Review—Prospects in Cancer Diagnosis: Exosome-Chip for Liquid Biopsy

A liquid biopsy combined with an exosome-chip (EC) is an important detection tool for early cancer diagnosis. Exosomes have a crucial function in the exchange of information between cells and are present in biological fluids. ECs are miniaturized microfluidic devices designed to isolate, capture, and analyze exosomes for analysis of patient samples. Such devices offer on-chip detection, high-throughput analysis, and multiplex measurements. Further, these chips can integrate with electrochemical and optical detectors, and mass spectrometry enabling comprehensive studies of diseases. This review will cover the outlook on chip-based diagnostics for liquid biopsy, detection, and isolation of exosomes to support cancer diagnostics.

Exosomes are extracellular nanovesicles and are actively released by the majority of living cells inside multivesicular endosomes through the creation of intraluminal vesicles. 1They have nanometerscaled size (50-100 nm in diameter), and exhibit a buoyant density ranging from 1.13 to 1.19 g ml −1 . 2 In 1997, exosomes were first discovered as an RNase in the budding yeast Saccharomyces cerevisiae and then were identified in a diverse array of mammalian cell classes. 3,4The core of the exosome comprises a ring structure of six proteins that belong to a similar class of RNases. 5Exosomes are released by all living cells into biological fluids including body plasma, urine, cerebrospinal fluid, and saliva, which are predominantly rich in membrane proteins. 6Exosome contains (ribonucleic acid) RNA species such as messenger RNA, microRNA, and other RNA's, lipid, and cell-specific proteins.][9][10] They significantly contribute to facilitating intercellular communication and the exchange of substrates, often inducing physiological alternates in target cells by transferring lipids, proteins, and nucleic acids.MicroRNA in exosomes can serve as a shuttle to transfer genetic materials between cells and regulate the gene expression to the target cells.Though many studies confirm that exosomes can interact with target cells, [11][12][13] but the mechanisn is still unclear.
Analyzing exosomes holds promise as a potential method for early cancer screening, providing valuable insights into the genetic makeup of tumors. 14This approach negates the need for invasive biopsies and holds promise for therapeutic applications. 157][18][19] Exosomes originating from malignant tumor cells are pivotal in shaping the tumor environment and facilitating the formation of pre-metastatic niches. 20Notably, cancerous cells tend to release a higher quantity of exosomes bearing specific biomarkers on their surface compared to normal cells. 21Consequently, tumor-derived exosomes serve as a crucial biomarker for both the analysis and detection of cancer at different stages, from a pathological to clinical standpoint.Also, recent studies have established the utility of exosomes for cancer detection before and after treatment, 12,22 where tumor-specific exosomes can provide a specific signature of transmembrane protein, 23 and changes in exosomal protein may correlate after treatment.
Liquid biopsy has significant benefits compared to traditional tumor biopsies. 24Body fluids including serum and plasma from cancer patients or urine, bronchoalveolar lavage fluid, synovial fluid, pleural effusions, amniotic fluid, saliva, etc. are widely employed for liquid biopsy. 25Liquid biopsy was established to define circulating tumor cells, exosomes, and DNA present in the bloodstream of individuals with cancer. 26,27In addition, membranous extracellular vesicles, including nanoscale exosomes (<50 nm) and other vesicles actively secreted from cancerous cells, have been identified in cancer bloodstream.Further, liquid biopsy offers rapid screening, direct detection, tracking treatment response for cancer patients, and detection of minimal residual after surgery.Exosomes have emerged as a new type of biomarker that will offer potential merits for liquid biopsy of cancer cells using biofluids. 28Being less costly and less risky than conventional tissue biopsies, a liquid biopsy can be performed much more frequently to provide up-to-date information about how a patient's cancer might be changing.Yoshioka et al., have developed a highly sensitive liquid biopsy method utilizing an ExoScreen tool to analyze circulating extracellular vesicles in patients with colorectal cancer. 29It has been demonstrated that ExoScreen can be finished off within 2 h and requires a low amount (5 ml) of serum samples whereas the conventional method required 12 h to distinguish the presence of proteins in circulating extracellular vesicles and immoderate volume of serum samples are needed. 29,30rotein biomarkers are invaluable tools for comprehending a wide range of diseases and finding applications in analytical epidemiology, screening, diagnosis, and prognosis. 313][34][35][36] However, these methods often encounter issues related to specificity, accuracy, reliability, extended processing times, and high rates of false positives, primarily due to the nonspecific binding of protein biomarkers.In contrast, exosomes offer distinctive advantages for ongoing monitoring and enable selective detection because of their unique surface markers that can be targeted by antibodies and their characteristic transmembrane protein signature. 37Various techniques have been employed for the detection of exosomes in biofluids.Nevertheless, due to the minuscule size of exosome nanoparticles, quantification poses a challenge.For instance, z E-mail: kamilreza@gmail.comand kamil.reza@woxen.edu.in;azahar@vt.edu9][40] Other molecular assay methods, such as ELISA and western blot, often require substantial sample volumes and pose constraints regarding available specimens in biorepositories, making them impractical for clinical research needs.To address these limitations, recent advancements in sensor technologies based on electrochemical and optical detection mechanisms have been leveraged for the rapid, on-site quantification of exosomes in bodily fluids.6][47] In the context of exosomes, lab-on-a-chip technology can isolate, capture, and analyze these minute circulating vesicles directly from cancer patient samples.The microfluidic channels of the chip allow us for in situ analysis of the exosome samples from patients to understand the complexity of disease by investigating the clinical sample in a dynamic state.The concept of an exosome-chip (EC) is quite new to the lab-on-a-chip technology.Previous reviews [48][49][50] have shown the emergence of EC as an innovative platform for exosome analysis providing versatile operations (screening, monitoring, detection) to be carried out on a single chip.Researchers have designed and fabricated various ECs to gain precious insights into diseases and potentially develop new diagnostic tools and treatments.This new generation ECs can transform healthcare diagnostics by providing precise exosome-based testing that provides vital information on cancer disease, and neurodegenerative disease, neuropsychiatric disorders. 48n this review, we cover the current scenario of exosome detection from research to clinical diagnosis for cancer or other diseases.Exosomes are excellent biomarkers for early cancer diagnostics.In addition, this review covers an overview of isolation, separation, and various detection methods of exosomes for cancer cells encompassing prognostic evaluation as well as the early identification of disease occurrence.It will also cover the recent advancement in point-of-care devices for the detection of exosomes and their challenges.Figure 1 depicts some of the current and future advancements in EC technology.The future of EC technology has great potential as the integration of Artificial Intelligence (AI) and Machine Learning (ML) will revolutionize this field by providing rapid and precise exosome data analysis of individuals.These technologies can extract valuable information from exosomes about heterogeneity, shape, size, etc. as biomarkers by analyzing complex patterns of data that current technology fails to provide.AI-enabled ECs can significantly improve the treatment strategy for a new age of personalized medicine and provide a smart healthcare system.

State-Of-The-Art-Detection Of Exosomes
Modern exosome detection systems utilize cutting-edge microfluidic and nanotechnology systems for multiplexed investigation of these tiny vesicles.This cutting-edge chip-based technology provides insights into the compositions, shapes, sizes, functions, and prospective diagnostic uses of exosomes through high-resolution imaging and single-particle tracking.Here, we discuss some of the state-of-the-art systems for exosome analysis.
There are various methods developed to isolate, detect, and characterize exosomes for disease screening, monitoring, and diagnostics.The morphology and size distribution of exosomes can be used as distinguishing factors.Some of the well-known techniques are western blot assay, enzyme-linked immunosorbent assay (ELISA), nanoparticle tracking analysis (NTA), etc. [49][50][51][52][53][54][55][56][57][58] Apart from that, transmission electron microscopy (TEM) and scanning electron microscopy (SEM) have provided high-resolution images of exosomes to distinguish them from other extracellular vesicles. 59urther, ECs have been explored by researchers to design versatile platforms for multiple analyses of exosomes.They could also provide morphology as well as size distribution of the exosomes for better clarity of extracellular vesicles for clinical application.
ELISA is one of the robust techniques to identify and confirm the protein receptors present in the exosomes. 60These immunoassays use specific antibodies against exosome surface markers to capture and quantify exosomes for high-throughput screening and analysis.Another established technique is known as Western blot assay which is used to confirm the exosome samples containing the protein associated with the cell.Then the protein concentration extracted from the exosome is determined by a protein assay kit. 61Similarly, flow cytometry is another widespread technique for cell and exosome analysis.Multiple fluorescents labeled antibodies can be used to identify and quantify exosomes based on their surface proteins to determine the heterogeneity of the biomarkers for disease identification.
Exosomes can also be identified by NTA which is generally used for nanoparticle tracking. 62It uses laser light scattering to track and analyze the Brownian motion of the exosomes providing real-time information on the concentration and size distribution of exosomes.4][65] This optical system works on the principle of total internal reflection which generates plasmons on the gold surface due to light-metal interaction causing a change in refractive index in the solution.This gold sensor chip will detect and quantify exosomes by monitoring changes in the refractive index upon capturing exosomes on a gold chip.
Microfluidic chip-based system has provided a versatile platform for exosome isolation, detection, and examination for clinical application.Some of the well-known chip-based methods developed for exosome analysis are viscoelastic flow sorting, acoustic nanofiltration, membrane-based filtration, immunoaffinity, trapping on nanowires, and deterministic lateral displacement (DLD) etc. 67 The purpose of developing these microfluidic systems is to achieve miniaturization, automation, and precise management of fluid flow, catering to exosome isolation and integrated analysis devices.Trau et al. introduced a SERS-based microfluidic chip for multiple exosome analyses from clinical samples to extract information for enhancing therapeutic outcomes and mitigating the advancement of drug resistance.(Fig. 2). 66Furthermore, the researchers successfully extracted exosomes from samples of melanoma cancer patients.They then conducted a comprehensive analysis of multiple exosomes to gain insights into their heterogeneity and diverse phenotypic reactions to treatment.To minimize nonspecific adsorption, a nanomixing strategy was employed, which proves especially advantageous when capturing exosomes directly from complex biological samples. 66Table I shows a list of different ECs with the sensing performances.
There are a few examples of exosome detection by using electrochemical sensor technology for early cancer diagnosis. 68,69 compact, eight-channel electrochemical sensor based on magneto--electrochemical assay was developed for efficient and real-time screening of exosomes at the site of collection. 68In this sensor, the exosomes were anchored on magnetic beads surface (immunomagnetically) which were conjugated with horseradish peroxidase (HRP) enzyme, and beads were immobilized with antibodies (anti-CD63) specific to CD63 antigen (exosome marker).This electrochemical technology was able to detect extracellular vesicles in ovarian cancer patients' specimens.In addition, this sensor enabled the concurrent profiling of numerous biomarkers, overcoming the limitations of conventional assays in terms of limit-of-detection (LOD) and sensitivity.An electrochemical micro-aptasensor was developed for the early detection of exosomes from lung cancer patients by integrating a micropatterned electrode geometry and dual amplification. 69This sensor provided a linear range of exosomes ECS Sensors Plus, 2023 2 043403 spanning from 2.5 × 10 3 to 1 × 10 7 exosomes ml −1 and a LOD of 5 × 10 2 exosomes ml −1 .A cost-effective, electrochemical biosensor based on paper was developed to detect exosomes derived from ovarian cancer cells in cell culture media.This sensor consists of a sandwich-type immunoassay wherein the exosomes are captured by the electrode-bound anti-CD9 antibodies and subsequently identified through the presence of CA125, which is derived from ovarian cancer cells.This sensor provided a LOD of 7.1 × 10 8 exosomes ml −1 . 70Unlike optical sensing, electrochemical sensing is a low-cost modality for the sensitive detection of exosomes owing to its considerable signal-to-noise ratio.Thus, electrochemical sensor technology could be an alternative point-of-care diagnostic tool for rapid examination of disease-specific exosomes in blood serum or other samples. 71y Exosome-Chips are Important?
EC plays an important role in biomedical research due to its versatility.They are capable of rapid and efficient isolation, profiling, and analysis of exosomes from biofluids.Scientists are exploring their potential to uncover various functions of the human body as valuable biomarkers, and early disease detection mechanisms, and advance our understanding of intercellular communication and disease mechanisms.
There are several advantages of ECs.EC-enhanced liquid biopsies may be able to identify diseases at an early stage when conventional imaging or clinical tests may not be sensitive enough. 79,80Through prompt intervention and the beginning of treatment, this early discovery can greatly enhance patient outcomes.These chips enable longitudinal monitoring of illness development, treatment response, and the formation of medication resistance or disease recurrence by the recurrent sampling of body fluids over time. 72,81This can offer insightful information for personalized medicine and treatment modifications.Further, ECs for liquid biopsies replace the need for risky and occasionally uncomfortable invasive tissue biopsies.Instead, the information can be obtained with a straightforward blood, urine, or saliva sample collection.Exosome technology is at a nascent stage with a lot of promise towards understanding disease biology, enabling early diagnostics, and developing targeted therapies for personalized medicine.However, ECs also have limitations.Some of the major limitations are the liquid biopsy sample complexity as they contain various extracellular vesicles, particles of similar size to exosomes that pose a huge challenge for isolation efficiency, low detection sensitivity (ultra-low concentration in the early stage of cancer), heterogeneous nature in size, surface markers, cargo, high cost of chips, less clinical testing of the patient sample, and so on.

Exosomes Isolation
There are several challenges for exosome isolation from body fluids due to its complexity in composition and functions.The composition of biofluids, such as blood or urine, can significantly impact the performance of detection techniques, especially affinitybased capture methods.The matrix effect refers to the impact of the complex mixture of proteins, lipids, nucleic acids, and other components present in biofluids on the accuracy and reliability of detection methods.The presence of these components can lead to various challenges, including, interference of molecules in biofluids that can lead to non-specific binding, interfering with the intended interaction between the target exosome biomarkers and the capture molecules, resulting in false positives or false negatives.Also, nonaffinity-based biosensors which are based on physicochemical properties, structural changes, or other non-binding interactions face similar challenges.Therefore, when designing and optimizing detection systems, it is crucial to consider the effect of matrix in both affinity-based and non-affinity-based biosensors.Additionally, background noise can create a high baseline signal, making it difficult to distinguish the signal from the target exosome biomarkers, and can impact the sensitivity of the measurement. 82Consequently, addressing the matrix effect is crucial for improving the accuracy, specificity, and reliability of exosome detection methods.Sample preparation and pre-processing steps are essential stages for exosome isolation for affinity and non-affinity-based biosensors.Generally, the common stages are centrifugation, filtration, or microfluidic platforms to remove or reduce interfering substances, such as proteins and lipids, from the biofluid samples (1-2).Furthermore, the integration of antibodies or aptamers can enhance the selectivity of the detection method, reducing the chances of false positives (3).Sometimes blocking agents can also reduce the interfering effects.Overall, a combination of careful sample preparation, tailored capture molecules, and innovative signal processing approaches is crucial in mitigating the matrix effect and enhancing the reliability of exosome detection methods in biofluids.4][85] Therefore, extracting exosomes from clinical fluids can heighten the sensitivity of biomarker amplification and prevent unreliable outcomes.Conventional techniques including ultracentrifugation and densitygradient separation can segregate exosomes according to exosomes' buoyant density and size from clinical biofluids. 86To effectively eliminate all larger entities, such as dead cells with a diameter exceeding 1 μm, the ultracentrifugation method employs a sequence of differential centrifugation steps at speeds of up to 200,000 × g.Nevertheless, this method has certain limitations.It does not yield highly pure isolates, and the process itself is time-consuming, typically taking 4-5 h.Additionally, it requires expensive equipment and has a relatively low recovery yield, ranging from 5% to 25%. 87he density-gradient separation provides an improved recovery rate and pureness over the ultracentrifugation.In the density-gradient separation method, samples are initially subjected to centrifugation in the presence of a viscous material with a density gradient.During this process, exosomes separate based on their isopycnic point, leading to their isolation.Nevertheless, this method is unable to effectively distinguish viruses' exosomes due to their similar buoyant densities.A schematic representation for exosome isolation using a ciliated micropillar array (Fig. 3).Recently, commercially available kits such as ExoQuick™ as well as Total Exosome Isolation™ were employed to isolate exosomes.Though these kits require only one or two steps and offer easy operation without using expensive equipment, they require an overnight incubation step.
Isolating exosomes from different cell sources, such as normal cells and cancer cells, can provide valuable insights into their distinct characteristics and potential diagnostic or therapeutic applications.Microfluidic systems offer a precise and efficient way to isolate exosomes based on their unique functionalities.Microfluidic devices are engineered systems that manipulate small volumes of fluids, such as blood or cell culture media, within microscale channels.They allow for precise control over fluid flow, enabling the separation and isolation of particles, including exosomes, based on their size, surface markers, and other properties.Exosomes from normal cells and cancer cells can exhibit differences in their surface protein markers, cargo contents, and lipid compositions.These differences form the basis for their separation using microfluidic systems.Exosomes carry distinct surface proteins that reflect their cell of origin.For instance, exosomes from cancer cells might express tumor-specific antigens or markers associated with oncogenic processes.Microfluidic devices can be functionalized with antibodies targeting these specific markers.By flowing the exosome-containing sample through these devices, exosomes with the desired markers can be selectively captured onto the device's surfaces.Additionally, exosomes contain various molecules such as nucleic acids, proteins, and lipids.In the case of cancer cells, exosomes may carry specific biomolecules associated with tumor progression or metastasis.Microfluidic systems can exploit these unique cargoes for isolation.As an example, certain microfluidic designs incorporate affinity-based capture mechanisms, where ligands specific to cancer-related exosome cargoes are immobilized on the channel walls.When the sample flows through the device, cancer-derived exosomes bind to these ligands, allowing other components to be washed away.Besides, exosome membranes are enriched with specific phospholipids that can vary based on the cell source.Microfluidic systems can exploit these lipid differences by incorporating lipid-binding moieties on the device's surface.This enables selective adhesion of exosomes with specific lipid compositions, aiding in their isolation. 88,89n affinity-based microfluidic device was developed to isolate exosomes. 90In this device, the microfluidic chamber has a dimension of 19 mm × 4.5 cm × 20 μm (H× L × W) along with 10 μm deep herringbone groves which increases the contact between the chip and the surface microparticles.After separation, the collected exosome particles are washed and characterized.In addition , an Exochip was developed to separate exosome particles.The device was constructed using a polydimethylsiloxane (PDMS) elastomeric material as well as incorporated functionalized antibodies specific to Figure 3.A schematic representation for exosome isolation using a ciliated micropillar array.This array is formed by a porous silicon nanowire-on-micropillar.A micropillar array used filter proteins, and cell debris from a liquid sample while exosomes were trapped within nanowires.Reproduced with permission. 88S Sensors Plus, 2023 2 043403 CD63, an antigen commonly found in exosomes.Additionally, a fluorescent carbocyanine dye was employed as part of the device design. 81These ExoChips are excellent platforms for exosomebased diagnostic tools for human cancer molecular investigation and screening.
The isolation and analysis of exosomes have a range of clinical applications in contemporary cancer treatment.These include earlystage disease diagnosis, precision therapy, and treatment monitoring.This is primarily due to the high abundance of exosomes in bodily fluids, their accessibility through liquid biopsy, and the presence of nucleic acid and protein cargo derived from their specific cell of origin. 91There are quite a few techniques available to isolate or separate exosomes from complex biological fluids for liquid biopsy application.These separation techniques are based on certain factors such as sample volume, high purity, scalability requirements, and downstream analysis of the sample.
As defined above, ultracentrifugation stands as a widely employed method for the purification and isolation of exosomes.In this process, samples undergo high centrifugal forces, typically ranging from 100,000 × g to 1,000,000 × g. 92 This results in the formation of exosome pellets based on their sedimentation characteristics.The method involves a series of sequential centrifugation steps, each at an increasing speed, aimed at pelleting exosomes while eliminating cell debris and larger vesicles.Size-exclusion chromatography (SEC) 93 represents an alternative technique that capitalizes on the disparities in size between exosomes and other particles, allowing for their separation from complex biofluids.SEC employs a column packed with a gel containing porous material.Large molecules, unable to traverse the pores in the packing material, move through the column more swiftly and are the first to be eluted.In contrast, exosomes, being smaller in size, become trapped within the pores, which slows down their movement.Consequently, exosomes are separated based on their size using this approach.
Immunocapture techniques rely on the use of specific antibodies to capture, identify, and measure extracellular vesicles, including exosomes.This method has demonstrated remarkable efficacy in screening, diagnosing, and even predicting the progression of tumors in samples collected from cancer patients.In this approach, specific antibodies are affixed to solid surfaces, such as magnetic beads or plates.This immobilization allows exosomes to bind to the antibodies, serving as surface markers, and selectively capturing exosomes from patient samples.As highlighted by Logozzi et al., the benefits of this method extend beyond its adaptability to enable the concurrent assessment of multiple markers expressed on exosomes derived from diverse patient samples.These advantages also encompass cost-effectiveness and ease of implementation in nearly all clinical and biological laboratories worldwide. 525][96] Sizebased filtration, affinity-based capture, and label-free isolation are some of the concepts being explored on chip technology for highthroughput and high-purity separation of exosomes.Chip-based technology is specifically beneficial for seamless integration with other detection systems for downstream analysis.
All the above-mentioned techniques have their pros and cons for exosome separation.However, each method has its advantages, limitations, and considerations for specific applications.Researchers are exploring and designing new separation techniques to tackle these challenges and trying to enhance the output and pureness of the isolated exosomes.Some of the common technical challenges faced by scientists in exosome separation are expensive instruments with complex analysis, time-consuming, repetitive procedures, large sample volume, and low yield.One of the biggest challenges for the exosomes is addressing the heterogeneity within exosome populations. 97These subtypes of exosomes are mainly due to the difference in size, functionality, biological origin, surface markers, cargo, and other aspects. 98,99The presence of an impurity in exosome samples is very common as other biological analytes such as cell debris, proteins, lipoproteins, nucleic acids, etc are hard to separate.Even goldstandard techniques inlcuding ultracentrifugation cannot eliminate the impurities completely.Therefore, the removal of non-specific analytes is very crucial for exosome isolation. 100Exosomes have similar characteristics such as microvesicles and apoptotic bodies, which poses challenges to achieving specificity and selectivity of the exosome sample.This is mainly due to the resemblance in size as well as the occurrence of analogous surface markers in all these vehicles which is difficult to extract pure exosome samples. 101nother issue in exosome isolation is its reproducibility 94 due to the dearth of standard protocols to maintain the quality control of clinical samples (blood, urine, saliva, and other body fluids).There is an abundant presence of proteins, nucleic acids, cell components, lipids, etc in plasma, serum, and urine samples of the patients which makes it a very challenging task to design a specific isolation protocol for exosomes.Apart from that, a few other challenges reported by the researchers are scalability issues for large exosome sample analysis along with preservation of the isolated sample for future analysis.
These exosomes provide myriad biological information including proteins, nucleic acids, and other biomolecules derived from their original cells. 17Without intrusive procedures like traditional tissue biopsies, it is feasible to learn more about the disease condition by extracting and analyzing exosomes from liquid samples using an EC.Exosomes from unhealthy cells, such as cancer cells or cells damaged by other diseases, can be found and examined on the chip for signs of disease.The exosomal payload, which may include proteins, nucleic acids, or other biomolecules, can be captured, and profiled by the chip to reveal details about the presence and features of the disease.Tumor heterogeneity is one of the biggest challenges in deciding disease diagnostics. 102Tumors frequently demonstrate tumor heterogeneity, with various tumor areas exhibiting different genetic mutations or protein expressions.ECs, which collect exosomes from biofluids, can offer a more thorough depiction of tumor heterogeneity than conventional biopsies, which are frequently restricted to a single tissue sample.The major advantages of ECs are as follows.(A)ECscan provide a rapid, simple, highpurity, and precise way for exosome analysis.(B)ECscan be used to track the effects of treatment over time.Real-time analysis of exosome cargo which act as carriers of biologically relevant molecules, including proteins and genetic material, can be indicative of treatment outcomes.Scientists can evaluate dynamic changes in the exosome cargo composition by analyzing EC at different time points during treatment. 103,104For example, the slight variations in specific proteins or genetic material associated with therapeutic targets or drug resistance mechanisms of any cancer patient exosome can provide the changes observed during treatment.(C)Further,exosome-based liquid biopsies can provide a less invasive option to conventional tissue biopsies as it will be a more practical and accommodating method for gathering diagnostic data.
The presence of such diverse biological information in exosomes presents an intriguing and cost-effective challenge for microfluidic technology.It also highlights the need for improved capabilities in terms of purity, diagnostic, and point-of-care acquisition.This review serves as a valuable resource for microfluidic experts, providing them with the opportunity to learn about this new class of biomarker-rich particles and the challenges associated with exosome enrichment.Additionally, biologists and clinicians familiar with exosome enrichment can evaluate the performance of novel microfluidic devices through this review.
Finally, it is noteworthy to mention that the exosome isolation from the complex environment of extracellular vesicles and bioparticles present in liquid biopsy samples is a difficult task.Due to the samples' heterogeneity, an isolation method that may selectively target exosomes while rejecting other vesicles and particles must be extremely specialized and effective.The similarity in size and surface characteristics of different extracellular components adds ECS Sensors Plus, 2023 2 043403 to this difficulty, necessitating creative approaches to identify exosomes in this complex environment.Exosome research and clinical diagnostics continue to face substantial challenges in the development of isolation techniques that guarantee high purity, yield, and repeatability while considering the inherently complicated nature of liquid biopsy samples.Therefore, a multidisciplinary strategy including expertise in cancer biology, biochemistry, microfluidics, and data analysis is necessary to address this challenge.More durable and dependable isolation techniques are anticipated to appear as technology develops and our understanding of exosomes grows.

Various High-Performance ECs for Exosome Analysis
Various high-performance ECs have emerged as powerful tools for exosome analysis.Such methods offer exceptional sensitivity, specificity, and scalability, making them invaluable for biomarker discovery, disease monitoring, and personalized medicine applications.6][107] These devices are highly efficient as they provide quick and automated sample processing, cutting down analysis time and enabling highthroughput screening.Moreover, the easy integration of DLD devices with various characterization systems is gaining attention as a multiple detection platform for exosome analysis. 108The DLD device consists of a fluidic platform that separates analytes based on their size and shape.The DLD chip is generally fabricated of micro or nanopillars or similar structures within a specific pattern inside a micro or nanochannel.The analytes which consist of exosomes, cell debris, nucleic acids, etc, have different shapes and sizes and are introduced in the channel using a combination of deterministic and inertial effects.Once the exosomes are inside the channel, they experience a hydrodynamic force that deflects them for a specific trajectory for separation from other analytes.Hydrodynamic force generates more force on the larger analytes like cell debris, and other vesicles deflecting them more from the main channel while exosomes being smaller in size are deflected less from the pillars.The exosomes get attracted to the pillars due to the size difference and fluid dynamics of the particles.The separated and extracted exosomes can be further utilized for downstream analysis involving a variety of characterizing techniques such as a nanoparticle tracking system for size distribution, a fluorescence-based technique for protein concentration, etc.In this way by integrating DLD platforms  91 (a) The schematic of the DLD chip features a pillar array with a specific gap size denoted as G.The pitch of the pillars, represented by λ, is approximately 400 nm.The row-to-row shift is indicated by δ, while the maximum geometric angle is denoted as θ max .The flow of particles within the chip is influenced by the array's geometry as well as the diameter of the particles being sorted.(b) A cross-sectional SEM image shows the pillar height, 1 μm, and gap, 225 nm (scale bar ∼400 nm).(c) The integrated nanoDLD device with zigzag outlet and SEM images of separated particles.Adapted with terms and conditions. 91S Sensors Plus, 2023 2 043403 with other analysis systems, we can extract information from exosomes to understand disease conditions.In their study, Wunsch et al. developed nanoscale deterministic lateral displacement (nano-DLD) arrays with consistent gap sizes ranging from 25 to 235 nm.These arrays were designed to sort exosomes based on both size and surface markers.The goal was to enable on-chip sorting and quantification of exosomes directly from biological samples.(Fig. 4). 109They demonstrated that this nano-DLD arrays-based chip can separate particle sizes from 20 to 110 nm with sharp resolution.In a similar vein, Smith et al. made further advancements by developing a system that incorporated nano-DLD arrays onto a single chip.This technology was specifically designed to separate exosomes from serum and urine samples. 91The isolated exosomes were analyzed for RNA sequencing obtained from patient serum samples for understanding the gene expression in prostate cancer.
Recently, graphene-based point-of-care devices are being explored for patient sample analysis.Yin et al. reported a graphenebased field effect sensor (GFET) platform for accurate and robust detection of pancreatic ductal adenocarcinoma (PDAC) in plasma samples obtained from patients through specific exosomes (GPC-1 expression).This new sensor consists of GFET sensor arrays with liquid gate electrodes integrated into the chip.A portable read-in/out electronic system was built to measure the real-time electrical response from the GFET sensors.This portable sensor can detect exosomes from plasma samples using a 20 μl drop within 45 min.Authors claimed that GFET technology has the potential to differentiate between exosome samples obtained from healthy individuals and those with PDAC patients.They noticed that cancer-derived exosomes significantly bind to the graphene sensor surface as compared to healthy exosome samples of the same volume.For the clinical validity of the work, they compared their GFET results with standard non-invasive tools like magnetic resonance imaging (MRI) and computer tomography (CT) scan data for early-stage pancreas cancer detection.Figure 5 describes the GFET sensor for point-of-care detection of pancreatic cancerous exosomes in patient plasma and demonstrates the test results in realtime display within 1 h.We need more such work in medical diagnostics for early-stage screening of cancer to reduce the mortality rate of the cancer.This portable sensor could replace the gold standard in cancer diagnostics as it demonstrated clinical testing in plasma samples. 110ne of the interesting works for multiple exosome detection in clinical patient samples has been investigated by Zhang et al.The exosome profiling platform (ExoProfile chip) was fabricated with the 3D nanostructures of patterned colloidal self-assembly for exosome immunophenotyping.This ExoProfile chip is multiplex and allows multiple analyte detection.The validation of this chip was realized by purified exosomes from ovarian cancer cell line (SKOV3) and simultaneously detecting eight exosome biomarkers.The whole experiment and analysis were performed within 3 h using only 10 μl ovarian cancer plasma.The circulating exosomes such as CA125, EGFR, CD24, HER2, FRα, EpCAM, and CD9 + CD63 were profiled from a number of plasma samples (∼15 ovarian cancer patients) including 5 controls (benign).Importantly, they were able to distinguish seven exosome biomarkers from a panel of 20 samples and categorized both the early-stage and late-stage ovarian cancer patients based on their analysis and observed significant heterogeneity in exosomal expression among patients.Figures 6a-6c depicts the schematic illustration of the ExoProfile chip, working principle, and fabrication mechanism of the chip using microfluidic colloidal self-assembly. 111

Challenges and Prospects
Notably, ECs can produce different types of data such as size, concentration, detection, separation, and analysis of biomarkers of Figure 5.A graphene-based field effect transistor for rapid detection of pancreatic cancer exosomes.The detection time of this device is about 45 min.The graphene was functionalized with antibodies of TCPP43 and GPC-1 (zoomed-in images).Fluorescence images exhibited a greater number of exosomes on the graphene surface for the patient sample compared to a healthy sample.The TEM images showed more immunogold of the GPC-1 patient sample compared to healthy.Adapted with terms and conditions. 105many target diseases.There are two main skills such as data analysis and pattern recognition that can be used to analyze the produced data from an EC.Further, the data produced by ECs may be quickly analyzed by applying an ML algorithm to find a specific trend or pattern that might be connected to a target disease.This can help in the identification of new biomarkers and more precise illness detection.Noted that the ML algorithms can establish correlations in huge datasets that human analysts might overlook.In addition, AI may identify potential biomarkers that denote particular diseases by tracking patterns and connections, making liquid biopsy more focused and efficient.Using the patterns from various clinical datasets, AI can classify and separate exosome profiles into disease-specific classifications.
The ML algorithms can monitor changes in exosome profiles over time, assisting in the monitoring of therapeutic effects.This dynamic analysis or results from ML models can assist clinicians in speeding up patient treatments.Also, AI and ML algorithms can assist in lowering the number of false positives and negatives in generating liquid biopsy data by integrating additional patient data from previous records.Hence, the sensitivity and selectivity of the test can be enhanced drastically.This all-encompassing method may result in more precise diagnoses and individualized treatment plans.Another major area of future work could be predictive modeling for treatment strategies for better patient outcomes.AI can be utilized to create predictive models that forecast the course or recurrence of disease with the help of previous exosome data.
ECs are widely used as next-generation biomarkers in POC diagnostics due to ongoing research and emerging technology developments that continue to overcome various issues related to screening, monitoring, and therapeutic aspects.ECs can greatly improve the accuracy and sensitivity of exosome analysis at the point of care for early disease detection, exploring personalized medicine, and thereby, improving patient outcomes.
EC development and commercialization for POC applications face quite a few challenges.The fabrication of chips should be standardized, exosome capture efficiency and specificity should be improved, portable detection technologies should be integrated, and large clinical cohorts should be used for validation.To ensure reproducibility and dependability, standardization of chip manufacture, capture procedures, and detection techniques are required.Large-scale clinical investigations will be required to confirm the clinical applicability of ECs and evaluate the outcome in comparison to traditional diagnostic techniques.
So far, nanomaterials' structural innovation, including screening printing electrodes, 112 has proven enormous success in biosensor development. 113However, their performance and sophistication depend on the manufacturing modalities as the geometry of the electrode plays an important role. 114Traditionally, photolithography (i.e.micro/nanofabrication) and electrochemical deposition are two excellent manufacturing modalities widely used to develop electrochemical and SPR biosensors.The commercially successful glucose sensor is an extensively adopted screening printing-based manufacturing modality to reduce cost per test. 115,116Further, the incorporation of nanomaterials for developing electrochemical ECs showed enhanced performance due to the high loading of antibodies (anti-CD63) specific to exosomes. 117The traditional EC developed for electrochemical nanosensing of exosomes (or antigens) provides limited sensitivities and limit-of-detection owing to the one and twodimensional geometries of the electrodes with limited reactive surfaces. 118It has been realized that nanostructuring of sensor surfaces alone is inadequate to detect low target concentration down to the femtomolar level.Recently, additively manufactured three-dimensional (3D) microelectrode geometries of the electrochemical sensors overcome these limitations for detecting antigens or antibodies due to the larger surface areas of the electrodes.Unlike traditional manufacturing, 3D printing provides customizability, complex geometries, and multiplexity of sensor construction. 119,120 3D micropillar array electrode called a "multi-length-scale electrode" of gold nanoparticles was coated with a thin layer of graphene to detect the attomole concentration of neurotransmitters. 121In this multi-length-scale electrode, the micropillar geometry of the electrode reduces the diffusion path of the target to interact with the electrode, while graphene accelerates the electrochemical reactions at the nanoscale due to their comparable size, thus accomplishing an attomolar sensitivity of a neurotransmitter (i.e., dopamine) detection. 121A similar mechanism was applied to build a 3D immunosensor to detect COVID-19 in seconds at a low concentration of antibodies (1 femtomolar). 122These applications demonstrated that the hierarchical architecture of 3D multi-length-scale electrodes opens the possibility of detecting target molecules including exosomes at ultralow concentrations.
There is increasing suggestion that ECs indicate cancer progression which will enable rapid and sensitive tools for quick analysis.As a result, current LOC techniques must be investigated to find better methods for isolating and locating malignant cell subpopulations that express proteins in a downregulated manner.In addition, by employing expression profiling to find organ-specific metastatic signals in exosomes, it might be possible to determine the tissue of origin of exosomes to determine the disease condition.
However, there are several challenges that must be overcome to commercialize EC technology.Though manufacturing of such devices may not be expensive, their standardized protocols for exosome isolation and analysis are still evolving, making it challenging to ensure consistent and reproducible results.It is noteworthy to mention that obtaining regulatory approvals for clinical use is a tedious and expensive process which is one of the major commercialization challenges of EC technology.Established liquid biopsy methods such as ctDNA analysis and circulating tumor cell (CTC) capture are already in the market, making it challenging for EC technology to gain market share. 123Compared to these methods, EC technology offers higher sensitivity and higher speed of analysis.Exosomes are often more abundant than ctDNA or CTCs and, hence, improved sensitivity.Besides, exosomes carry a diverse range of biomolecules, providing a more comprehensive picture of the tumor's biology.In addition, exosomes can be detected before ctDNA mutations become apparent.
The use of EC technology in cancer diagnosis raises ethical concerns.For instance, patients must be informed about the potential for incidental findings, as exosome analysis may uncover unrelated health issues.Highly sensitive tests may lead to the overdiagnosis of indolent cancers, raising questions about the need for treatment.Additionally, records of patient data, especially genetic information, require privacy safeguards.

Conclusions
This review article has covered the outlook of ECs with recent advancements for liquid biopsy.We have summarized various applications of ECs as diagnostic tools, exosome isolation, separation, and electrochemical detection.Unlike ultracentrifugation, though magnetic bead-based immuno-separation is an inexpensive process, it is not feasible for cross-reactivity.With structural innovation in chip fabrication technology, various lab-on-a-chip platforms including POC sensors show huge potential for exosome analysis, however, there are still challenges to overcome.Integration of AI and ML with ECs would further improve cancer diagnostics for better patient outcomes.It is essential to optimize device design and operation parameters and validate through clinical investigations for high-precision ECs.

Figure 1 .
Figure 1.Schematic illustration of the current and future of EC technology.

Figure 2 .
Figure 2. Schematic of a multiplex extracellular vesicles phenotype analyzer chip (EPAC). 66(a)-(c) Capture of exosomes on EPAC from melanoma cells and multiplex EV phenotype analysis using SERS nanotags.(d) Examining EV samples before, during, and following BRAF inhibitor treatment enables the monitoring of phenotypic changes and provides valuable insights into treatment responses and early indications of drug resistance in melanoma patient samples.Reproduced with permission.66

Figure 4 .
Figure 4.A size-based nano-DLD chip (22 mm × 30 mm) to separate exosome particles.91(a) The schematic of the DLD chip features a pillar array with a specific gap size denoted as G.The pitch of the pillars, represented by λ, is approximately 400 nm.The row-to-row shift is indicated by δ, while the maximum geometric angle is denoted as θ max .The flow of particles within the chip is influenced by the array's geometry as well as the diameter of the particles being sorted.(b) A cross-sectional SEM image shows the pillar height, 1 μm, and gap, 225 nm (scale bar ∼400 nm).(c) The integrated nanoDLD device with zigzag outlet and SEM images of separated particles.Adapted with terms and conditions.91

Figure 6 .
Figure 6.Schematics of ExoProfile chips.(a) An ExoProfile chip has a pneumatic and a fluidic layer along with 3D serpentine nanostructures and was used for the immunophenotyping of exosomes.(b) The ExoProfile chip along with flow manipulation showed an immuno-reaction and the multiplexed detection.The arrows showed the flow direction of exosomes.(c) Schematic presentation of the ExoProfile chip manufacturing.Adapted with terms and conditions. 106

Table I .
List of different ECs with their sensing performances.