Molybdenum Disulfide-Integrated Iron Organic Framework Hybrid Nanozyme-Based Aptasensor for Colorimetric Detection of Exosomes

Tumor-derived exosomes are considered as a potential marker in liquid biopsy for malignant tumor screening. The development of a sensitive, specific, rapid, and cost-effective detection strategy for tumor-derived exosomes is still a challenge. Herein, a visualized and easy detection method for exosomes was established based on a molybdenum disulfide nanoflower decorated iron organic framework (MoS2-MIL-101(Fe)) hybrid nanozyme-based CD63 aptamer sensor. The CD63 aptamer, which can specifically recognize and capture tumor-derived exosomes, enhanced the peroxidase activity of the hybrid nanozyme and helped to catalyze the 3,3′,5,5′-tetramethylbenzidine (TMB)-H2O2 system to generate a stronger colorimetric signal, with its surface modification on the hybrid nanozyme. With the existence of exosomes, CD63 aptamer recognized and adsorbed them on the surface of the nanozyme, which rescued the enhanced peroxidase activity of the aptamer-modified nanozyme, resulting in a deep-to-moderate color change in the TMB-H2O2 system where the change is visible and can be monitored with ultraviolet-visible spectroscopy. In the context of optimal circumstances, the linear range of this exosome detection method is measured to be 1.6 × 104 to 1.6 × 106 particles/μL with a limit of detection as 3.37 × 103 particles/μL. Generally, a simple and accessible approach to exosome detection is constructed, and a nanozyme-based colorimetric aptamer sensor is proposed, which sheds light on novel oncological biomarker measurements in the field of biosensors.


Introduction
Malignant tumor is one of the main causes of lethal casualty worldwide, presenting with escalating morbidity among the developing and underdeveloped countries. Besides effective therapy, early screening also plays an important role in the fight against malignant tumors, which raises a huge demand for a large volume of precise and repeatable detection [1,2]. However, the common screening strategies for malignant tumors, including positron emission tomography, magnetic resonance imaging, computed tomography, X-ray, and endoscopy, are not suitable for large-volume screening and are not accessible in primary clinics. In addition to radiological imaging examinations, tumoral biomarkers detections in liquid samples are widely used in tumor screening, diagnosis, and follow-up [3]. Although a variety of novel potential biomarkers have been identified for different cancers, methodological and technical obstacles still exist in terms of biomarker detection and limit its further applications, such as low concentration or poor stability of biomarkers in human body fluid samples, which caused attention in sensitive and stable biomarker-detecting techniques [4]. hybridization regulation strategy could considerably enhance the catalytic activity of the nanozyme due to their synergistic effects of hybrid materials beneficial for the improvement of dispersion, conductivity, and specific surface area.
In terms of molybdenum disulfide (MoS 2 ), a transition metal dihalide compound, presented as a two-dimensional, flake, and graphene-like structure, exhibits abundant catalytic-active edges and advanced specific surface area [31]. Recently, MoS 2 nanosheets have been reported as a promising mimic of peroxidase [32]. To date, in several functional materials [33,34], MoS 2 has been adopted as the subunit to mimic natural enzymes and achieve enhanced catalytic activity. Therefore, MoS 2 is an ideal alternative for the construction of MOF-based nanozyme.
In this work, a visible exosome detection technique was developed based on an artificial, mimetic nanozyme of peroxidase, the MoS 2 -MIL-101(Fe) hybrid nanozyme was constructed and was proved to possess superior peroxidase enzymatic activity. The configuration of MIL-101(Fe) provided MoS 2 with a large specific surface area, which is conducive to the absorption of substrates on the exterior of the hybrid nanozyme. Additionally, modified on the exterior of the hybrid nanozyme via electrostatic interaction, the CD63 aptamers not only specifically recognize and capture exosomes but also enhances the affinity of the hybrid nanozyme to its substrates and further improve its catalytic activity with the aptamer's single-strand DNA configuration [35,36]. Generally, with these synergistic effects, the MoS 2 -MIL-101(Fe) hybrid nanozyme-based aptamer sensor is assumed to have an ideal detection limit. And a multi-purpose design of the nanozyme-based colorimetric aptamer sensor is proposed.

The Preparation of MoS 2 -MIL-101(Fe)
MoS 2 nanoflowers were synthesized by the hydrothermal method as previously reported [37]. In brief, 50 mg of (NH 4 ) 2 MoS 4 was dispersed in 30 mL of DMF under ultrasonic treatment for 15 min. Then, the solution was transferred into a Teflon liner and was kept at 200 • C for 10 h. After being cooled down, the raw product was washed with DMF and ethanol several times. After being dehydrated at 60 • C in a vacuum drier, MoS 2 nanoflowers were purified and collected.
MoS 2 -MIL-101(Fe) nanocomposites were synthesized via heat treatment of a mixture of FeCl 3 ·6H 2 O, terephthalic acid, and MoS 2 nanoflowers. In detail, under ultrasonic treatment, 20 mg of as-prepared MoS 2 nanoflowers were dispersed in 15 mL of DMF to form a homogeneous solution. Then 0.206 g of terephthalic acid and 0.675 g of FeCl 3 ·6H 2 O were dissolved in the abovementioned solution by continuous stirring. The mixture was transferred into a Teflon liner and was kept at 110 • C for 20 h. After being cooled down to room temperature, the raw product was separated, and washed by DMF and ethanol three times. Finally, after being dehydrated at 60 • C in a vacuum drier, MoS 2 -MIL-101(Fe) nanocomposites were collected ( Figure 1A). In addition, MIL-101(Fe) nanocomposites were synthesized through the same processes described above except for the introduction of MoS 2 nanoflowers [38]. synthesized through the same processes described above except for the introduction of MoS2 nanoflowers [38].

Cell Culture and Exosomes Preparation
The HGC-7901 cells and LO2 cells were cultured in RMPI-1640 supplemented with 10% FBS at 37 °C in 5% CO2. After the cells proliferated to 80% confluence, the culture medium was replaced with RMPI-1640 supplemented with 10% Exofree-FBS. After 48 h incubation of exosome-free medium, the cell culture supernatant was collected to harvest tumor-derived exosomes, and the cells were passaged and cultured. Exosomes were isolated from the cell culture supernatant by the standard ultracentrifugation method with slight modifications [39]: (1) 1500× g centrifugation for 10 min to eliminate dead cells; (2) 1000× g for 20 min to eliminate cellular debris and the acquired supernatant was filtrated by a 0.22 μm filter; (3) 100,400× g for 4 h to precipitate exosomes.

Simulation of Peroxidase Activity in Nanocomposites
In the presence of a given concentration of H2O2 in a pH 4.0 HAc-NaAc buffer, the simulation of peroxidase activity in MoS2-MIL-101(Fe) nanocomposite was evaluated by introducing MoS2-MIL-101(Fe) hybrids to a TMB solution. The total volume of the reaction solution was set to 4 mL. The solution was composed of 1 mL of pH 4.0 HAc-NaAc buffer, 1 mL of MoS2-MIL-101(Fe) solution (50 mg/L), 1 mL of H2O2 solution (10 mM), and 1 mL of TMB solution (5 mM). Next, the reaction solution was incubated at 40 °C for 5 min and the absorbance was measured by an ultraviolet-visible (UV-vis) spectrophotometer.
Kinetic experiments of MoS2-MIL-101(Fe) hybrids were performed by measuring the initial rate of the reaction in the first 5 min, with one of the concentrations of H2O2 and TMB fixed, and the other varied. The H2O2 concentration was set to 4 mmol/L and the fixed TMB concentration was set to 2.5 mmol/L.
The kinetic parameters were fitted by the following equation: V0 = V max . After being fitted, k1 is Vmax and k2 is Km [38].

Cell Culture and Exosomes Preparation
The HGC-7901 cells and LO2 cells were cultured in RMPI-1640 supplemented with 10% FBS at 37 • C in 5% CO 2 . After the cells proliferated to 80% confluence, the culture medium was replaced with RMPI-1640 supplemented with 10% Exofree-FBS. After 48 h incubation of exosome-free medium, the cell culture supernatant was collected to harvest tumor-derived exosomes, and the cells were passaged and cultured. Exosomes were isolated from the cell culture supernatant by the standard ultracentrifugation method with slight modifications [39]: (1) 1500× g centrifugation for 10 min to eliminate dead cells; (2) 1000× g for 20 min to eliminate cellular debris and the acquired supernatant was filtrated by a 0.22 µm filter; (3) 100,400× g for 4 h to precipitate exosomes.

Simulation of Peroxidase Activity in Nanocomposites
In the presence of a given concentration of H 2 O 2 in a pH 4.0 HAc-NaAc buffer, the simulation of peroxidase activity in MoS 2 -MIL-101(Fe) nanocomposite was evaluated by introducing MoS 2 -MIL-101(Fe) hybrids to a TMB solution. The total volume of the reaction solution was set to 4 mL. The solution was composed of 1 mL of pH 4.0 HAc-NaAc buffer, 1 mL of MoS 2 -MIL-101(Fe) solution (50 mg/L), 1 mL of H 2 O 2 solution (10 mM), and 1 mL of TMB solution (5 mM). Next, the reaction solution was incubated at 40 • C for 5 min and the absorbance was measured by an ultraviolet-visible (UV-vis) spectrophotometer.
Kinetic experiments of MoS 2 -MIL-101(Fe) hybrids were performed by measuring the initial rate of the reaction in the first 5 min, with one of the concentrations of H 2 O 2 and TMB fixed, and the other varied. The H 2 O 2 concentration was set to 4 mmol/L and the fixed TMB concentration was set to 2.5 mmol/L.
The kinetic parameters were fitted by the following equation Here, K m stands for Mi's constant, [S] for substrate concentration, V 0 for initial reaction rate, and V max for maximum reaction rate. For each preset H 2 O 2 and TMB concentrations, K m and V max were calculated by Hyperbola curve fit using the OriginPro 2019 (OriginLab Corporation, Northampton, MA, USA) after measuring V 0 . The hyperbolic function is also the Michaelis-Menten model in enzyme kinetics, and its formula is y = k 1 x k 2 +x corresponding to the Michaelis-Menten equation V 0 = V max . After being fitted, k 1 is V max and k 2 is K m [38].

Exosomes Detection
Ten microliters of CD63 aptamer (10 µM) and 200 µL MoS 2 -MIL-101(Fe) nanocomposites (100 mg/L) were mixed and blended by vortex. Then the different solutions with different concentrations of exosomes (10 µL) were added to the mixtures and were blended by vortex. After 30 min of incubation, 100 µL TMB (2.5 mM) solution and 100 µL H 2 O 2 (4 mM) solution were added to the mixtures, and then HAc-NaAc buffer was added to fill up the volume of mixtures to 1000 µL. The mixtures were incubated at 40 • C for 5 min in the dark, and then the absorbances were measured by a UV-vis spectrophotometer with a 1.0 cm quartz cell.  Figure 1B. The chromogenic reaction was introduced to evaluate and compare the peroxidase catalytic activities of the hybrid nanozyme, as well as its precursors. MoS 2 -MIL-101(Fe) nanocomposite was suggested to have a higher peroxidase activity in contrast to both MoS 2 and MIL-101(Fe) (Figure 2A), which catalyzed TMB to transform from the colorless substrates to the deep blue substances in the presence of H 2 O 2 . Modified by CD63 aptamers, the peroxidase catalytic activity of the MoS 2 -MIL-101(Fe) was magnified, which is attributed to π-π stacking between single-stranded DNA and the substrate ( Figure 2B). In detail, the bases of DNA aptamer facilitate the bindings between the substrates, especially through hydrogen bonding between DNA bases and the amino groups of TMB, as well as the nucleobase interacting between DNA bases and the benzene rings of TMB via π-π stacking, which led to the increased substrate affinity and further enhanced the catalytic activity of MoS 2 -MIL-101(Fe) [35,40]. In the presence of exosomes, the specific ligand-receptor recognition between CD63 aptamers and the CD63 proteins on the exterior of exosomes confined exosomes within the external surface of MoS 2 -MIL-101(Fe)@Aptamer and rescued the enhanced peroxidase catalytic activity of MoS 2 -MIL-101(Fe)@Aptamer, where MoS 2 -MIL-101(Fe)@Aptamer@Exosomes exhibited an even weaker peroxidase catalytic activity than MoS 2 -MIL-101(Fe) ( Figure 2B). With various concentrations of exosomes, the hybrid nanozyme exhibited different degrees of peroxidase catalytic activities, so as to present different absorbances of the TMB colorimetric reaction, which could be obviously visualized and measured by UV-vis spectrometer. Therefore, the exosome-detection aptamer sensor was constructed based on the integration of CD63 aptamer and the hybrid nanozyme, which was verified with the following zeta potential measurements. As shown in Figure 3, the zeta potential of MoS 2 -MIL-101(Fe) is 21.0 mV, which shows that the composite material was positively charged. After incubating with the aptamer, the zeta potential of the composite material changed to 1.95 mV, which suggested that the CD63 aptamers were modified on the surface of MoS 2 -MIL-101(Fe) through electrostatic interaction. In addition, while the CD63 aptamers combined with the exosomes, the overall zeta potential of MoS2-MIL-101(Fe)@Aptamer@Exosomes turned to −7.65 mV, which continued to decline; the nanocomposite was negatively charged (Figure 3).      Figure 4B). The successful preparation of the hybrid nanozyme was observed by TEM ( Figure 4C) and further identified with X-ray diffraction (XRD) (Figure 4D), depicting a characteristic peak at 9.4 • which is attributed to the (001) reflection of MoS 2 .

Results and Discussion
successful preparation of the hybrid nanozyme was observed by TEM ( Figure 4C) and further identified with X-ray diffraction (XRD) (Figure 4D), depicting a characteristic peak at 9.4° which is attributed to the (001) reflection of MoS2.  Figure 5A,B), which showed an average diameter of 100 nm that coincided with the previous studies. The exosome counting was carried out by NTA, and the results were further used as the standard of exosomes. (Figure 5C). The expression of typical labeled proteins [41] (transmembrane proteins CD9, CD63 and CD81) of exosomes derived from HCG-7901 was directly verified by Western Blots (WB) ( Figure 5D).  Figure 5A,B), which showed an average diameter of 100 nm that coincided with the previous studies. The exosome counting was carried out by NTA, and the results were further used as the standard of exosomes. (Figure 5C). The expression of typical labeled proteins [41] (transmembrane proteins CD9, CD63 and CD81) of exosomes derived from HCG-7901 was directly verified by Western Blots (WB) ( Figure 5D).

Optimization of Experimental Conditions
The optimal experimental condition of the TMB reaction under MoS2-MIL-101(Fe)@Aptamer was then investigated, with the total reaction volume set to 4 mL. The activity of the nanozyme active site is affected by pH. Generally, the POD-like activity of the metal-based nanozyme is more efficient in an acidic environment while its catalaselike (CAT-like) activity is more efficient in an alkaline environment, where the transferred domination between POD-and CAT-like activities were driven through different reactant decomposition pathways at different pH [42]. The peroxidase catalytic activities of MoS2-MIL-101(Fe) at various pH conditions (from pH 2 to pH 8) were first investigated. The results showed that the catalytic activity was correlated with pH value, which presented the maximum activity at pH 4 ( Figure S1A). Therefore, pH 4 was selected as the ideal pH

Optimization of Experimental Conditions
The optimal experimental condition of the TMB reaction under MoS 2 -MIL-101(Fe)@Aptamer was then investigated, with the total reaction volume set to 4 mL. The activity of the nanozyme active site is affected by pH. Generally, the POD-like activity of the metal-based nanozyme is more efficient in an acidic environment while its catalase-like (CAT-like) activity is more efficient in an alkaline environment, where the transferred domination between POD-and CAT-like activities were driven through different reactant decomposition pathways at different pH [42]. The peroxidase catalytic activities of MoS 2 -MIL-101(Fe) at various pH conditions (from pH 2 to pH 8) were first investigated. The results showed that the catalytic activity was correlated with pH value, which presented the maximum activity at pH 4 ( Figure S1A). Therefore, pH 4 was selected as the ideal pH value. Temperature also can regulate the catalytic activity of nanozymes. With increasing temperatures, the reactants' thermal motions in the vicinity of active sites can be further activated, thus enhancing the catalytic activities of each individual active site. Therefore, the probability of molecular collisions between nanozymes and substrates is greatly proposed, thereby lifting the reaction rate [43]. Since enzymatic catalytic activity can be affected by temperature, the catalytic activities under different temperatures were also explored, which showed that the catalytic activity reaches to maximum at 40 • C ( Figure S1B). Therefore, 40 • C was selected as the optimal temperature. Then, the effect of different CD63 aptamer concentrations (from 2 µM to 20 µM) on the absorbances at 653 nm (A653) that reflect the concentrations of oxidized TMB was investigated. The A653 gradually increased as the CD63 aptamer concentration increased from 2 µM to 10 µM, achieved the highest value at 10 µM, and then gradually fell off when the concentration was above 10 µM ( Figure S1C), which suggested that the optimal concentration of CD63 aptamer was 10µM. Similarly, the effects of different TMB concentrations, different H 2 O 2 concentrations, and different MoS 2 -MIL-101(Fe) concentrations on A653 were monitored. It is reported [44] that the colorific reaction of benzidine under one-electron, or two-electron oxidation is blue or yellow, respectively, where the corresponding absorption peak is 653 nm or 450 nm. With increasing concentrations of TMB or hydrogen peroxide, the nanozyme-catalytic reaction rate, as well as the reaction rate of one-electron benzidine oxidation are lifted. Meanwhile, as a substrate, the product of one-electron benzidine oxidation also promotes two-electron oxidation, which causes the blue to yellow-green transformation of the solution system, leading to the decrease in the absorption peak at 653 nm. And it is consistent with the experimental phenomenon. The optimal conditions of these variables were shown as the followings: 2.5 mM TMB

Analytical Performance in Determination of Exosomes
Further, the absorbances at 653 nm (A653) of the TMB under diverse exosome concentrations were measured under the optimal conditions. As shown in Figure 7A, A653 decreased proportionally with the increase in exosome concentration. Correspondingly, color changes of the solutions with different exosome concentrations were obviously visible and comparable (a-h, Figure 7A). The linear range between A653 and the logarithm of exosome concentration is 1.6 × 10 4~1 .6 × 10 6 particles/μL, where the correlation equation is y = −0.73081 × lg[c(exosomes)] + 4.94426, and the squared correlation coefficient is 0.996 (y reflects A653, and c(exosomes) reflects the exosome concentration, Figure 7B). As reported [45,46], a cancer cell can secrete more than 10 4 vesicles in 24 h in contrast to a nor-

Analytical Performance in Determination of Exosomes
Further, the absorbances at 653 nm (A653) of the TMB under diverse exosome concentrations were measured under the optimal conditions. As shown in Figure 7A, A653 decreased proportionally with the increase in exosome concentration. Correspondingly, color changes of the solutions with different exosome concentrations were obviously visible and comparable (a-h, Figure 7A). The linear range between A653 and the logarithm of exosome concentration is 1.6 × 10 4~1 .6 × 10 6 particles/µL, where the correlation equation is y = −0.73081 × lg[c(exosomes)] + 4.94426, and the squared correlation coefficient is 0.996 (y reflects A653, and c(exosomes) reflects the exosome concentration, Figure 7B). As reported [45,46], a cancer cell can secrete more than 10 4 vesicles in 24 h in contrast to a normal epithelial cell. In the clinical approach of exosome detection [47,48], our work could reach the level of detecting tumor-derived exosomes. Designed on the basis of the 3σ method (σ is the blank standard deviation), this aptamer sensor exhibited a promising limit of detection (LOD) of 3.37 × 10 3 particles/µL, which is lower than that of other methods reported and is attributed to the high POD-like catalytic activity of the hybrid nanozyme (Table 1) [49][50][51][52][53][54][55][56].  The specificity was a pivotal issue for developing a novel exosome-detection aptamer sensor. Heterogeneous exosomes with different CD63 protein expressions were adopted to verify the specificity of this aptamer sensor. As previously reported [36], the CD63 expressions of exosomes derived from human gastric cancer cell line HCG-7901 cells were lower than those derived from normal human liver cell line LO2. As shown in Figure 8A, the stronger signals were observed in the group of CD63-low HGC-7901 cells-derived exosomes, while the much weaker signals were observed in the group of CD63-high LO2 cells-derived exosomes with the same concentration of exosomes. In accordance with the previous studies, the results demonstrated that this aptamer sensor had a good selectivity on various exosomes based on the specificity of the CD63 aptamer.  The specificity was a pivotal issue for developing a novel exosome-detection aptamer sensor. Heterogeneous exosomes with different CD63 protein expressions were adopted to verify the specificity of this aptamer sensor. As previously reported [36], the CD63 expressions of exosomes derived from human gastric cancer cell line HCG-7901 cells were lower than those derived from normal human liver cell line LO2. As shown in Figure 8A, the stronger signals were observed in the group of CD63-low HGC-7901 cells-derived exosomes, while the much weaker signals were observed in the group of CD63-high LO2 cells-derived exosomes with the same concentration of exosomes. In accordance with the previous studies, the results demonstrated that this aptamer sensor had a good selectivity on various exosomes based on the specificity of the CD63 aptamer. The reproducibility of this aptasensor has also been explored. As shown in Figure S2, the exosome detection effectiveness of the aptasensor after 30-day storage at 4 °C exhibited a good similarity with that of synthesized on the first day as its RSD values still presented consistencies less than 5%, which indicated favorable reproducibility, stability, and the shelf-life of this aptasensor.

Detection of Exosomes in Human Serum Sample
To explore the applicability of this hybrid nanozyme-based aptamer sensor on a clinical approach, HGC-7901-derived exosomes were added to human serum to establish artificial samples for detection. The results showed that the calculated recoveries ranged from 95% to 103% (Table S1). As shown in Figure 8B, exosome detections of the artificial samples presented results consistent with those of PBS samples with the same concentration of exosomes added. These results clearly indicated that exosome detection based on this aptasensor is accurate and suitable for detection in complex systems, providing an important tool for exosome detection in clinical applications.

Conclusions
In conclusion, a novel, sensitive, visible and simple approach to exosome detection is developed by means of the MoS2-MIL-101(Fe) hybrid nanozyme-based aptasensor. Excellent linearity was obtained for exosome detection within the extent of 1.6 × 10 4 to 1.6 × 10 6 particles/μL, as well as a LOD of 3.37 × 10 3 particles/μL was achieved. The aptamer sensor possesses equal applicability in complex biological samples in clinical approach, which exhibited a cutting-edge economic efficiency and accessibility that can be potentially applied for portable exosome-detection devices. Nonetheless, in this study, there are limitations that need to be pointed out that the unsolved defects of nanozyme, such as the relatively low substrate selectivity and catalytic efficiency, still impede the further enhancement of the sensitivity of this aptasensor. In addition, the single CD63 DNA aptamer design of this aptasensor also hinders the further improvement of its exosome specificity.
Supplementary Materials: The following supporting information can be downloaded at: www.mdpi.com/xxx/s1, Figure S1: Influences of (A) pH, (B) temperature, (C) aptamer concentration, (D) TMB concentration, (E) H2O2 concentration, (F) MoS2-MIL-101(Fe) concentration for the MoS2-MIL-101(Fe) hybrid nanozyme-based aptasensor; Figure S2: Comparison of absorbances between measurement at first-day synthesis and measurement after 30-day room-temperature storage with The reproducibility of this aptasensor has also been explored. As shown in Figure S2, the exosome detection effectiveness of the aptasensor after 30-day storage at 4 • C exhibited a good similarity with that of synthesized on the first day as its RSD values still presented consistencies less than 5%, which indicated favorable reproducibility, stability, and the shelf-life of this aptasensor.

Detection of Exosomes in Human Serum Sample
To explore the applicability of this hybrid nanozyme-based aptamer sensor on a clinical approach, HGC-7901-derived exosomes were added to human serum to establish artificial samples for detection. The results showed that the calculated recoveries ranged from 95% to 103% (Table S1). As shown in Figure 8B, exosome detections of the artificial samples presented results consistent with those of PBS samples with the same concentration of exosomes added. These results clearly indicated that exosome detection based on this aptasensor is accurate and suitable for detection in complex systems, providing an important tool for exosome detection in clinical applications.

Conclusions
In conclusion, a novel, sensitive, visible and simple approach to exosome detection is developed by means of the MoS 2 -MIL-101(Fe) hybrid nanozyme-based aptasensor. Excellent linearity was obtained for exosome detection within the extent of 1.6 × 10 4 to 1.6 × 10 6 particles/µL, as well as a LOD of 3.37 × 10 3 particles/µL was achieved. The aptamer sensor possesses equal applicability in complex biological samples in clinical approach, which exhibited a cutting-edge economic efficiency and accessibility that can be potentially applied for portable exosome-detection devices. Nonetheless, in this study, there are limitations that need to be pointed out that the unsolved defects of nanozyme, such as the relatively low substrate selectivity and catalytic efficiency, still impede the further enhancement of the sensitivity of this aptasensor. In addition, the single CD63 DNA aptamer design of this aptasensor also hinders the further improvement of its exosome specificity.

Institutional Review Board Statement:
The study was conducted in accordance with the Declaration of Helsinki, and approved by the Ethics Committee of Shanghai Normal University.