Electrochemical Micro-Immunosensor of Cubic AuPt Dendritic Nanocrystals/Ti3C2-MXenes for Exosomes Detection

Exosomes are extracellular vesicles that exist in body circulation as intercellular message transmitters. Although the potential of tumor-derived exosomes for non-invasive cancer diagnosis is promising, the rapid detection and effective capture of exosomes remains challenging. Herein, a portable electrochemical aptasensor of cubic AuPt dendritic nanocrystals (AuPt DNs)/Ti3C2 assisted in signal amplification, and aptamer CD63 modified graphene oxide (GO) was immobilized on a screen-printed carbon electrode (SPCE) as the substrate materials for the direct capture and detection of colorectal carcinoma exosomes. Cubic AuPt DNs/Ti3C2 was synthesized according to a simple hydrothermal procedure, and the AuPt DNs/Ti3C2-Apt hybrid demonstrated an efficient recognition of exosomes. Under optimal conditions, a detection limit of down to 20 exosomes µL−1 was achieved with the linear range from 100 exosomes μL−1 to 5.0 × 105 exosomes μL−1. The proposed immunosensor could be suitable for the analysis of exosomes and has clinical value in the early diagnosis of cancer.


Introduction
Malignant tumors are dangerous to human health due to their high morbidity and mortality. The non-invasive early diagnosis of malignant tumors has attracted attention to improve the survival rates of patients in clinical research. Exosomes, lipid-membranebound nanovesicles [1,2], have attracted much attention for early cancer screening technologies to provide intercellular communication with large numbers of macromolecules involving specific proteins, lipids, DNA, mRNA, and microRNAs [3]. Exosomes act as a potential tumor biomarker, which is one of the most efficient prognostic indicators for early cancer diagnosis in clinical research [4,5]. They are derived from tumor cells and mediate messages between donor cells and recipient cells, and alter immune response regulation and intracellular bioactive molecule transmission [6].
Several quantitative methods, such as enzyme-linked immunosorbent analysis [7], dynamic light scattering [8], flow cytometry [9], and nanoparticle tracking [10], have been reported for recognizing and detecting exosomes. These methods can detect exosome concentration and size as well as provide new pathways for extracellular vesicles; they can provide further utility due to their special structures and functions. However, they all have limitations such as complicated procedures, long detection time, and the difficulty in quantifying smaller exosomes. New detection techniques, such as colorimetry, fluorescence, and electrochemical techniques, have been developed for exosome identification and measurement [11][12][13]. Among these strategies, biosensors have be regarded as a valuable analytical platform in many fields, especially in the detection and monitoring of disease diagnosis and treatment, displaying the advantages of being highly selective, providing fast responses, high sensitivity [14,15], and efficient use of the analyte. The use of biosensors has attracted is 100-5 × 10 5 exosomes /µL, and the detection limit is 20 exosomes /µL (S/N = 3). The developed immunosensor was tested for exosome detection in human serum samples, which can provide a new promising method to create a miniaturized portable potentiostat working with microvolumes and for point-of-care analysis in clinical settings.
Micromachines 2023, 14, x FOR PEER REVIEW 3 of 13 optimization of detection conditions (such as the incubation time, the Apt concentration, and the concentration of KCL) for the detection of exosomes was performed. The linear range of the sensor is 100-5 × 10 5 exosomes /µL, and the detection limit is 20 exosomes /µL (S/N = 3). The developed immunosensor was tested for exosome detection in human serum samples, which can provide a new promising method to create a miniaturized portable potentiostat working with microvolumes and for point-of-care analysis in clinical settings.

Materials and Methods
The main fabrication of the sandwich-type electrochemical biosensor is the synthesis of Ti3C2 MXenes, cubic Au/Pt DNs, and Au/Pt/Ti3C2 MXenes, which we performed as described below.

Apparatus and Characterization
The X-ray diffraction (XRD) patterns were collected using a D8 ADVANCE X-ray diffractometer. The transmission electron microscopy (TEM) images were obtained with a JEOL 2010 transmission electron microscope. A JEOL 5160LV electron microscope was used to acquire scanning electron microscopy (SEM) images. Electrochemical detection was performed on disposable screen-printed electrodes (SPCEs) with a 3 mm diameter

Materials and Methods
The main fabrication of the sandwich-type electrochemical biosensor is the synthesis of Ti 3 C 2 MXenes, cubic Au/Pt DNs, and Au/Pt/Ti 3 C 2 MXenes, which we performed as described below.

Apparatus and Characterization
The X-ray diffraction (XRD) patterns were collected using a D8 ADVANCE X-ray diffractometer. The transmission electron microscopy (TEM) images were obtained with a JEOL 2010 transmission electron microscope. A JEOL 5160LV electron microscope was used to acquire scanning electron microscopy (SEM) images. Electrochemical detection was performed on disposable screen-printed electrodes (SPCEs) with a 3 mm diameter working area (Zensor research & development, Taiwan) connected with a CHI 660E workstation (Chenhua, Shanghai, China).

Cell Culture, Exosome Extraction, and Counting
HCT116 human colon cancer cell lines (HCT-1165FR) obtained from the American Type Culture Collection were maintained in MacCoy's 5A medium (Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS) (GIBCOBRL Laboratories, Grand Island, NY, USA) and 1% penicillomycin into DMEM in a humidified incubator at 37 • C in an atmosphere of 5% CO 2 (Thermo Scientific, Waltham, MA, USA) [31]. When the cell concentration reached about 70%, the culture medium was replaced with a serum-free medium. After 24 h, the cell medium was collected and exosomes were separated by performing differential centrifugation. The specific steps were as follows: The culture medium (300 mL) was centrifuged at 500× g (g represents gravitational acceleration) for 15 min, and the supernatant was collected. The supernatant was centrifuged at 2000× g for 20 min and filtrated by membrane. The precipitates were then centrifuged at 3000× g for 15 min twice and 100,000× g for 90 min, before being collected and dispersed in PBS solution. Exosomes were stored at −20 • C for further use. The particle size distribution of exosomes was measured with the Malvern particle size analyzer, and the results showed that the size of exosomes was about 120 nm. The concentration of exosomes was determined with the BCA method, and the results show that the concentration of exosomes was 2.2 × 10 9 exosomes /µL.

Synthesis of Ti3C2 MXenes
The multilayered Ti 3 C 2 T X was synthesized using the HF etching method according to a previously reported procedure with simple modification [32,33]. A total of 1 g of Ti 3 AlC 2 powder and 20 mL of HF (40 wt%) solution was mixed at 0 • C and stirred for 10 min. The above mixture was then stirred at 35 • C for 24 h until the reaction was completed. After that, the mixture was centrifuged at a speed of 8000 rpm and washed with deionized water until the pH was no less than 6. The precipitate Ti 3 C 2 MXene was dispersed in deionized water by performing ultrasound and freeze-dried for 12 h.

Preparing Au/Pt DNs
The Au/Pt DNs were prepared according to a previously published method [34]. Firstly, 10 mL of HAuCl 4 (0.25 mM) and CTAB (75 mM) aqueous solution were prepared in a flask, following which 0.6 mL of ice NaBH 4 (10 mM) solution was rapidly injected, and stirred slowly for 3 h. Then, the hydrosol prepared at 1 mL was diluted to 100 mL and used as the seed solution. The colorless mixture of 0.1 mL HAuCl 4 (10 mM), 2 mL CTAB (0.2 M), and 1.5 mL AA (ascorbic acid) (0.1 M) was diluted to 25 mL, and 0.3 mL of the prepared seed solution was added. The reaction mixture was shaken at room temperature for 8 h and a colloid containing pale purple cubic gold was obtained. Finally, AuPt DNs were prepared by adding 2 mL AA (0.1 M) and 1 mL H 2 PtCl 6 (10 mM) to the third gold solution, successively, and washed with ultra-pure water at 60 • C for 12 h.

Synthesis of Au/Pt/Ti 3 C 2 MXene
The Au/Pt/Ti 3 C 2 MXene nanocomposite was synthesized as follows [35]: Under ultrasonic conditions, Ti 3 C 2 MXene powder and Au/Pt powder were dispersed in DMF solution. Next, the DMF solution of Au/Pt and Ti 3 C 2 MXene were mixed together and ultrasonicated for 3 h. The mixed solution was further stirred for 20 h. The Au/Pt/Ti 3 C 2 MXene was collected by performing centrifugation, washed with DMF and ethanol, and then dried in vacuum at 60 • C for 12 h.

Preparation of Au/Pt/Ti 3 C 2 MXene Nanoprobe
A total of 5 mg of Gly was dissolved in 5 mL of deionized water and stirred at room temperature. Then, 10 mL 1.0 mg/ mL Au/Pt-Ti 3 C 2 MXene dispersion was added drop by drop, stirred at room temperature for 24 h, centrifuged for washing (12,000 rpm, 20 min), that is, Pt DNS-Ti 3 C 2 MXene-Gly was prepared. After 150 µL EDC (400 mM), NHS (100 mM) and 100 µL Aptamer (-COOH) mixtures were activated at 37 • C for 1 h, 200 µL Au/Pt DNS-Ti 3 C 2 MXene-St. Louis Gly was added to the above solution, and the reaction continued for 1 h at 37 • C. Centrifugal washing (1200, 10 min) was performed, as well as deionized water dispersion, for Au/Pt DNS-Ti 3 C 2 MXene-GLY-Apt. Figure 1 shows a schematic of representation of fabrication process of the electrochemical biosensor. This article uses disposable screen-printed electrodes (SPCEs) for detection. In total, 6 µL of GO (0.25 mg/L) was dropped on the cleaned SPCE surface to gain GO/SPCE. After being dried, the electrode was immersed in EDC/NHS(100 µM/300 µM), and incubated at 37 • C for 1 h [36]. The CD63 aptamer stock solution (100 µM) was mixed with 10 mM tris(2carboxyethyl) phosphine (TCEP, Sigma-Aldrich, St. Louis, MO, USA) for 1 h to cleave the disulfide bonds, and then 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES, Sigma-Aldrich, St. Louis, MO, USA) buffer was used to further dilute the CD63 aptamers to 1 µM. Then, 40 µL of the prepared solution of CD63 aptamers was placed on the microelectrodes for 2 h to obtain Apt/GO/SPCEs. After incubation, the Apt/GO/SPCEs were washed with deionized (DI) water. Next, 30 µL of 1 mM 6-mercapto-1-hexanol (MCH, Sigma-Aldrich, St. Louis, MO, USA) was added to the Apt/GO/SPCEs for 15 min. To remove the excess MCH, the Apt/GO/SPCEs were washed thoroughly with DI water [37]. The Apt/GO/SPCEs were immersed in the exosomes derived from colorectal cells at certain concentrations. Then, the exosomes/Apt/GO/SPCEs were incubated with MXenes-Apt for 2 h at 37 • C.

Results and Discussion
Electrochemical immunosensors have been a tool in the early monitoring and diagnosis of cancer. Our group demonstrated the use of this method as a label-free detection route of the cancer biomarker by dendritic tri-fan blade-like PdAuCu nanoparticles/aminefunctionalized graphene oxide [30]. In order to obtain a high sensitivity in the electrochemical immunosensors, trimetallic nanomaterials have broad applications due to their excellent properties, e.g., superior catalytic activity, better durability, and larger surface-active areas.
The major accomplishment of this work is detecting potential tumor-biomarker exosomes with nanomaterials of cubic AuPt dendritic nanocrystals/Ti 3 C 2 . Herein, the electrochemical immunosensor of cubic AuPt dendritic nanocrystals/Ti 3 C 2 -MXene are reported. The properties of the used materials are displayed first, and the characterization of the immunosensor was followed by its further application in a real sample.

Characterization of Ti 3 C 2 MXenes, Ti 3 C 2 MXenes, and Cubic AuPt DNs/Ti 3 C 2 MXenes
The morphological structure of the as-prepared MXene-Ti 3 C 2 T x and Ti 3 C 2 MXenes was analyzed using SEM. The SEM image of Ti 3 AlC 2 ( Figure 2A) shows a multilayered structure with a typical accordion-like morphology. Figure 2C shows the morphology of the prepared MXene-Ti 3 C 2 T x with thin and transparent nanoflakes stacked on each other. The layer spacing is wider than before the etching [24,25]. The structural characteristics of the as-prepared MXene-Ti 3 C 2 T x and Ti 3 C 2 MXenes composite structures were investigated by performing XRD analysis, as shown in Figure 2B. Visibly, following etching, the characteristic peak of Ti 3 AlC 2 at 39 • disappeared [26]. This proves that MXene-Ti 3 C 2 T x was successfully prepared. In the pattern of Ti 3 C 2 MXenes, the sharp peak located at ≈32 • can be assigned to Ti 3 C 2 T x , confirming the existence of Ti 3 C 2 T x support [22]. The high-resolution TEM (HRTEM) image reveals a highly crystalline lattice of the MXene basal plane without evident defects ( Figure 2D), which is consistent with a previous report on MXene nanosheets prepared using LiF-HCl etchant. Figure 3A shows the TEM images of cubic Au, indicating that the obtained Au NPs h formed a cubic morphology with an average size of 30 nm. Figure 3B shows the diffrac pattern of cubic Au, which further confirms the successful preparation of cubic Au. Fig  3C shows the TEM image of AuPt DNs, indicating that AuPt DNs form a highly unif dendrite form with a regular size. Figure 3D shows a visible modification of cubic A DNs on the Ti3C2 MXene lamellar, which proves the successful preparation of the com site materials. TEM was used to characterize Au, cubic AuPt DNs, and cubic AuPt-TI 3 C 2 MXene. Figure 3A shows the TEM images of cubic Au, indicating that the obtained Au NPs have formed a cubic morphology with an average size of 30 nm. Figure 3B shows the diffraction pattern of cubic Au, which further confirms the successful preparation of cubic Au. Figure 3C shows the TEM image of AuPt DNs, indicating that AuPt DNs form a highly uniform dendrite form with a regular size. Figure 3D shows a visible modification of cubic AuPt DNs on the Ti 3 C 2 MXene lamellar, which proves the successful preparation of the composite materials.

Electrochemical Characterizations of the Assembly Processes of the Biosensor
CV and EIS were used to study the modified process of the electrochemical biosensor. Figure 4A shows the CVs of [Fe(CN) 6 ] 3− /[Fe(CN) 6 ] 4− in PBS using the bare (curve a), GO modified (curve b), GO/Apt (curve c), GO/Apt/exosomes (curve d), and GO/Apt/exosome /Ti 3 C 2 -Apt-cubic AuPt DNs (curve e) modified SPCEs. The changes in the amperometric response correspond to the obstruction of the electron transfer kinetics of the Fe(CN) 6 3− /Fe(CN) 6 4− probe. An electrochemical signal was detected using bare SPCEs, as shown in curve a. When GO and Apt composite materials (curves b/c) are modified on SPCEs, the peak current after modification of GO and Apt gradually decreases due to the electronic inertness. After exosomes are loaded, due to the specific binding of exosomes and aptamers, the peak current is further reduced. Due to the electronic inertness of Apt and exosomes, the electron transfer of the substrate ions on the electrode interface is hindered. Therefore, after Ti 3 C 2 MXene modification, the peak current will also decrease. Due to the negative charge of GO, the transfer of the substrate at the electrode interface was blocked, resulting in a further reduction in the peak current.

Electrochemical Characterizations of the Assembly Processes of the Biosensor
CV and EIS were used to study the modified process of the electrochemical biosensor Figure 4A shows An electrochemical signal was detected using bare SPCEs, a shown in curve a. When GO and Apt composite materials (curves b/c) are modified on SPCEs, the peak current after modification of GO and Apt gradually decreases due to the electronic inertness. After exosomes are loaded, due to the specific binding of exosome and aptamers, the peak current is further reduced. Due to the electronic inertness of Ap and exosomes, the electron transfer of the substrate ions on the electrode interface is hin dered. Therefore, after Ti3C2 MXene modification, the peak current will also decrease. Du to the negative charge of GO, the transfer of the substrate at the electrode interface wa blocked, resulting in a further reduction in the peak current.
The EIS measurement was more sensitive in monitoring the changes in the interface features in the [Fe(CN)6] 3− /[Fe(CN)6] 4− probes system of the modified electrodes. Figure 4B is the EIS diagram of the modified electrode. The impedance spectrum of EIS is divided into two parts, a semicircular part and a linear part. The semicircle part represents the electron transfer process, and the diameter of the semicircle is equal to the electron trans fer resistance; the linear part corresponds to the diffusion process. After the electrode sur face is assembled, the resistance increases, and the obtained result corresponds to the CV diagram, which proves that the sensor is successfully assembled.

Optimization of the Detection Conditions
In order to obtain the best analytical performance of the biosensor, the influence of the incubation time of the exosomes, the concentration of Apt, and the concentration of KCL in the substrate were detected. It can be seen from Figure 5A that the performance of this biosensor is related to the incubation time of the exosomes. The optimal incubation time of the exosomes, as shown in Figure 5A, is 120 min, when the peak current is the largest. For this reason, a 120 min incubation time was used in the following experiments.
The concentration of Apt also affects the performance of this biosensor. As shown in Figure 5B, when the concentration of Apt increased from 0.6 µM to 1.0 µM, the peak current gradually increased and did not change drastically when the Apt concentration exceeded 1.0 µM. This result shows that when the concentration of Apt is under 1.0 µM, there is not enough Apt to combine the exosome; when the concentration of Apt is 1.0 µM, Apt reaches the point of saturation on the surface of the exosome, and the ability to combine exosomes is optimal. Therefore, 1.0 µM of Atp was used for electrochemical measurement [23].
In addition, the performance of the biosensor is also related to the concentration of KCL in the substrate ( Figure 5C). Before the concentration of KCL in the base solution is 0.10 M, the peak current increases, which is conducive to the transfer of electrons; however, if the concentration exceeds this, the peak current will appear to decrease, which is not conducive to the transfer of electrons. Therefore, 0.10 M KCL was used for the preparation of the biosensors.   Figure 4B is the EIS diagram of the modified electrode. The impedance spectrum of EIS is divided into two parts, a semicircular part and a linear part. The semicircle part represents the electron transfer process, and the diameter of the semicircle is equal to the electron transfer resistance; the linear part corresponds to the diffusion process. After the electrode surface is assembled, the resistance increases, and the obtained result corresponds to the CV diagram, which proves that the sensor is successfully assembled.

Optimization of the Detection Conditions
In order to obtain the best analytical performance of the biosensor, the influence of the incubation time of the exosomes, the concentration of Apt, and the concentration of KCL in the substrate were detected. It can be seen from Figure 5A that the performance of this biosensor is related to the incubation time of the exosomes. The optimal incubation time of the exosomes, as shown in Figure 5A, is 120 min, when the peak current is the largest. For this reason, a 120 min incubation time was used in the following experiments.
Apt reaches the point of saturation on the surface of the exosome, and the ability to combine exosomes is optimal. Therefore, 1.0 µM of Atp was used for electrochemical measurement [23].
In addition, the performance of the biosensor is also related to the concentration of KCL in the substrate ( Figure 5C). Before the concentration of KCL in the base solution is 0.10 M, the peak current increases, which is conducive to the transfer of electrons; however, if the concentration exceeds this, the peak current will appear to decrease, which is not conducive to the transfer of electrons. Therefore, 0.10 M KCL was used for the preparation of the biosensors. The concentration of Apt also affects the performance of this biosensor. As shown in Figure 5B, when the concentration of Apt increased from 0.6 µM to 1.0 µM, the peak current gradually increased and did not change drastically when the Apt concentration exceeded 1.0 µM. This result shows that when the concentration of Apt is under 1.0 µM, there is not enough Apt to combine the exosome; when the concentration of Apt is 1.0 µM, Apt reaches the point of saturation on the surface of the exosome, and the ability to combine exosomes is optimal. Therefore, 1.0 µM of Atp was used for electrochemical measurement [23].
In addition, the performance of the biosensor is also related to the concentration of KCL in the substrate ( Figure 5C). Before the concentration of KCL in the base solution is 0.10 M, the peak current increases, which is conducive to the transfer of electrons; however, if the concentration exceeds this, the peak current will appear to decrease, which is not conducive to the transfer of electrons. Therefore, 0.10 M KCL was used for the preparation of the biosensors.

The Sensitivity of the Biosensor
The designed electrochemical biosensor was used in this study strategy to detect exosomes through the DPV response curve under optimal conditions. Figure 6A illustrates the electrochemical signal intensity of the biosensor in different concentrations of colorectal cancer cell exosome solutions. These curves, from curve a to curve h in Figure 6A, represent the current responses of the as-prepared immunosensor incubated with different concentration of exosomes: 1.0 × 10 2 , 5.0 × 10 2 , 1.0 × 10 3 , 2.5 × 10 3 , 1.0 × 10 4 , 2.5 × 10 4 , 5.0 × 10 4 , and 5.0 × 10 5 exosomes µL −1 .
At the same time, it can be seen that the current response increases as the concentration of exosomes increased. Figure 6B shows the relationship between the logarithm of the exosome concentration and the current corresponding with what was shown in Figure 6A, and the peak current height was linear with the logarithm of the exosomes concentration in the range from 100 to 5.0 × 10 5 exosomes µL −1 . The linear regression equation is I = 1.726 × 10 −5 logC + 1.420 × 10 −5 (exosomes µL −1 ), and the correlation coefficient is 0.9971. The detection limit is 20 exosomes µL −1 (S/N = 3). These data show that this biosensor can be used to detect exosomes with high sensitivity. Table 1 shows the comparison between the proposed electrochemical biosensor and other technologies in the electrochemical detection of exosomes. These comparisons indicated that this method is significantly better than other similar methods. This explains that the designed biosensor has good signal amplification performance.
The designed electrochemical biosensor was used in this study strategy to detect exosomes through the DPV response curve under optimal conditions. Figure 6A illustrates the electrochemical signal intensity of the biosensor in different concentrations of colorectal cancer cell exosome solutions. These curves, from curve a to curve h in Figure 6A, represent the current responses of the as-prepared immunosensor incubated with different concentration of exosomes: 1.0 × 10 2 , 5.0 × 10 2 , 1.0 × 10 3 , 2.5 × 10 3 , 1.0 × 10 4 , 2.5 × 10 4 , 5.0 × 10 4 , and 5.0 × 10 5 exosomes µL −1 . At the same time, it can be seen that the current response increases as the concentration of exosomes increased. Figure 6B shows the relationship between the logarithm of the exosome concentration and the current corresponding with what was shown in Figure  6A, and the peak current height was linear with the logarithm of the exosomes concentration in the range from 100 to 5.0 × 10 5 exosomes µL −1 . The linear regression equation is I = 1.726 × 10 −5 logC + 1.420 × 10 −5 (exosomes µL −1 ), and the correlation coefficient is 0.9971. The detection limit is 20 exosomes µL −1 (S/N = 3). These data show that this biosensor can be used to detect exosomes with high sensitivity. Table 1 shows the comparison between the proposed electrochemical biosensor and other technologies in the electrochemical detection of exosomes. These comparisons indicated that this method is significantly better than other similar methods. This explains that the designed biosensor has good signal amplification performance.

Specificity and Reproducibility of the Sensor
Specificity is an important parameter of electrochemical biosensors. The specificity between aptamers and exosomes was analyzed. As shown in Figure 7A, the peak current without exosomes and random Apt is lower than under normal conditions. This is because the colorectal cancer exosomes are incubated in the exosome solution, so that the exosomes are captured on the electrode; therefore, more cubic AuPt DNs-Ti 3 C 2 -Apt nanoprobes can be modified on the electrode to generate a larger current signal. At the same time, it can also be attributed to the modification of the CD63 aptamer on the electrode to capture more exosomes, so that a large number of cubic AuPt DNs-Ti 3 C 2 -Apt nanoprobes are modified on the electrode to generate a stronger current. These results show that this biosensor has good specificity. Figure 7B shows the reproducibility test of the immunosensor. Specifically, five immunosensors fabricated independently were applied to detect the target exosome (5.0 × 10 3 exosomes µL −1 ) under the optimized conditions. The relative standard deviation (RSD) was approximately 2.8%, displaying acceptable reproducibility.

Detection of Cancerous Exosomes Isolated from Human Serum Samples
In order to investigate the practical application of the proposed electrochemical biosensor in biological samples, different concentrations of colorectal-derived exosomes were added to the serum for measurement. The experimental results detected are shown in Table 2, and the recoveries were in the range of 98-108%. This indicates that the developed electrochemical sensor has high accuracy in the detection of exosomes in complex samples.
time, it can also be attributed to the modification of the CD63 aptamer on the electr capture more exosomes, so that a large number of cubic AuPt DNs-Ti3C2-Apt nano are modified on the electrode to generate a stronger current. These results show th biosensor has good specificity. Figure 7B shows the reproducibility test of the immu sor. Specifically, five immunosensors fabricated independently were applied to det target exosome (5.0 × 10 3 exosomes µL −1 ) under the optimized conditions. The r standard deviation (RSD) was approximately 2.8%, displaying acceptable reproduc

Detection of Cancerous Exosomes Isolated from Human Serum Samples
In order to investigate the practical application of the proposed electrochemic sensor in biological samples, different concentrations of colorectal-derived exosome added to the serum for measurement. The experimental results detected are shown ble 2, and the recoveries were in the range of 98-108%. This indicates that the dev electrochemical sensor has high accuracy in the detection of exosomes in comple ples.

Conclusions
In summary, an MXene electrochemical biosensor platform based on cubic DNs-Ti3C2 MXene was built for the detection of exosomes using an aptamer-assiste

Conclusions
In summary, an MXene electrochemical biosensor platform based on cubic AuPt DNs-Ti 3 C 2 MXene was built for the detection of exosomes using an aptamer-assisted amplification strategy. The GO/SPEC interface modified by the CD63 aptamer can specifically bind to the CD63 protein on the surface of exosomes to improve their capture ability. Ti 3 C 2 can specifically bind to a large number of CD63 aptamers and can efficiently recognize exosomes and serve as nanocarriers. The cubic AuPt DNs-Ti 3 C 2 MXene-Apt hybrid also improves electrocatalytic activity. The fabricated electrode shows a wider linear detection range of 100~5.0 × 10 5 exosomes µL −1 with a limit of detection of as low as 20 exosomes µL −1 . In addition, this method can also be effectively applied to detect exosomes in complex serum samples. It is believed that the design of the functionalized MXene membranes expands possibilities for the implementation of 2D membrane platforms for reliable biomarker diagnosis and for the monitoring of clinical applications, such as tumor cell-promoting miRNA detection, tumor cell/exosome enrichment, and drug screening.  Data Availability Statement: The data of this work are available from the corresponding author upon reasonable request.

Conflicts of Interest:
The authors declare no conflict of interest.