM target proteins on the surface of CRC exosomes, and then are released from the surface of GO. DNase I promotes the digestion of aptamers and induces the release of exosomes to release more aptamers, thereby achieving signal amplification. Few exosomes cause the release of large amounts of dye and the restoration of fluorescence. Li et al. developed a homogeneous magnetic fluorescence nanosensor for exosomal analysis, using GO as a quencher to reduce background signal. 103 As shown in Figure 4B, after the exosomes were isolated by MB coated with GPC-1 antibody, the expanded CD63 aptamer was used to label the exosomes and the extended ends were used as a foothold for initiating strand replacement, resulting in the formation of a large number of DNA three-way connection (TWJ). After magnetic separation, the DNA TWJ in the supernatant can adsorb a large number of positively charged tetrastyrene (TPE) aggregation-inducing luminophores (AIEgens) derivatives through electrostatic interaction. As a result, an enhanced fluorescent signal was observed. At the same time, GO was added to quench the fluorescence of ssDNA stained with AIEgens. The new method achieves a wide linear detection range, and the calculated detection limit is 6.56×104 particles/μL. In addition, MoS2-multi-walled carbon nanotube nanocomposites are used to quench the fluorescence of the dye-labeled CD63 antibody, which can be restored after the immune reaction between the exosomes and the antibody. 104 Figure 4 (A) Schematic diagram of GO-based enzyme-assisted fluorescence amplification-DNA aptamer interaction for exosome detection. Reprinted with permission from Wang H, Chen H, Huang Z, Li T, Deng A, Kong J. DNase I enzyme-assisted fluorescence signal amplification based on graphene oxide-DNA aptamer interaction is used for colorectal cancer exosomes detection. Taranta. 2018; 184: 219-226. Copyright 2018 Elsevier BV102 (B) Schematic diagram of homogeneous magnetic fluorescence nanosensor for tumor-derived exosomes separation and analysis. (a) Tumor-derived exosomes are specifically captured by GPC-1 antibody-coated magnetic beads, and then combined with the extended CD63 aptamer to form a bead-exosomal-aptamer complex. (b) Through the DNA TWJ cycle assembly strategy triggered by the aptamer and the TPE-TA and GO-based "on" fluorescence system, the captured exosomes are detected in the homogeneous solution. Reprinted with permission from Li B, Pan Wei, Liu C and others. Homogeneous magnetic fluorescence nanosensor for separation and analysis of tumor-derived exosomes. ACS Sensor 2020; 5: 2052-2060. Copyright 2020 American Chemical Society. 103 (C) Schematic diagram of mixing Cy3-CD63 aptamer with MXenes aqueous solution and adding exosomes. Reprinted with permission from Zhang Q, Wang F, Zhang H, Zhang Y, Liu M, Liu Y. Self-standard ratio fluorescence resonance energy transfer platform based on universal Ti3C2 MXenes, used for high-sensitivity detection of exosomes. Anal Chemistry 2018; 90: 12737–12744. Copyright 2018 American Chemical Society. 105

Figure 4 (A) Schematic diagram of enzyme-assisted fluorescence amplification based on GO-DNA aptamer interaction for exosomes detection. Reprinted with permission from Wang H, Chen H, Huang Z, Li T, Deng A, Kong J. DNase I enzyme-assisted fluorescence signal amplification based on graphene oxide-DNA aptamer interaction is used for colorectal cancer exosomes detection. Taranta. 2018; 184: 219-226. Copyright 2018 Elsevier BV102 (B) Schematic diagram of homogeneous magnetic fluorescence nanosensor for tumor-derived exosomes separation and analysis. (a) Tumor-derived exosomes are specifically captured by GPC-1 antibody-coated magnetic beads, and then combined with the extended CD63 aptamer to form a bead-exosomal-aptamer complex. (b) Through the DNA TWJ cycle assembly strategy triggered by the aptamer and the TPE-TA and GO-based "on" fluorescence system, the captured exosomes are detected in the homogeneous solution. Reprinted with permission from Li B, Pan Wei, Liu C and others. Homogeneous magnetic fluorescence nanosensor for separation and analysis of tumor-derived exosomes. ACS Sensor 2020; 5: 2052-2060. Copyright 2020 American Chemical Society. 103 (C) Schematic diagram of mixing Cy3-CD63 aptamer with MXenes aqueous solution and adding exosomes. Reprinted with permission from Zhang Q, Wang F, Zhang H, Zhang Y, Liu M, Liu Y. Self-standard ratio fluorescence resonance energy transfer platform based on universal Ti3C2 MXenes, used for high-sensitivity detection of exosomes. Anal Chemistry 2018; 90: 12737–12744. Copyright 2018 American Chemical Society. 105

As a sub-category of two-dimensional transition metal carbide and carbonitride materials, ultra-thin MXenes have attracted much attention in biomedical applications due to their excellent properties similar to those of GO. Based on its excellent quenching efficiency, MXenes has been widely used to construct fluorescent biosensors for detecting targets, including DNA, RNA and proteins. Recently, Liu and colleagues reported a self-standard ratio FRET platform based on Ti3C2 MXenes for the detection of exosomes (Figure 4C). 105 In this work, Cy3-labeled CD63 (Cy3-CD63) aptamer is adsorbed on MXenes through hydrogen bond and metal chelate interaction. The fluorescence of Cy3-CD63 aptamer is quenched by FRET, and the intrinsic fluorescence of MXenes is almost unchanged as a standard reference. Exosomes can specifically bind to aptamers and induce their release from the surface of MXenes, resulting in the recovery of fluorescence signals.

Colorimetric biosensors have received widespread attention due to their low cost and convenient reading. The results can be quickly observed with the naked eye. Therefore, colorimetric measurement is very important for real-time detection in environments with limited facilities. Generally, enzymes are needed to catalyze the color reaction in colorimetric assays. 106,107 In the traditional ELISA used to detect exosomes, horseradish peroxidase (HRP) linked to the detection antibody is always used to catalyze the reaction between H2O2 and the colorimetric substrate 3,3', 5,5' -Tetramethylbenzidine (TMB). 108,109 Then, the color of the solution changed from colorless to blue. However, they face the problem of low reproducibility and sensitivity (minimum 3 μg purified sample). 110 Hemin/G-quadruplex with HRP mimic catalytic activity is also widely used in signal amplification bioassays. 111,112 In order to improve sensitivity, several signal amplification strategies have been proposed, such as the use of immunomagnetic nanoparticles (MNPs) to enrich exosomes and the use of NPs to increase the number of enzymes used for signal output. 113-115 For example, He et al. reported the quantification of exosomes directly based on HCR and HRP-mediated signal amplification. 116 As shown in Figure 5A, after the exosomes are captured by the MB coated with CD9 antibody, a DNA probe labeled with divalent cholesterol is added to identify the lipid membrane of the exosomes. Hydrophobic cholesterol part. The DNA probe triggers HCR, and then many SA-HRP conjugates are captured by the DNA polymer to catalyze the color reaction. The recommended assay shows a detection limit of 2.2×103 particles/μL. In addition, DNA nanoflowers are also used to encapsulate HRP, thereby increasing the number of enzymes loaded in signal output. 117 Yang et al. proposed a pH-responsive paper-based bioassay to detect exosomes. 118 As shown in Figure 5B, after SA-coated MNP captures exosomes, HRP is coupled with CD63 antibody to catalyze the formation of a polydopamine membrane on the surface of exosomes, thereby allowing the binding of urease. 119 The captured urease can hydrolyze urea into ammonia and carbon dioxide, causing the pH of the solution to change from 5 to 10 and the color of commercially available pH test papers. However, the use of natural enzymes faces serious shortcomings such as low stability, high cost, and complex preparation processes. Figure 5 (A) A schematic diagram of colorimetric detection of exosomes by a combination of immunoaffinity separation and cholesterol-based signal amplification. Reprinted with permission from He Fei, Liu Hai, Guo X, Yin BC, Ye BC. Quantification of exosomes is performed directly through a bivalent cholesterol-labeled DNA anchor for signal amplification. Anal Chemistry 2017; 89: 12968-12975. Copyright 2017 American Chemical Society. 116 (B) Schematic diagram of magnetic capture of exosomes, HRP-mediated exosomal PDA engineering and immobilization of urease for immediate detection. Reprinted with permission from Yang Y, Li C, Shi H, Chen T, Wang Z, Li G. A pH-responsive bioassay uses mussel-inspired surface chemistry for paper-based diagnosis of exosomes. Taranta. 2019; 192: 325-330. Copyright 2019 Elsevier BV118

Figure 5 (A) A schematic diagram of colorimetric detection of exosomes by a combination of immunoaffinity separation and cholesterol-based signal amplification. Reprinted with permission from He Fei, Liu Hai, Guo X, Yin BC, Ye BC. Quantification of exosomes is performed directly through a bivalent cholesterol-labeled DNA anchor for signal amplification. Anal Chemistry 2017; 89: 12968-12975. Copyright 2017 American Chemical Society. 116 (B) Schematic diagram of magnetic capture of exosomes, HRP-mediated exosomal PDA engineering and immobilization of urease for immediate detection. Reprinted with permission from Yang Y, Li C, Shi H, Chen T, Wang Z, Li G. A pH-responsive bioassay uses mussel-inspired surface chemistry for paper-based diagnosis of exosomes. Taranta. 2019; 192: 325-330. Copyright 2019 Elsevier BV118

Au and Ag NPs with local surface plasmon resonance (LSPR) characteristics exhibit higher extinction coefficients than organic chromogens. Such NPs have been widely used as alternative substrates to develop plasma colorimetry for biological assays. The detection principle of the colorimetric strategy based on NPs can be divided into two sub-categories: aggregation/deaggregation and etching/growth. Generally, Maiolo et al. proposed a simple plasma colorimetric strategy for the determination of exosomes. 120 As shown in Figure 6A, AuNP can accumulate on the lipid membrane of exosomes, causing the shift and broadening of the LSPR absorption spectrum and the color change of the solution from red to blue. However, in the presence of exosomes and protein contaminants, the formation of protein corona around AuNPs prevented aggregation, and LSPR absorption remained unchanged. Tan and colleagues developed a colorimetric aptamer sensor for the analysis of exosomal proteins (Figure 6B). 121 In this work, the combination of aptamer and AuNP can prevent NP from accumulating in high-salt solutions. However, the specific interaction between the aptamer and exosomes caused the aptamer to leave the AuNPs surface and caused the AuNPs to aggregate, changing the color from red to blue. Liu et al. reported a fast and convenient colorimetric method that combines target-induced proximity ligation analysis (PLA) with recombinase polymerase amplification (RPA) and transcription-mediated amplification (TMA) to detect ultra-low concentrations Exosomes. 122 as shown in the figure. As shown in Figure 6C, after the two PLA probes bind to the LMP1 protein on the surface of the exosomes, the two DNA probes hybridize to each other. Under RPA and TMA amplification, multiple copies of RNA transcripts are produced, which can induce the aggregation of DNA-modified AuNPs and the color change of the solution. In addition, the principle of color measurement based on AuNPs has been introduced into LFA for rapid and sensitive analysis of exosomes. 123-126 In addition, since AuNR is more sensitive to changes in the local medium environment, the color change based on AuNR is more realistic. The colorimetric determination based on AuNR shows better performance. Zhang et al. reported a multicolor visual measurement of exosomes by enzymatically catalyzed metalization amplified by AuNR and HCR. 127 As shown in Figure 7A, after capturing exosomes and labeling them with cholesterol-modified DNA probes, the terminal DNA probes initiate HCR assembly. A large number of alkaline phosphatase (ALP) molecules are loaded on the surface of exosomes to catalyze ascorbic acid ( The production of AA) and the in-situ formation of the silver shell on the AuNRs, while the color of the solution changes sharply. In addition, Au [email protected]2 nanosheets are also used as a substrate for colorimetric detection of exosomes. 128 In the competitive reaction induced by exosomes, a small amount of exosomes release a large number of ALP molecules into the solution, and free ALP catalyzes the formation of AA to etch Au [email protection]2 nanosheets, accompanied by multicolor changes. Figure 6 (A) Schematic diagram of nanoplasma analysis for detection of ocular protein contaminants (single and aggregated exogenous proteins, SAP) in EV preparations. Reprinted with permission from Maiolo D, Paolini L, Di Noto G, etc. Colorimetric nanoplasma assay to determine purity and titrate extracellular vesicles. Anal Chemistry 2015; 87: 4168-4176. Copyright 2015 American Chemical Society. 120 (B) Schematic diagram of the aptamer/AuNP complex used for molecular analysis of exosomal proteins. (A) Schematic diagram of replacement of aptamers from gold nanoparticles by binding to exosomal surface proteins and concomitant aggregation of gold nanoparticles. (b) Use the aptamer/AuNP complex to analyze different exosomal surface proteins. Reprinted by Jiang Yu, Shi Ming, Liu Yu, etc. with permission. Aptamer/gold nanoparticle biosensor for colorimetric analysis of exosomal proteins. Angu Chemical 2017;129:12078-12082. Copyright 2017 Wiley-VCH.121 (C) PLA-RPA-TMA detection diagram. Reprinted with permission from Liu Wen, Li Jie, Wu Yan, etc. The adjacent connection induced by the target triggers recombinase polymerase amplification and transcription-mediated amplification to detect tumor-derived exosomes in nasopharyngeal carcinoma with high sensitivity. Biosens Bioelectronics. 2018; 102: 204-210. Copyright 2018 Elsevier BV122 Figure 7 (A) Schematic diagram of the multicolor visual detection mechanism of exosomes based on HCR and Au NRs enzyme-catalyzed metallization. Reprinted with permission from Zhang Y, Wang D, Yue S, etc. The dual-signal amplification strategy of enzymatically catalyzed gold nanorod metallization and hybrid chain reaction is used for sensitive multi-color visual detection of exosomes. ACS Sensor 2019; 4:3210-3218. Copyright 2019 American Chemical Society. 127 (B) Schematic diagram of the visible detection of exosomes based on ssDNA-enhanced Fe3O4 NPs nanozyme activity. Reprinted with permission from Chen J, Xu Y, Lu Y, Xing W. Separate and visually detect tumor-derived exosomes from plasma. Anal Chemistry 2018; 90: 14207–14215. Copyright 2018 American Chemical Society. 131 (C) Schematic diagram of DNA aptamer accelerating the intrinsic peroxidase-like activity of g-C3N4 NSs to detect exosomes. Reprinted with permission from Wang YM, Liu JW, Adkins GB, etc. ssDNAs enhances the intrinsic peroxidase-like activity of graphite carbon nitride nanosheets and its application in the detection of exosomes. Anal Chemistry 2017; 89: 12327-12333. Copyright 2017 American Chemical Society. 132 (D) Schematic diagram of the detection mechanism for the visible detection of exosomes based on the Fe3O4 NPs nanoenzyme activity enhanced by ssDNA. Reprinted with permission from Zhang Y, Su Q, Song D, Fan J, Xu Z. Label-free detection of exosomes based on the mimic activity of CuCo2O4 nanorod oxidase regulated by ssDNA. Journal of Anal Chim. 2021; 1145: 9-16. Copyright 2021 Elsevier BV134

Figure 6 (A) Schematic diagram of nanoplasma analysis for detection of ocular protein contaminants (single and aggregated exogenous proteins, SAP) in EV preparations. Reprinted with permission from Maiolo D, Paolini L, Di Noto G, etc. Colorimetric nanoplasma assay to determine purity and titrate extracellular vesicles. Anal Chemistry 2015; 87: 4168-4176. Copyright 2015 American Chemical Society. 120 (B) Schematic diagram of the aptamer/AuNP complex used for molecular analysis of exosomal proteins. (A) Schematic diagram of replacement of aptamers from gold nanoparticles by binding to exosomal surface proteins and concomitant aggregation of gold nanoparticles. (b) Use the aptamer/AuNP complex to analyze different exosomal surface proteins. Reprinted by Jiang Yu, Shi Ming, Liu Yu, etc. with permission. Aptamer/gold nanoparticle biosensor for colorimetric analysis of exosomal proteins. Angu Chemical 2017;129:12078-12082. Copyright 2017 Wiley-VCH.121 (C) PLA-RPA-TMA detection diagram. Reprinted with permission from Liu Wen, Li Jie, Wu Yan, etc. The adjacent connection induced by the target triggers recombinase polymerase amplification and transcription-mediated amplification to detect tumor-derived exosomes in nasopharyngeal carcinoma with high sensitivity. Biosens Bioelectronics. 2018; 102: 204-210. Copyright 2018 Elsevier BV122

Figure 7 (A) Schematic diagram of multicolor visual detection mechanism of exosomes based on HCR and enzyme-catalyzed Au NRs metallization. Reprinted with permission from Zhang Y, Wang D, Yue S, etc. The dual-signal amplification strategy of enzymatically catalyzed gold nanorod metallization and hybrid chain reaction is used for sensitive multi-color visual detection of exosomes. ACS Sensor 2019; 4:3210-3218. Copyright 2019 American Chemical Society. 127 (B) Schematic diagram of the visible detection of exosomes based on ssDNA-enhanced Fe3O4 NPs nanozyme activity. Reprinted with permission from Chen J, Xu Y, Lu Y, Xing W. Separate and visually detect tumor-derived exosomes from plasma. Anal Chemistry 2018; 90: 14207–14215. Copyright 2018 American Chemical Society. 131 (C) Schematic diagram of DNA aptamer accelerating the intrinsic peroxidase-like activity of g-C3N4 NSs to detect exosomes. Reprinted with permission from Wang YM, Liu JW, Adkins GB, etc. ssDNAs enhances the intrinsic peroxidase-like activity of graphite carbon nitride nanosheets and its application in the detection of exosomes. Anal Chemistry 2017; 89: 12327-12333. Copyright 2017 American Chemical Society. 132 (D) Schematic diagram of the detection mechanism for the visible detection of exosomes based on the Fe3O4 NPs nanoenzyme activity enhanced by ssDNA. Reprinted with permission from Zhang Y, Su Q, Song D, Fan J, Xu Z. Label-free detection of exosomes based on the mimic activity of CuCo2O4 nanorod oxidase regulated by ssDNA. Journal of Anal Chim. 2021; 1145: 9-16. Copyright 2021 Elsevier BV134

Since Fe3O4NPs are reported to exhibit peroxidase-like activity, more and more nanomaterials called nanozymes have been shown to have catalytic ability and have been integrated into colorimetric bioassays. 129 Compared with natural enzymes, nanoenzymes show improved stability, low cost and easy storage. Surface charge and composition are two key roles in regulating the catalytic activity of nanozymes. ssDNA can increase the peroxidase mimic activity of nanozymes. 130 Chen et al. reported the colorimetric analysis of exosomes for enhancing the peroxidase activity of Fe3O4NPs through ssDNA aptamers (Figure 7B). 131 They found that aptamers attached to the surface of Fe3O4NPs can increase the affinity between NPs and TMB, resulting in an increase in peroxidase activity. In this work, an anion exchange method was first designed to extract exosomes from plasma. Then, the captured exosomes bind to aptamers from NPs, resulting in a decrease in catalytic activity. In addition, Wang et al. developed a ssDNA-enhanced nanozyme-based colorimetric method for exosome detection (Figure 7C). 132 They demonstrated that ssDNA can electrostatically accelerate the intrinsic peroxidase of graphite carbon nitride nanosheets (g-C3N4 NSs) to mimic the aromatic stacking interaction between active ssDNA and TMB. However, CD63 on the surface of exosomes can competitively bind to ssDNA aptamers and reduce the enhancement of peroxidase mimic activity. This method is highly sensitive and can measure exosomes in the range of 1.9×106 to 3.38×107 particles/μL. Xia et al. used ssDNA-modified single-walled carbon nanotubes to detect exosomes through the same principle. 133 In contrast, Zhang et al. reported on the non-labeled colorimetric assay of nanorods (NRs) of exosomes based on the CuCo2O4 oxidase-like activity inhibited by ssDNA. 134 As shown in Figure 7D, CuCo2O4 NRs can catalyze the oxidation of ABTS, with O2 acting as an electron acceptor instead of volatile H2O2. The negatively charged CD63 aptamer is adsorbed on CuCo2O4 NRs through electrostatic interaction, and the oxidase-like activity of NRs is inhibited by hindering the electron transfer between NRs and the substrate. However, in the presence of exosomes, the aptamer is released from CuCo2O4 NRs and the oxidase-like activity is restored.

Surface Plasmon Resonance (SPR) is a label-free real-time sensing technology used to study and quantify biomolecular interactions. 135 It can monitor changes in refractive index caused by binding events close to the gold surface (within 200 nm)-resulting in an increase in thickness. In addition, it has the advantages of high signal-to-noise ratio, good compatibility with microfluidic technology and advanced surface modification. Therefore, SPR can detect exosomes with a size of approximately 100 nm and a large mass, which is suitable for the depth of the surface plasmon wave. So far, a series of label-free SPR biosensors for the detection of exosomes have been developed by modifying the sensor surface with membrane protein-specific antibodies on the exosomes. 136-145 In order to overcome the slow diffusion-limited mass transfer, magnetic nanoparticles can be used to pre-concentrate exosomes on the sensor surface under an external magnetic field gradient. 146 However, the poor sensitivity of these methods limits their further application for the analysis of trace targets in complex samples.

AuNPs can enhance SPR signal through plasma coupling. 147-150 Wang et al. proposed an SPR aptamer sensor to quantify cancerous exosomes through dual AuNPs auxiliary signal amplification. 151 As shown in Figure 8, the gold chip is functionalized with aptamers to capture exosomes. Aptamer/T30 modified AuNPs are further used to label exosomes on the Au membrane. Due to the electronic coupling between the gold film and AuNPs, the sensitivity of the biosensor is very high. In order to improve specificity, they further developed a dual aptamer-based SPR strategy for the detection of human liver cancer (SMMC-7721) exosomes. 152 In addition, combined with DNA-based signal amplification, Ding's team reported a hydrogel-AuNP supramolecular sphere-based (H-Au) SPR biosensor for the analysis of prostate cancer-derived exosomes. The 153 H-Au network is prepared by self-assembly of DNA strands and DNA-modified AuNPs. PSMA-specific aptamer functionalized MB is used to transduce target exosomes into triple DNA, which can initiate terminal transferase to catalyze the generation of polyA tails, which can then be combined with the H-Au network on the surface of the SPR. The biosensor has a wide linear range from 1.00×102 to 1.00×104 particles/μL. Figure 8 Schematic diagram of double AuNP auxiliary signal amplification for SPR determination of exosomes. Reprinted with permission from Wang Q, Zou Li, Yang X, etc. Directly quantify cancerous exosomes through surface plasmon resonance and double gold nanoparticle-assisted signal amplification. Biosens Bioelectronics. 2019;135:129-136. Copyright 2019 Elsevier BV151

Figure 8 Schematic diagram of double AuNP auxiliary signal amplification for SPR determination of exosomes. Reprinted with permission from Wang Q, Zou Li, Yang X, etc. Directly quantify cancerous exosomes through surface plasmon resonance and double gold nanoparticle-assisted signal amplification. Biosens Bioelectronics. 2019;135:129-136. Copyright 2019 Elsevier BV151

Raman spectroscopy can provide characteristic fingerprint spectra. However, the signal strength is always too weak to distinguish. The discovery of the SERS effect has aroused great interest in SERS research. Generally, metal nanostructures or nanomaterials can be used to amplify signals through chemical and electromagnetic field enhancement. SERS spectroscopy has been used to design biosensors through label-free analysis and SERS tag-based methods. 154

Label-free SERS analysis is mainly based on the use of coarse or nano-sized SERS substrates to enhance the weak Raman vibration signals of fingerprint-like exosomal biomolecules. For example, Avella-Oliver et al. reported the unmarked SERS analysis of exosomes on recordable optical discs, which is to coat recordable optical discs with silver. 155 This cost-effective technology provides an alternative solution for SERS bioassays in non-specialized areas. environment. Inspired by the concept of beehives, Dong et al. proposed that Au-coated TiO2 macroporous inverse opal (MIO) structure can be used as a SERS substrate for label-free detection of exosomes. 156 As shown in Figure 9A, different from the traditional SERS MIO structure, it can capture exosomes through its interconnected nano-scale pore network, showing a prominent "slow light effect" and enhancing exosomes through the SERS effect of the Au layer. Raman signal. The SERS intensity of 1087 cm-1 of the PO bond in the phosphoprotein on the surface of the exosomes was used as the detection standard. Due to the heterogeneity, the Raman spectroscopy of exosomes shows that the data is complex and inconsistent, making it difficult to classify. For this point of view, principal component analysis was used to monitor Raman signals, and a meaningful pattern of exosomal analysis was obtained. 157-159 Figure 9 (A) Schematic diagram of the detection process and design inspiration of the Au-coated TiO2 MIO SERS probe. Reprinted with permission from Dong Si, Wang Yao, Liu Z, etc. The honeycomb type macroporous SERS probe captures and analyzes exosomes in plasma for cancer detection. ACS application program interface. 2020; 12: 5136-5146. Copyright 2020 American Chemical Society. 156 (B) Using SERS nanotags and CD63 antibody to functionalize MB to prepare three types of SERS nanotags and a schematic diagram of the molecular phenotype analysis of exosomes. Reprinted with permission from Zhang Wen, Jiang L, Diefenbach RJ and others. Use surface-enhanced Raman spectroscopy nanotags for sensitive phenotyping of cancer-derived small extracellular vesicles. ACS Sensor 2020; 5: 764-771. Copyright 2020 American Chemical Society. 164 (C) Schematic diagram of exosome engineering based on hydrophobic insertion strategy and DSPE-PEG-Mal. Reprinted with permission from Di H, Zeng E, Zhang P, etc. The general method of engineering extracellular vesicles for biomedical analysis. Anal Chemistry 2019; 91: 12752–12759. Copyright 2019 American Chemical Society. 165 (D) Schematic diagram of the assembly of AuNP in triangular pyramid DNA. Reprinted with permission from Zhang X, Liu C, Pei Y, Song W, Zhang S. Preparation of a new type of Raman probe and its application in the detection of circulating tumor cells and exosomes. ACS application program interface. 2019; 11:28671–28680. Copyright 2019 American Chemical Society. 169

Figure 9 (A) Schematic diagram and design inspiration of Au-coated TiO2 MIO SERS probe detection process. Reprinted with permission from Dong Si, Wang Yao, Liu Z, etc. The honeycomb type macroporous SERS probe captures and analyzes exosomes in plasma for cancer detection. ACS application program interface. 2020; 12: 5136-5146. Copyright 2020 American Chemical Society. 156 (B) Using SERS nanotags and CD63 antibody to functionalize MB to prepare three types of SERS nanotags and a schematic diagram of the molecular phenotype analysis of exosomes. Reprinted with permission from Zhang Wen, Jiang L, Diefenbach RJ and others. Use surface-enhanced Raman spectroscopy nanotags for sensitive phenotyping of cancer-derived small extracellular vesicles. ACS Sensor 2020; 5: 764-771. Copyright 2020 American Chemical Society. 164 (C) Schematic diagram of exosome engineering based on hydrophobic insertion strategy and DSPE-PEG-Mal. Reprinted with permission from Di H, Zeng E, Zhang P, etc. The general method of engineering extracellular vesicles for biomedical analysis. Anal Chemistry 2019; 91: 12752–12759. Copyright 2019 American Chemical Society. 165 (D) Schematic diagram of the assembly of AuNP in triangular pyramid DNA. Reprinted with permission from Zhang X, Liu C, Pei Y, Song W, Zhang S. Preparation of a new type of Raman probe and its application in the detection of circulating tumor cells and exosomes. ACS application program interface. 2019; 11:28671–28680. Copyright 2019 American Chemical Society. 169

Au and Ag nanomaterials with LSPR have been used as SERS active nanotags to enhance the signal intensity of Raman dyes. Using immunomagnetic beads and capturing exosomes, several aptamers or antibody-modified SERS nanotags have been developed to detect exosomes by forming antibody-exosomes-aptamer sandwich immune complexes. The 160-163 microfluidic Raman biochip has also been manufactured for isolation and in situ determination of exosomes. 43 Generally, Wang's group reports an effective method for detecting exosomes by analyzing multiple protein biomarkers on the surface at the same time. 164 As shown in Figure 9B, three specific nanotags for antibody modification were prepared, and the exosomes-suspension medium were labeled under filtration conditions. Then, antibody-modified CD63-conjugated MB was added for sandwich-like immunoassay. The heterogeneous antigens expressed on different exosomes limit the application of methods based on antigen-antibody/aptamer interactions. Liu's team proposed a general, simple, and robust strategy for labeling maleimide (Mal)-tagged exosomes through hydrophobic insertion. 165 As shown in Figure 9C, maleimide-terminated DSPE-PEG (DSPE-PEG-Mal) is inserted into the lipid membrane as a labeled probe. The Mal group can be combined with a thiol-containing substance (1,6-hexanedithiol) through click chemistry, and further combined with bare gold nanoparticles for SERS analysis. Wang's team developed a SERS biosensor for multiple detection of exosomes, in which a gold-coated MB was used as a SERS probe, which was modified with three different types of Raman reporter genes and aptamers. 166 In addition, Kwizera et al. proposed a method for detecting exosomes using cationic AuNR. The SERS label uses electrostatic attraction to label exosomes. 167 The "hot spot" generated at the AuNP-AuNP junction due to the plasmonic coupling effect can enhance the Raman signal of the SERS molecule. For this reason, Ning et al. reported multiple SERS analyses of exosomes using gold-silver bimetallic nano sea cucumbers, in which different Raman reporter molecules were confined in the interface between the gold core and the silver shell. 168 Zhang et al. designed a new Raman probe to detect exosomes by assembling AuNP in triangular pyramid DNA (TP-DNA). 169 As shown in Figure 9D, TP-DNA is prepared by the hybridization of four X-shaped DNA sequences, and then the positively charged AuNP and Raman reporter molecules are captured through electrostatic interaction. In addition, laser tweezers Raman spectroscopy has also been used to identify exosomes with single nanoparticles to enhance the signal. 170

Electrochemical biosensors are recognized as an excellent platform for biological sample analysis due to their high sensitivity, low cost, fast response speed and small sample volume. 171,172 Several classic electrochemical techniques are often used in biological assays, including current method, voltammetry, impedance method, and electric field method. Effect transistor. Nanomaterials mainly play two important roles in these technologies: as an electrode substrate for improving electron transfer and as a functional nanotag for signal amplification. 173

Direct electrochemical detection is achieved by monitoring the change of electrode conductivity through the change of target-induced electrical signal. 174-177 This method can quantify exosomes without a labeling step, thereby reducing response time. For example, Tan and his colleagues proposed an electrochemical aptamer sensor that uses DNA nanotetrahedrons to fix the aptamer on the electrode surface to increase the accessibility of exosomes, thereby directly measuring cancerous exosomes. 178 Davis’ group reported on an immunosensor for the analysis of exosomes by electrochemical impedance.179 Vaidyanathan developed a multiplexed device to detect exosomes through nano-shear induced by AC electrohydrodynamics. 180

AuNPs with good conductivity and easy functionalization have been widely used to modify sensor electrodes. Cucurbit[7] urea has excellent supramolecular recognition ability for ferrocene (Fc), and has been widely used as a receptor in electrochemical analysis. Liu et al. reported a label-free electrochemical aptamer sensor for exosome detection based on the host-guest interaction between cucurbit[7] uril and Fc.181. As shown in Figure 10A, cucurbit[7] uril is fixed. Used to capture Fc-labeled CD63 aptamers on AuNPs modified electrodes. The aptamer that binds to the target exosomes with high affinity can be released from the electrode surface, resulting in a decrease in electrochemical signal. Sun et al. developed a dual-signal and intrinsic self-calibrating aptamer sensor for direct detection of exosomes. 182 As shown in Figure 10B, the ITO slices were modified (BPNS) by electrodeposition and black phosphorous nanosheets with a metal organic framework (ZIF-67) doped with Fc. Then, the methylene blue labeled ssDNA aptamer was adsorbed on the surface of the electrode. This platform exhibits a dual redox signal response from methylene blue and Fc. In the presence of exosomes, the aptamer desorbs from the electrode surface, resulting in a decrease in methylene blue redox current. During this process, no significant changes in Fc current were observed. The inherent self-calibrating aptamer sensor shows a detection limit as low as 0.1 particles/μL. Figure 10 (A) A schematic diagram of an electrochemical aptamer sensor for capturing and releasing exosomes based on the specific host-guest interaction between cucurbit[7] uril and Fc. Reprinted with permission from Liu Q, Yue X, Li Y, etc. A new type of electrochemical aptamer sensor for the determination and release of exosomes based on the specific host-guest interaction between cucurbitaceae [7] uril and ferrocene. Taranta. 2021; 232: 122451–122458. Copyright 2021 Elsevier BV181 (B) The construction process and application diagram of the exosomal dual-signal and intrinsic self-calibrating aptamer sensor based on the functional hybrid thin-film sensing platform aptamer-BPNSs/Fc/ZIF-67/ITO. Sun Y, Jin H, Jiang X, Gui R. Assemble black phosphorous nanosheets and MOF to form a functional hybrid film for precise protein capture, dual signal and intrinsic self-calibration sensing of cancer-derived exosomes. Anal Chemistry 2020 ; 92: 2866-2875. Copyright 2020 American Chemical Society. 182

Figure 10 (A) A schematic diagram of an electrochemical aptamer sensor for capturing and releasing exosomes based on the specific host-guest interaction between cucurbit[7] uril and Fc. Reprinted with permission from Liu Q, Yue X, Li Y, etc. A new type of electrochemical aptamer sensor for the determination and release of exosomes based on the specific host-guest interaction between cucurbitaceae [7] uril and ferrocene. Taranta. 2021; 232: 122451–122458. Copyright 2021 Elsevier BV181 (B) The construction process and application diagram of the exosomal dual-signal and intrinsic self-calibrating aptamer sensor based on the functional hybrid thin-film sensing platform aptamer-BPNSs/Fc/ZIF-67/ITO. Sun Y, Jin H, Jiang X, Gui R. Assemble black phosphorous nanosheets and MOF to form a functional hybrid film for precise protein capture, dual signal and intrinsic self-calibration sensing of cancer-derived exosomes. Anal Chemistry 2020 ; 92: 2866-2875. Copyright 2020 American Chemical Society. 182

Field-effect transistor (FET) biosensor is a promising unmarked detection tool, which can monitor the micro-electric signal caused by the interaction between the target and the identification element on the sensing interface. 183 Yu et al. designed a FET biosensor based on reduced graphene oxide (rGO) for the electrical and label-free quantification of exosomes. 184 In this article, 1-pyrene butyrate succinimidyl ester was modified on the surface of rGO through the π-π stacking interaction between pyrene and graphene. Then, the CD63 antibody is covalently immobilized on the surface of the FET. With the introduction of negatively charged exosomes, the net carrier density changes, causing the Dirac point to shift to the left.

Although the label-free electrochemical method is simple, its sensitivity and selectivity are poor. Therefore, different types of sandwich-like methods have been developed for bioassays. Usually, the electrode is modified with a biological recognition element to capture exosomes, and another biological recognition element modified with a signal reporter gene is added to recognize the captured exosomes and generate electrical signals. Enzymes and electroactive molecules are commonly used as signal reporters for signal amplification. 185 For example, Doldan et al. reported an electrochemical immunosensor for the detection of exosomes using HRP-conjugated antibodies. 186 An et al. designed an electrochemical aptamer sensor to detect tumor exosomes through HCR assembly to connect many HRP molecules to carry out catalytic redox reactions. 187 He and colleagues reported an electrochemical aptamer sensor for analyzing exosomes with heme/G-quadruplex assisted rolling circle amplification. 188 Generally, nanomaterials can be used as nanocarriers, nanoelectrocatalysts and electroactive tags for signal amplification in sandwich-like electrochemical analysis.

Due to its excellent biocompatibility and large surface area, AuNPs have been widely used to carry various biomolecules (such as proteins, DNA and RNA) for different biological applications. Jiang et al. reported an electrochemical aptamer sensor for exosome detection using AuNP and enzymes for signal amplification. 189 As shown in Figure 11A, the aptamer-modified DNA nanotetrahedron (NTH) is used to modify the electrode to avoid the aptamer and steric hindrance effects. The aptamer and biotin were modified on AuNPs through the interaction between polyA10 and AuNPs. After the sandwich-like complex is formed, many avidin-HRP conjugates are immobilized on biotin-labeled AuNP to catalyze the reduction of TMB and H2O2. Figure 11 (A) A schematic diagram of an electrochemical aptamer sensor for exosome detection using AuNPs and enzymes for amplification. Reprinted with permission from Jiang Jie, Yu Y, Zhang Hai, Cai C. An electrochemical aptamer sensor based on DNA nanotetrahedron combined with enzymatic signal amplification for analysis of exosomal proteins. Journal of Anal Chim. 2020;1130:1-9. Copyright 2020 Elsevier BV189 (B) [Email protection] Schematic diagram of the manufacturing process of nanoprobes and electrochemical biosensors for detecting GBM-derived exosomes. Reprinted with permission from Sun Zhi, Wang Li, Wu Sheng, etc. The electrochemical biosensor designed using Zr-based metal organic framework is used to detect glioblastoma-derived exosomes, which has practical application value. Anal Chemistry 2020; 92: 3819-3826. Copyright 2020 American Chemical Society. 190 (C) Schematic diagram of PD-L1 exosome identification based on HRCA response [email protection]@ZIF-8. Reprinted with permission from Cao Y, Wang Y, Yu X, Jiang X, Li G, Zhao J. Identification of Breast Cancer Programmed Death Ligand 1 Positive Exosomes Based on DNA Amplification in Response to Metal-Organic Frameworks Biosens Bioelectronics. 2020;166:112452–112460. Copyright 2020 Elsevier BV192 (D) Schematic diagram of the manufacturing process of COF-based nanoprobes and the mechanism of EC biosensors for exosome detection. Reprinted with permission from Wang Min, Pan Yu, Wu Family, etc. Detection of colorectal cancer-derived exosomes based on a covalent organic framework. Biosens Bioelectronics. 2020; 169: 112638–112645. Copyright 2020 Elsevier BV193

Figure 11 (A) A schematic diagram of an electrochemical aptamer sensor for exosome detection using AuNPs and enzymes for amplification. Reprinted with permission from Jiang Jie, Yu Y, Zhang Hai, Cai C. An electrochemical aptamer sensor based on DNA nanotetrahedron combined with enzymatic signal amplification for analysis of exosomal proteins. Journal of Anal Chim. 2020;1130:1-9. Copyright 2020 Elsevier BV189 (B) [Email protection] Schematic diagram of the manufacturing process of nanoprobes and electrochemical biosensors for detecting GBM-derived exosomes. Reprinted with permission from Sun Zhi, Wang Li, Wu Sheng, etc. The electrochemical biosensor designed using Zr-based metal organic framework is used to detect glioblastoma-derived exosomes, which has practical application value. Anal Chemistry 2020; 92: 3819-3826. Copyright 2020 American Chemical Society. 190 (C) Schematic diagram of PD-L1 exosome identification based on HRCA response [email protection]@ZIF-8. Reprinted with permission from Cao Y, Wang Y, Yu X, Jiang X, Li G, Zhao J. Identification of Breast Cancer Programmed Death Ligand 1 Positive Exosomes Based on DNA Amplification in Response to Metal-Organic Frameworks Biosens Bioelectronics. 2020;166:112452–112460. Copyright 2020 Elsevier BV192 (D) Schematic diagram of the manufacturing process of COF-based nanoprobes and the mechanism of EC biosensors for exosome detection. Reprinted with permission from Wang Min, Pan Yu, Wu Family, etc. Detection of colorectal cancer-derived exosomes based on a covalent organic framework. Biosens Bioelectronics. 2020; 169: 112638–112645. Copyright 2020 Elsevier BV193

Due to its high specific surface area, flexible porosity and adjustable framework structure, metal organic framework (MOF) has attracted widespread attention in comprehensive applications including catalysis, sensors, and energy conversion and storage. Porosity gives MOF the ability to carry a large number of enzymes or electroactive molecules. Sun et al. reported a label-free, enzyme-free electrochemical biosensor for the detection of glioblastoma-derived exosomes using Zr-based MOF. 190 As shown in Figure 11B, Zr-MOF (UiO-66) prepared from metal ions and organic ligands is used to load a large number of electroactive methylene blue molecules by hydrothermal method. After the peptide modified electrode captures the exosomes, the Zr-MOF loaded with methylene blue is anchored by the exosomes. Zr-MOFs interact with the phosphate groups in the phospholipid bilayer of exosomes with high affinity by forming Zr-OP bonds. The concentration of exosomes can be determined by measuring the electrochemical signal of methylene blue inside the MOF. Recently, Gu et al. proposed a biofuel cell-based self-powered biosensor for exosome detection, in which two MOFs (ZIF-8 and UiO-66-NH2) are used to load glucose dehydrogenase and electricity. Active molecule (K3[Fe(CN)6]). 191 Using the characteristics of exogenous stimulus response, Cao et al. reported an electrochemical biosensor for detecting programmed death ligand-1 positive (PD-L1) exosomes based on DNA amplification in response to MOF. 192 As shown in Figure 11C, HRP-encapsulated ZIF-8 is prepared by a biomineralization promotion method, and then coated with PVP. The PVP remains intact at a weakly alkaline pH and will be destroyed at an acidic pH. After immunizing MB to capture PD-L1 exosomes, add anti-PD-L1 linked DNA strands to mark the exosomes, and the DNA part initiates hyperbranched rolling circle amplification (HRCA). The released H ions cause the pH of the environment to become weakly acidic, which leads to the decomposition of MOF. Release HRP molecules to increase electrochemical response.

As an emerging porous crystalline material, covalent organic frameworks (COFs) have shown great application potential in bioassays. Li's team reported an aptamer sensor based on COFs for the analysis of CRC exosomes. 193 As shown in Figure 11D, spherical COFs with high porosity are used to load p-thiocalix[4] arene hydrate (pSC4) modified AuNPs and a large number of HRP molecules to form HRP-pSC4[email protected] pSC4 can interact with external Various amino acid residues on the surface of the body bind specifically. AuNPs can accelerate the charge transfer of carriers. The CD63 aptamer is anchored on the surface of the Au electrode to capture exosomes. Then, add HRP-pSC4[email protected] to identify the captured exosomes. HRP catalyzes the oxidation of TMB by H2O2, generating a high electrochemical signal.

Nanozymes can catalyze the redox reaction between H2O2 and the substrate in colorimetric analysis, which can be converted into electrochemical analysis to improve sensitivity. For example, Boriachek et al. reported the direct separation and subsequent detection of exosomes using gold-loaded iron oxide nanocubes (Au-NPFe2O3NC). 194 As shown in Figure 12A, Au-NPFe2O3NC was modified with CD63 antibody to capture exosomes. After magnetic separation, the complex of Au-NPFe2O3NC and exosomes was attached to a screen-printed electrode functionalized with placental alkaline phosphatase (PLAP) antibody. The signal is measured by the reaction of TMB and H2O2 catalyzed by Au-NPFe2O3NC. Figure 12 (A) A schematic diagram of the assay for the direct isolation and detection of exosomes from cell culture media based on Au-NPFe2O3NC. Reprinted with permission from Boriachek K, Masud MK, Palma C, etc. Avoid the pre-separation step in the analysis of exosomes: use gold-loaded nanoporous iron oxide nanozymes to directly separate and sensitively detect exosomes. Anal Chemistry 2019; 91: 3827-3834. Copyright 2019 American Chemical Society. 194 (B) Schematic diagram of an electrochemical biosensor using in-situ generation of Prussian blue exosomal activity detection signal amplification strategy. Reprinted with permission from Zhang H, Wang Z, Wang F, Zhang Y, Wang H, Liu Y. Ti3C2 MXene-mediated Prussian blue in situ hybridization and electrochemical signal amplification are used to detect exosomes. Taranta. 2021; 224: 121879–121886. Copyright 2021 Elsevier BV195 (C) Two-step separation and analysis of exosomes and microsomes: (a) Capture step, where vesicles are immobilized on an aptamer-modified sensor, and (b) captured exosomes/particles Body electrochemical detection of Cu and AgNPs. Reprinted with permission from Zhou YG, Mohamadi RM, Poudineh M, etc. Use metal nanoparticles to interrogate circulating microsomes and exosomes. small. 2016; 12: 727-732. Copyright 2016 Wiley-VCH.197

Figure 12 (A) A schematic diagram of the assay for the direct isolation and detection of exosomes from cell culture media based on Au-NPFe2O3NC. Reprinted with permission from Boriachek K, Masud MK, Palma C, etc. Avoid the pre-separation step in the analysis of exosomes: use gold-loaded nanoporous iron oxide nanozymes to directly separate and sensitively detect exosomes. Anal Chemistry 2019; 91: 3827-3834. Copyright 2019 American Chemical Society. 194 (B) Schematic diagram of an electrochemical biosensor using in-situ generation of Prussian blue exosomal activity detection signal amplification strategy. Reprinted with permission from Zhang H, Wang Z, Wang F, Zhang Y, Wang H, Liu Y. Ti3C2 MXene-mediated Prussian blue in situ hybridization and electrochemical signal amplification are used to detect exosomes. Taranta. 2021; 224: 121879–121886. Copyright 2021 Elsevier BV195 (C) Two-step separation and analysis of exosomes and microsomes: (a) Capture step, where vesicles are immobilized on an aptamer-modified sensor, and (b) captured exosomes/particles Body electrochemical detection of Cu and AgNPs. Reprinted with permission from Zhou YG, Mohamadi RM, Poudineh M, etc. Use metal nanoparticles to interrogate circulating microsomes and exosomes. small. 2016; 12: 727-732. Copyright 2016 Wiley-VCH.197

Wang and colleagues developed a sensitive electrochemical biosensor to detect exosomes by generating electroactive Prussian blue (Fe[Fe(CN)6]) in situ on the surface of MXenes. 195 As shown in Figure 12B, the CD63 aptamer-modified poly(amidoamine)-AuNP electrode is used to capture exosomes. Then, the aptamer-conjugated MXene is used to recognize the captured exosomes. The MXene on the surface of the exosomes acts as a reducing carrier to induce in situ Prussian blue production, simplifying the synthesis process. Prussian blue can generate electrochemical signals at low potential without being interfered by electroactive substances. The detection limit of this method is 229 particles/μL. Quantum dots containing a large amount of metal ions can also be used as signal transduction markers for exosomal analysis. 196 After acid-assisted dissolution, a large amount of Cd2 ions are released, which can be quantified by anodic stripping voltammetry (ASV). In addition, metal nanoparticles such as Ag and Cu can be used as signal reporter molecules because they can be directly electrochemically oxidized to generate typical electrochemical peaks. Kelley's group reported the electrochemical detection of exosomes/microsomes using AgNP modified with anti-EpCAM aptamers and CuNP modified with anti-PSMA aptamers (Figure 12C). 197 After capturing exosomes and microsomes from VCaP cells by a simple centrifugation procedure, the aptamer is functionalized with NPs added to label the captured exosomes. Then, linear sweep voltammetry (LSV) is used to directly electrochemically oxidize AgNPs or CuNPs.

Fixing the recognition probe on the electrode may inhibit the effective recognition between exosomes and the probe, thereby reducing sensitivity. MBs have been widely used to capture exosomes and can be integrated into electrochemical biosensors. MBs can not only simplify the detection procedure, but also concentrate the captured exosomes on the electrode. MB modified with antibodies (immune MB) has been used to isolate and enrich exosomes. An electrochemical technique for the detection of exosomes without immobilization based on MBs has been developed. 198,199 Lee's team designed an integrated immunomagnetic electrochemical sensor for exosome detection (Figure 13A), 200 of which MBs were modified with CD63 antibody to directly capture exosomal plasma. Next, the captured exosomes are recognized by the HRP-labeled detection antibody. HRP can catalyze the reaction between TMB and H2O2, thereby generating a strong electrochemical signal. In order to meet the needs of portability and sensitivity, Ye's team reported a two-stage magnetic microfluidic platform for on-chip separation and detection of exosomes. 201 As shown in Figure 13B, a staggered Y-shaped microcolumn mixing pattern is applied to create an anisotropic flow that improves capture efficiency. The tumor-derived exosomes captured by the MB coated with Tim4 were fixed on the ITO electrode. The ssDNA in the hairpin structure is composed of aptamers, which mimic DNA enzyme sequences for labeling exosomes. After identification, the development clip is opened, and the G-quadruplex formed with hemin is used as NADH oxidase and HRP mimic DNA enzyme at the same time. In addition, CdSe QD is used as a signal marker instead of an unstable enzyme for the detection of exosomes by anodic stripping voltammetry. 196 Figure 13 (A) Schematic diagram of an integrated immunomagnetic electrochemical sensor for exosome detection. Reprinted with permission from Jeong S, Park J, Pathania D, etc. Integrated magneto-electrochemical sensor for exosomal analysis. ACS nano. 2016; 10: 1802-1809. Copyright 2016 American Chemical Society. 200 (B) Schematic diagram of Exo PCD chip and electrochemical sensor on the surface of ITO electrode. Reprinted with permission from Xu Hua, Liao C, Zuo Ping, Liu Z, Ye BC. Magnetic-based microfluidic device for on-chip separation and detection of tumor-derived exosomes. Anal Chemistry 2018; 90: 13451–13458. Copyright 2018 American Chemical Society. 201

Figure 13 (A) Schematic diagram of an integrated immuno-magnetic-electrochemical sensor for exosome detection. Reprinted with permission from Jeong S, Park J, Pathania D, etc. Integrated magneto-electrochemical sensor for exosomal analysis. ACS nano. 2016; 10: 1802-1809. Copyright 2016 American Chemical Society. 200 (B) Schematic diagram of Exo PCD chip and electrochemical sensor on the surface of ITO electrode. Reprinted with permission from Xu Hua, Liao C, Zuo Ping, Liu Z, Ye BC. Magnetic-based microfluidic device for on-chip separation and detection of tumor-derived exosomes. Anal Chemistry 2018; 90: 13451–13458. Copyright 2018 American Chemical Society. 201

Due to its flexible structure, aptamers can hybridize with other DNA sequences that can initiate DNA-based signal amplification or DNA nanomachines. 202,203 Therefore, the detection of exosomes can be transformed into analysis of DNA, the number of which is directly proportional to the number of exosomes. This strategy avoids the direct detection of exosomes on electrodes, and many methods can be developed to sensitively measure DNA. 204 For example, Dong et al. reported a highly sensitive electrochemical biosensor based on multiple DNA release induced by aptamer recognition and detection of exosomes. Cyclic enzymatic amplification. 205 As shown in Figure 14A, the aptamer-messenger DNA (mDNA) complex is first modified on the MB. The exosomes derived from LNCaP cells bind to the aptamer with high affinity, resulting in the release of three mDNA sequences. After magnetic separation, the mDNA released in the supernatant initiates a cyclic enzymatic amplification reaction assisted by Exo III, resulting in a sharp drop in the content of Ru(NH3)63 on the surface of the Au electrode. Zhao et al. reported a ratio electrochemical biosensor for the detection of exosomes through target-triggered 3D DNA walker and exonuclease III-assisted cyclic enzymatic amplification. 206 As shown in Figure 14B, MB is modified with high-density DNA into a 3D DNA walker scaffold. The DNA sequence is composed of CD63 aptamer and DNAzyme substrate. In the presence of exosomes, the recognition of the CD63 aptamer on MBs and the EpCAM aptamer on the swing arm simultaneously binds to different target proteins on the same exosome, resulting in a close proximity effect between the DNAzyme and the substrate. After hybridization, the DNAwalker is started to release a large number of oligonucleotide fragments, which can be sensitively detected by Exonuclease III-assisted cyclic enzymatic amplification. Figure 14 (A) Schematic diagram of a high-sensitivity electrochemical biosensor for exosome detection based on multiple DNA release induced by aptamer recognition and cyclic enzymatic amplification. Reprinted with permission from Dong H, Chen H, Jiang J, Zhang H, Cai C, and Shen Q. Highly sensitive electrochemical detection of tumor exosomes based on aptamer recognition inducing multiple DNA release and cyclic enzymatic amplification. Anal Chemistry 2018; 90: 4507–4513. Copyright 2018 American Chemical Society. 205 (B) Schematic diagram of a ratio electrochemical biosensor used to detect exosomes through the target-triggered 3D DNA walker and Exo III auxiliary cycle enzyme amplification. Reprinted with permission from Zhao L, Sun R, He P, Zhang X. The three-dimensional DNA walking machine triggered by the target and exonuclease III assisted electrochemical ratio biosensing for ultra-sensitive detection of exosomes. Anal Chemistry 2019; 91: 14773-14779. Copyright 2019 American Chemical Society. 206 (C) In the absence of (a) and (b) tumor exosomes, a schematic diagram of a non-proportional immobilized electrochemical sensing system for tumor exosomes detection. Reprinted with permission from Yang L, Yin X, An B, Li F. Accurately capture and directly quantify tumor exosomes through an efficient dual aptamer identification assisted ratio measurement non-immobilized electrochemical strategy. Anal Chemistry 2021;93:1709-1716. Copyright 2021 American Chemical Society. 207

Figure 14 (A) Schematic diagram of a high-sensitivity electrochemical biosensor for exosome detection based on multiple DNA release induced by aptamer recognition and cyclic enzymatic amplification. Reprinted with permission from Dong H, Chen H, Jiang J, Zhang H, Cai C, and Shen Q. Highly sensitive electrochemical detection of tumor exosomes based on aptamer recognition inducing multiple DNA release and cyclic enzymatic amplification. Anal Chemistry 2018; 90: 4507–4513. Copyright 2018 American Chemical Society. 205 (B) Schematic diagram of a ratio electrochemical biosensor used to detect exosomes through the target-triggered 3D DNA walker and Exo III auxiliary cycle enzyme amplification. Reprinted with permission from Zhao L, Sun R, He P, Zhang X. The three-dimensional DNA walking machine triggered by the target and exonuclease III assisted electrochemical ratio biosensing for ultra-sensitive detection of exosomes. Anal Chemistry 2019; 91: 14773-14779. Copyright 2019 American Chemical Society. 206 (C) In the absence of (a) and (b) tumor exosomes, a schematic diagram of a non-proportional immobilized electrochemical sensing system for tumor exosomes detection. Reprinted with permission from Yang L, Yin X, An B, Li F. Accurately capture and directly quantify tumor exosomes through an efficient dual aptamer identification assisted ratio measurement non-immobilized electrochemical strategy. Anal Chemistry 2021;93:1709-1716. Copyright 2021 American Chemical Society. 207

In order to improve the capture efficiency, MB modified with dual aptamers was used to effectively capture exosomes. 207 As shown in Figure 14C, after capturing tumor exosomes, cholesterol-modified DNA probes are anchored on the exosomal membrane through hydrophobic interactions, triggering the hyperbranched HCR based on DNA tetrahedrons to generate sandwich complexes. The complex can chelate a large amount of Ru(NH3)63 through electrostatic interaction, thereby reducing the amount of Ru(NH3)63 in the solution after magnetic separation. This causes the current ratio of [Fe(CN)6]3- to Ru(NH3)63 to change.

As a powerful technology, ECL has been used in various biological analyses due to its significant advantages such as low background signal, high sensitivity, and wide detection range. In order to meet the needs of ultra-high sensitivity, loading a large number of luminous bodies into nanomaterials can improve ECL efficiency and increase analytical sensitivity. 208 Feng et al. designed an aptamer-binding DNA walking machine, which used Ru(bpy) to detect tumor exosomes sensitively by ECL detection with 32-loaded silica nanoparticles as signal reporter molecules. 209 So far, several nanomaterials have been used as ECL emitters for various biosensing. For example, Sheng and his colleagues developed an ECL aptamer sensor for the analysis of exosomes by G-quadruplex/heme DNase-induced RCL signal quenching of Eu3-doped CdSQDs. 210 Zhang et al. designed a sensitive g-C3N4 coated liquid based on the metal nanoprobe ECL sensing strategy for the detection of exosomes on the multivalent interface (Figure 15A). 211 In the nanoprobe, Galinstan NPs accelerate the electrode transfer and inhibit the passivation of g-C3N4 during the electrochemical reduction process, thereby enhancing the ECL signal. In addition, antibody-modified PAMAM-Au NPs are used to modify electrodes to recognize exosomes multivalently with high capture efficiency. This method achieves a detection limit of 31 particles/μL and is used to determine exosomes derived from HeLa cells. In order to simplify the operating procedures and reduce pollution, Guo et al. reported the QDs-based homogeneous ECL sensing of exosomes with stimulus-responsive DNA microcapsules and a target recovery system. 212 As shown in Figure 15B, the CaCO3 microcapsules loaded with CdS QDs respond to the shell by the stimulation of DNA assembly. Then, the core CaCO3 is removed by treatment with EDTA, and the DNA shell-coated CdS QDs microcapsules are formed. The presence of exosomes initiates nicking endonuclease (Nt.BbvCI) assisted target recovery, and the cross-linked DNA shell is broken down. The released quantum dots are determined by ECL technology. The established double amplification method has a wide detection range from 5×104 to 1×108 particles/μL. Figure 15 (A) A schematic diagram of an exosomal ECL biosensor based on the multivalent recognition and signal amplification strategy of the anti-GPC1-g-C3N4@Galinstan-PDA nanoprobe. Reprinted with permission from Zhang Y, Wang F, Zhang H, Wang H, Liu Y. Multivalent interface and g-C3N4 coated liquid metal nanoprobe signal amplification, used for sensitive electrochemiluminescence detection of exosomes and their surface proteins. Anal Chemistry 2019; 91: 12100-12107. Copyright 2021 Elsevier BV211 (B) A schematic diagram of the preparation process of DNA microcapsules loaded with CdS QDs combined with target recovery and amplification, used for homogeneous ECL detection of tumor exosomes. Reprinted with permission from Guo Y, Cao Q, Zhao C, Feng Q. Stimulus response DNA microcapsules for homogeneous electrochemiluminescence sensing of tumor exosomes. Sens Actuat B Chemistry 2021; 329: 129136–129142. Copyright 2019 American Chemical Society. 212 (C) Schematic diagram of a dual-mode biosensor for exosome detection based on MXenes and black phosphorous quantum dots. Reprinted with permission from Fang D, Zhao D, Zhang S, Huang Y, Dai H, Lin Y. Black phosphorus quantum dots functionalized MXenes as enhanced dual-mode probes for exosome sensing. Sens Actuat B Chemistry 2020; 305: 127544–127552. Copyright 2020 Elsevier BV214

Figure 15 (A) A schematic diagram of an exosomal ECL biosensor based on the multivalent recognition and signal amplification strategy of the anti-GPC1-g-C3N4@Galinstan-PDA nanoprobe. Reprinted with permission from Zhang Y, Wang F, Zhang H, Wang H, Liu Y. Multivalent interface and g-C3N4 coated liquid metal nanoprobe signal amplification, used for sensitive electrochemiluminescence detection of exosomes and their surface proteins. Anal Chemistry 2019; 91: 12100-12107. Copyright 2021 Elsevier BV211 (B) A schematic diagram of the preparation process of DNA microcapsules loaded with CdS QDs combined with target recovery and amplification, used for homogeneous ECL detection of tumor exosomes. Reprinted with permission from Guo Y, Cao Q, Zhao C, Feng Q. Stimulus response DNA microcapsules for homogeneous electrochemiluminescence sensing of tumor exosomes. Sens Actuat B Chemistry 2021; 329: 129136–129142. Copyright 2019 American Chemical Society. 212 (C) Schematic diagram of a dual-mode biosensor for exosome detection based on MXenes and black phosphorous quantum dots. Reprinted with permission from Fang D, Zhao D, Zhang S, Huang Y, Dai H, Lin Y. Black phosphorus quantum dots functionalized MXenes as enhanced dual-mode probes for exosome sensing. Sens Actuat B Chemistry 2020; 305: 127544–127552. Copyright 2020 Elsevier BV214

Nanomaterials with excellent catalytic properties are increasingly used in the development of ECL biosensors. For example, MXenes has attracted attention in the fields of catalysis, biosensors and supercapacitors due to its excellent electron transfer ability and unique catalytic ability. Wang and colleagues reported an ECL biosensor catalyzed by MXenes for exosome detection. 213 Fang et al. proposed a self-enhanced ECL and photothermal dual-mode biosensor for exosome detection. 214 As shown in Figure 15C, MXenes is used to carry black phosphorus (BP) quantum dots (BPQD), Ru(dcbpy)32 and CD63 antibodies. In nanocomposites, BPQDs not only catalyze the oxidation of Ru(dcbpy)32, but also act as co-reactants. Integrating BPQD and Ru(dcbpy)32 into MXenes can shorten the distance and amplify the ECL signal. In addition, both MXenes and BPQDs with good photothermal properties can be used as photothermal probes. The linear range of the dual-mode biosensor is 1.1×102 ~ 1.1×107 particles/μL. However, stabilizers used to adjust structure and morphology and biomolecules used for specific recognition may block the active site and hinder electron transfer. Therefore, Zhang et al. reported an ECL biosensor for exosome detection based on the formation of AuNP in situ on aptamer-modified MXenes (Figure 16). 215 MXenes is modified with CD63 aptamer to recognize exosomes. After the exosomes are captured, the MXenes bound by the aptamer are adsorbed on the surface of the exosomes. MXenes with large surface area and strong reducing ability can induce the formation of AuNP in situ on the surface without adding additional reducing agent. AuNPs with catalytic surface greatly enhance the ECL signal of luminol. The proposed biosensor shows a detection limit as low as 30 particles/μL. In recent years, g-C3N4 not only serves as a carrier for luminol, but also as a catalyst to promote the reaction of luminol with H2O2, thereby amplifying the ECL signal for exosomal detection. 216 Figure 16. Schematic diagram of exosomal ECL biosensor. Detection of MXenes nanoprobes decorated with AuNPs formed in situ. Reprinted with permission from Zhang H, Wang Z, Wang F, Zhang Y, Wang H, and Liu Y. Exosomes and their surface proteins. Anal Chemistry 2020; 92: 5546–5553. Copyright 2020 American Chemical Society. 215

Figure 16 Schematic diagram of ECL biosensor based on in-situ formation of AuNPs-modified MXenes nanoprobes. MXenes nanoprobes are used for high-sensitivity electrochemiluminescence detection of exosomes and their surface proteins. Anal Chemistry 2020; 92: 5546–5553. Copyright 2020 American Chemical Society. 215

The photoelectrochemical (PEC) process involves photocurrent as the detection signal generated by photoelectrochemically active materials under light. The separation of excitation source (light) and detection signal (current) makes the PEC biosensor have low background signal, high signal-to-noise ratio and excellent stability. Photosensitive materials play a vital role in the manufacture of PEC biosensors. Usually, Li's team designed a cathodic PEC aptamer sensor to detect exosomes using a p-type NiO/BiOI/AuNP composite material sensitized by CdSe QD. 217 In this work, a narrow band gap (1.8 eV) BiOI was immobilized on NiO-modified ITO to sensitize the wide band gap (3.6–4.0 eV) of NiO. Then, AuNPs are deposited on the surface and bind to EpCAM aptamers by forming Au-S bonds. The DNA2-modified quantum dots are combined with the electrode surface through hybridization, making the nanocomposite sensitive and increasing the intensity of the photocurrent. In the presence of exosomes, the aptamer binds to the EpCAM protein on the surface of the exosomes, resulting in the release of quantum dots and the reduction of photocurrent. At the same time, the exosomes on the electrode surface hinder the electron transfer between the electrode and the electron acceptor, causing the photocurrent signal to be further attenuated.

Chemiluminescence is produced by an exothermic chemical reaction, in which intermediate molecules in a singlet excited state undergo radiative decay. The chemiluminescence biosensor can sensitively detect the target of interest in the dark without any additional input energy. 218 Zhong’s group reported a chemiluminescence strategy based on CuS sealed microgels for rapid separation and quantification of exosomes. 219 As shown in Figure 17, CuS NPs were generated in situ in the porous microgel and were further modified with streptavidin and antibodies. After interacting with exosomes, the microgel promotes the separation of exosomes through membrane filtration. Release a large amount of Cu2 ions, catalyze the reaction of H2O2 with luminol derivatives, and generate a strong chemiluminescence signal. In addition, Wang et al. reported the use of antibody-conjugated superparamagnetic iron oxide particles (SIOP) and acridinium ester as signal markers for chemiluminescence immunoassay for exosomal detection. 220 Figure 17 CuS-microgel synthesis schematic and CuS-microgel-based analysis of exosomes quantification. Reprinted with permission from Jiang Q, Liu Y, Wang L, Adkins GB, Zhong W. The rapid enrichment and detection of extracellular vesicles realized by the cus sealed microgel. Anal Chemistry 2019; 91: 15951-15958. Copyright 2019 American Chemical Society. 219

Figure 17 Schematic diagram of CuS-microgel synthesis and schematic diagram of quantitative analysis of exosomes based on CuS-microgel. Reprinted with permission from Jiang Q, Liu Y, Wang L, Adkins GB, Zhong W. The rapid enrichment and detection of extracellular vesicles realized by the cus sealed microgel. Anal Chemistry 2019; 91: 15951-15958. Copyright 2019 American Chemical Society. 219

Mass spectrometer (MS) is an effective tool to characterize the content of biomolecules with high throughput. 221 However, low sensitivity limits its application in the early detection of low concentrations of exosomes in cancer. The element labeling strategy gives MS the advantages of high selectivity and signal amplification. In this detection principle, nanomaterials can not only be used as a matrix material to capture exosomes, but also because they contain a large number of elements, they can also amplify MS signals. 222-227 Recently, Zhang et al. reported an ultra-sensitive inductively coupled plasma mass spectrometry (ICP-MS) method that uses UCNPs as elemental markers to detect exosomes. -Similar to nano-components. Exosomes with surface proteins can trigger the release of corresponding aptamers coupled to UCNPs from AuNPs. Then, the UCNP released in the solution is detected by ICP-MS. Finally, this sensitive and high-throughput method can distinguish exosomes from seven different cell lines. Figure 18 Schematic diagram of ultrasensitive inductively coupled plasma mass spectrometry for detecting exosomes using UCNP as an element label. Reprinted from Zhang Xinwen, Liu Meisi, He MQ, Chen Si, Yu Yuling, Wang Jianhua. The UCNP-AuNP nanosatellite module programmed by DNA integrates multi-element signals for ultra-sensitive ICP-MS detection of exosomal proteins and cancer identification. Anal Chemistry 2021;93:6437-6445. Copyright 2021 American Chemical Society. 228

Figure 18 Schematic diagram of ultrasensitive inductively coupled plasma mass spectrometry for detecting exosomes using UCNP as an element label. Reprinted from Zhang Xinwen, Liu Meisi, He MQ, Chen Si, Yu Yuling, Wang Jianhua. The UCNP-AuNP nanosatellite module programmed by DNA integrates multi-element signals for ultra-sensitive ICP-MS detection of exosomal proteins and cancer identification. Anal Chemistry 2021;93:6437-6445. Copyright 2021 American Chemical Society. 228

In this review, we systematically reviewed various exosomes detection techniques based on nanomaterials. The unique characteristics of nanomaterials make them fascinating materials for signal transduction and biosensor development. In addition, through integration with various complex DNA or enzyme-based signal amplification strategies, the sensitivity and selectivity of biosensors for exosome detection have been greatly improved. Although significant progress has been made in the detection of exosomes, there are still some challenges that need to be resolved. For example, the stability of nanomaterials and the selectivity for complex samples should be improved by using biocompatible polymers or other materials for appropriate modification. The biometric elements used in bioassays may be affected by the digestion of enzymes in the actual sample. The key obstacle to the transformation of laboratory research into clinical practice is the development of standardized technical specifications, including the collection of biological samples, the separation of exosomes in liposomes, and the detection operations. It is helpful to enhance the comparability of results and establish a reliable and comprehensive data set for future exosomes research. The cost-effective and high-throughput separation and detection of exosomes help to identify a large number of clinical samples at the same time. We believe that with the joint efforts and unremitting efforts of different fields such as chemistry, materials science and clinical diagnosis, the detection of exosomes based on nanomaterials will make greater progress.

This research was supported by the Henan University Science and Technology Innovation Research Team Project (21IRTSTHN005), the National Natural Science Foundation of China (21804085), and the Henan Provincial Key Laboratory of Biomolecular Recognition and Sensing Research Fund (HKLBRSK1902).

The author declares that there is no conflict of interest.

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