"Extracellular vesicles (EVs) have emerged as key mediators of intercellular communication and valuable biomarkers for various diseases. However, traditional EV isolation and detection methods often struggle with efficiency, scalability, and purity, limiting their clinical utility. Recent advances in microfluidic and paper-based technologies offer innovative solutions that enhance EV isolation and detection by reducing sample volume, accelerating processing times, and integrating multiple analytical steps into compact platforms. These technologies hold significant promise for advancing point-of-care diagnostics, enabling rapid disease detection, personalized treatment monitoring, and better patient outcomes. For example, early detection of cancer biomarkers through EVs can facilitate timely intervention, potentially improving survival rates, while rapid infectious disease diagnostics can support prompt treatment. Despite their potential, challenges such as standardization, scalability, and regulatory hurdles remain. This review discusses recent advancements in microfluidic and paper-based EV diagnostic technologies, their comparative advantages over traditional methods, and their transformative potential in clinical practice."

Plant-based extracellular vesicles (PBEVs) have been recognized for their wide range of applications in drug delivery however, the extent of their medicinal applicability depends on how well they are preserved and stored. Assessing their physicochemical properties, such as size, particle concentration, shape, and the activity of their cargo, forms the foundation for determining their stability during storage. Moreover, the evaluation of PBEVs is essential to ensure both safety and efficacy, which are critical for advancing their clinical development. Maintaining the biological activity of EVs during storage is a challenging task, similar to the preservation of cells and other cell-derived products like proteins. However, despite limited studies, it is expected that storing drug-loaded EVs may present fewer challenges compared to cell-based therapies, although some limitations are inevitable. This article provides a comprehensive overview of current knowledge on PBEVs preservation and storage methods, particularly focusing on their role as drug carriers. PBEVs hold promise as potential candidates for oral drug administration due to their effective intestinal absorption and ability to withstand both basic and acidic environments. However, maintaining their preservation and stability during storage is critical. Moreover, this review centers on the isolation, characterization, and storage of PBEVs, exploring the potential advantages they offer. Furthermore, it highlights key areas that require further research to overcome existing challenges and enhance the development of effective preservation and storage methods for therapeutic EVs.

Wide-field inspection, nano detection, and real-time observation are essential for investigating biomolecular interaction processes. Surface plasmon resonance microscopy (SPRM) is a label-free, real-time, and nano-imaging method that is widely employed for the dynamic detection of nanoscale biomolecules. The field of view (FOV) of SPRM is limited by the usage of high NA objectives, and a scanning SPRM is required to obtain a large FOV. However, during the scanning, the focus drift introduced by the mechanical vibrations blurs the imaging quality of SPRM, making the detection deviate from the true status. To this end, this paper presents the development of autofocus scanning SPRM (AFS-SPRM) that is capable of performing automated real-time focus drift correction during auto-scanning, thereby enabling high-quality SPRM imaging with large FOV. Only 80 ms is taken to process each defocusing event, and the ability to maintain focus has been improved by 30 times by comparison with SPRM. The AFS-SPRM was successfully employed to distinguish nanoparticles of different sizes and to observe the changes of macrophages in a culture medium containing nanoparticles. This investigation illustrates the superior imaging capabilities of AFS-SPRM and demonstrates its potential for observing interactions between biomolecules at the nanoscale.

Accurate diagnosis of early Parkinson's disease requires platforms suitable for detecting minute amounts of neuronally derived biomarkers in the massive protein excess of easily accessible biofluids such as blood. Here, we describe an on-chip droplet-confined fluorescence reporting assay that identified α-synuclein on the membrane of L1CAM+ extracellular vesicles (EVs) immunocaptured from human serum and corroborate this finding by super-resolution direct stochastic optical reconstruction microscopy (dSTORM) microscopy. Using conditioned media from neuroblastoma cells expressing α-synuclein mutants or patient-derived induced pluripotent stem cell (iPSC) neurons with α-synuclein gene triplication, we found that association of α-synuclein with the L1CAM+ EV surface is increased under pathological conditions. Accordingly, this readout, as measured by the droplet-based assay, is an improved predictive biomarker in the prodromal phase (area under the receiver operating characteristic curve [AUC] = 0.93) or diagnostic biomarker in the clinical phase (AUC = 0.95) of Parkinson's disease. More broadly, our platform will simplify the assessment of EV membrane proteins and facilitate their application as diagnostic biomarkers across diverse clinical indications.

Tumor-derived circulating small extracellular vesicles (sEVs) is a promising non-invasive biomarker for disease diagnosis. However, their quantitative detection remains challenging due to their small size and the complexity of blood plasma. Typically, sample preparation like purification is required. This study presents a purification-free approach using a microfluidic chip integrated with PANORAMA (Plasmonic nano-aperture label-free imaging) for label-free single sEV counting in plasma. CD63, CD9, and CD81 antibodies, specific biomarkers for most sEVs, are functionalized on AGNIS (arrayed gold nanodisks on invisible substrate) for selective capture. The automated microfluidic platform minimizes manual errors and allows precise programming of flow rates, directions, and media for optimization. Only 20 µL of plasma is required, and the analysis is completed within 60 minutes. This platform shows great potential as a sensitive and efficient tool for detecting circulating sEVs without purification or labeling.

Liver cancer is a significant global health concern due to its poor prognosis, often resulting from late-stage diagnosis and limited treatment options. While non-invasive methods such as ultrasound, blood tests (like AFP and PIVKA-II), CT scans, and MRIs are commonly employed in liver cancer diagnosis, they can occasionally be limited in sensitivity or associated with high costs. This has heightened the demand for innovative, non-invasive biomarkers that enable early and accurate diagnosis, leading to increased interest in the potential of exosomes. Exosomes are small vesicles released by cells and have the potential to serve as biomarkers for liver cancer. They contain a variety of biomolecules, including nucleic acids, proteins, and lipids, which can offer important information about cell health and disease progression. Developing fast, accurate, sensitive, and reliable techniques for detecting exosomes is essential. Biosensors, analytical tools for biological samples, have emerged as powerful instruments for analyzing exosomes. This review focuses on recent advancements in biosensor technology for exosome detection and explores future perspectives. The goal is to promote the development of innovative biosensor-based methods for detecting exosomes to enable earlier diagnosis and better clinical management of liver cancer.

Extracellular vesicles (EVs) are heterogeneous lipid containers carrying complex molecular cargoes, including proteins, nucleic acids, glycans, etc. These vesicles are closely associated with specific physiological characteristics, which makes them invaluable in the detection and monitoring of various diseases. However, traditional isolation methods are often labour-intensive, inefficient, and time-consuming. In addition, single biomarker analyses are no longer accurate enough to meet diagnostic needs. Routine isolation and molecular analysis of high-purity EVs in clinical applications is even more challenging. In this review, we discuss a promising solution, microfluidic-based techniques, that combine efficient isolation and multiplex detection of EVs, to further demystify EV heterogeneity. These microfluidic-based EV multiplexing platforms will hopefully facilitate development of liquid biopsies and offer promising opportunities for personalised therapy.

Exosomes, nanoscale extracellular vesicles secreted by various cells, are abundantly present in biological fluids. They have been identified as carriers of specific molecules, suggesting their potential role in early disease detection. However, their clinical application is hindered by several challenges, including the need for large sample volumes for enrichment, limitations of traditional detection methods, and the complexity involved in phenotype analysis and separation.

This review aims to explore the application of Surface-Enhanced Raman Scattering (SERS) technology in exosome detection. SERS, known for its unique photonic properties and high sensitivity, offers a promising solution for detecting exosomes without the need for large sample volumes or extensive phenotypic analysis. This review focuses on the real-time and non-invasive assessment capabilities of SERS in exosome detection, providing insights into its potential for early disease diagnosis.

The review concludes by emphasizing the potential of SERS-based exosome detection in advancing early disease diagnosis. By overcoming existing challenges, SERS technology offers a promising approach for the development of sensitive and specific diagnostic assays, contributing to better patient outcomes and personalized medicine.

Aim: Cells in the human body release extracellular vesicles (EVs) into fluids, such as plasma, urine, and cerebrospinal fluid. EVs express tetraspanin family proteins (e.g., CD63, CD9, and CD81) and cell-specific antigens on their surface as common and specific markers, respectively. In this study, we hypothesized that the profile of blood cell-derived circulating EVs could reveal both common and specific pathophysiology in atherogenic diseases. Methods: Using surface plasmon resonance imaging (SPRi), we analyzed EVs surface molecules and identified circulating EVs in healthy controls (n = 18), patients with type 2 diabetes mellitus (T2DM; n = 71), and those with hypertension (HT; n = 47). Results: Patients with T2DM and HT exhibited distinct EV profiles: (i) CD9, CD110, CD20, activin receptor type-2A (AcvRIIA), Duffy antigen receptor for chemokine, and CD44 positive EVs were upregulated in T2DM; (ii) CD9, Maackia amurensis agglutinin lectin binding molecules (MBM), CD20, AcvRIIA, and CD44 positive EVs were upregulated in HT. By analyzing an appropriate set of three antigens or using dimensional reduction clustering, we were able to clearly differentiate between T2DM, HT, and control groups. In some patients, disease severity correlated with CD44 and CD20 in T2DM and MBM and AcvRIIA in HT. Conclusion: Our findings demonstrate that profiling of circulating EVs via the SPRi method offers a novel approach for diagnosing and monitoring human diseases.

Tumor exosomes, known as maternal cell messengers, play an important role in cancer occurrence, proliferation, metastasis, immune escape, drug resistance, and other processes and are an entry point for cancer research. However, there is still a lack of an efficient detection technology for exosomes. In this study, the ultrahigh sensitivity SERS nanoprobe with a three dimensional (3D) magnetic core/Au nanocolumn/Au nanoparticles shell strongly coupling multistage structure (Fe3O4@NR-NPs) was constructed by crystal growth of nanocrystals in the confined space of a central radiating single particle mesoporous molecular sieve channel and strong coupling secondary growth of gold particles. The exosomes were confined onto the "hot spot" of plasmonic nanoparticles and rapidly enriched by CD63 antibody functional-Fe3O4@NR-NPs to achieve high sensitivity detection, with the limit of detection of 1 × 103 particles/mL (S/N = 3). The spectral data set of different exosomes is applied to train for multivariate classification of cell types and to estimate how the normal exosome data resemble cancer cell exosomes by principal component analysis (PCA). Finally, this detection method has also been successfully employed for the detection of exosomes in complex samples; this proves that the proposed SERS-based method is a promising tool for clinical cancer screening.

Exosomes, as next-generation biomarkers, has great potential in tracking cancer progression. They face many detection limitations in cancer diagnosis. Plasmonic biosensors have attracted considerable attention at the forefront of exosome detection, due to their label-free, real-time, and high-sensitivity features. Their advantages in multiplex immunoassays of minimal liquid samples establish the leading position in various diagnostic studies. This review delineates the application principles of plasmonic sensing technologies, highlighting the importance of exosomes-based spectrum and image signals in disease diagnostics. It also introduces advancements in miniaturizing plasmonic biosensing platforms of exosomes, which can facilitate point-of-care testing for future healthcare. Nowadays, inspired by the surge of artificial intelligence (AI) for science and technology, more and more AI algorithms are being adopted to process the exosome spectrum and image data from plasmonic detection. Using representative algorithms of machine learning has become a mainstream trend in plasmonic biosensing research for exosome liquid biopsy. Typically, these algorithms process complex exosome datasets efficiently and establish powerful predictive models for precise diagnosis. This review further discusses critical strategies of AI algorithm selection in exosome-based diagnosis. Particularly, we categorize the AI algorithms into the interpretable and uninterpretable groups for exosome plasmonic detection applications. The interpretable AI enhances the transparency and reliability of diagnosis by elucidating the decision-making process, while the uninterpretable AI provides high diagnostic accuracy with robust data processing by a "black-box" working mode. We believe that AI will continue to promote significant progress of exosome plasmonic detection and mobile healthcare in the near future.

Cell-derived extracellular vesicles (EVs) have emerged as a promising non-invasive liquid biopsy technique due to their accessibility and their ability to encapsulate and transport diverse biomolecules. EVs have garnered substantial research interest, notably in cardiovascular diseases (CVDs), where their roles in pathophysiology and as diagnostic and prognostic biomarkers are increasingly recognized. This review provides a comprehensive overview of EVs, starting with their origins, followed by the techniques used for their isolation and characterization. We explore the diverse cargo of EVs, including nucleic acids, proteins, lipids, and metabolites, highlighting their roles in intercellular communication and as potential biomarkers. We then delve into the application of genomics, transcriptomics, proteomics, and metabolomics in the analysis of EVs, particularly within the context of CVDs. Finally, we discuss how integrated multi-omics approaches are unveiling novel biomarkers, offering fresh insights into the diagnosis and prognosis of CVDs. This review underscores the growing importance of EVs in clinical diagnostics and the potential of multi-omics to propel future advancements in CVD biomarker discovery.

Extracellular vesicles (EVs) are secreted by cells with a membrane structure and complex components such as DNA, RNA and proteins. These biomolecules play an important role in cell communication, cell proliferation, cell migration, vascularization, immune response and other physiological and pathological processes. Most current research on EVs focused on populations of EVs. Heterogeneity of EVs is neglected. Considering the heterogeneity of single EVs may offer critical molecular insights into cell-cell interactions, it is necessary to enhance our understanding about molecular characteristics from EVs derived from cell population to a single EV of derived from a single cell. This transformation is expected to provide a new insight into the understanding of cellular biology and the accurate description of the law of disease progress. In this article, we review the current research progress of single EV analysis technology for single EVs derived from cell population (SECP) and discuss its main applications in biological and clinical medicine research. After that, we propose the development direction, main difficulties and application prospect of single EV analysis technology for single EVs derived from single cells (SESC) according to our own research work, to provide new perspectives for the field of EV research.

Endocytosis-derived extracellular vesicles (EVs), which can be as small as 100 nm, are useful for disease prediction. However, very small EVs are below the optical diffraction limit and are difficult to visualize with conventional fluorescence microscopy. In this study, single EVs captured on a plasmonic chip, where fluorescently labeled antibodies were bound over the EV surface, were detected as bright spots using plasmon-field enhanced fluorescence without any pretreatment of isolating labeled EVs, followed by analyzing the full width at half-maximum and the fluorescence peak value for each enhanced fluorescence bright spot. Bright spots smaller than the threshold determined by the observation of the fluorescent nanospheres were attributed to single EVs. The number of single EVs was quantitatively evaluated against the concentration of EV solution injected in the 1.4 pM-95 fM range. Furthermore, single EVs were detected by labeling two different membrane proteins. A molecularly imprinted polymer was applied to a capture interface on a plasmonic chip, and it is found that nonspecific adsorption of aggregates was suppressed. To accurately distinguish single EVs from aggregates of labeled antibodies, the fluorescence microscopy with transmitted light was superior to the epifluorescence method. Finally, single EVs were successfully detected with multiple targets at multiple wavelengths by using different fluorescently labeled antibodies.

Liquid biopsy technology provides invaluable support for the early diagnosis of tumors and surveillance of disease course by detecting tumor-related biomarkers in bodily fluids. Currently, liquid biopsy techniques are mainly divided into two categories: biomarker and label-free. Biomarker liquid biopsy techniques utilize specific antibodies or probes to identify and isolate target cells, exosomes, or molecules, and these techniques are widely used in clinical practice. However, they have certain limitations including dependence on tumor markers, alterations in cell biological properties, and high cost. In contrast, label-free liquid biopsy techniques directly utilize physical or chemical properties of cells, exosomes, or molecules for detection and isolation. These techniques have the advantage of not needing labeling, not impacting downstream analysis, and low detection cost. However, most are still in the research stage and not yet mature. This review first discusses recent advances in liquid biopsy techniques for early tumor diagnosis and disease surveillance. Several current techniques are described in detail. These techniques exploit differences in biomarkers, size, density, deformability, electrical properties, and chemical composition in tumor components to achieve highly sensitive tumor component identification and separation. Finally, the current research progress is summarized and the future research directions of the field are discussed.

The vascular network plays an essential role in the maintenance of all organs in the body via the regulated delivery of oxygen and nutrients, as well as tissue communication via the transfer of various biological signaling molecules. It also serves as a route for drug administration and affects pharmacokinetics. Due to this importance, engineers have sought to create physiologically relevant and reproducible vascular systems in tissue, considering cell-cell and extracellular matrix interaction with structural and physical conditions in the microenvironment. Extracellular vesicles (EVs) have recently emerged as important carriers for transferring proteins and genetic material between cells and organs, as well as for drug delivery. Vascularized platforms can be an ideal system for studying interactions between blood vessels and EVs, which are crucial for understanding EV-mediated substance transfer in various biological situations. This review summarizes recent advances in vascularized platforms, standard and microfluidic-based techniques for EV isolation and characterization, and studies of EVs in vascularized platforms. It provides insights into EV-related (patho)physiological regulations and facilitates the development of EV-based therapeutics.

To present the recent update on the isolation, engineering techniques for extracellular vesicles, limitations associated with different isolation techniques, different biomedical applications, and challenges of engineered extracellular vesicles for the benefit of researchers from academic, industry, etc. MATERIALS AND METHODS: Peer-reviewed articles from most recognized journals were collected, and presented information was analyzed to discuss collection, chemical, electroporation, cellular, and membrane surface engineering to design extracellular vesicles for various therapeutic applications. In addition, we present the applications and limitations of techniques for the collection of extracellular vesicles.

There is a need for isolation techniques with the gold standard. However, advanced extracellular vesicle isolation techniques showed improved recovery, and purity of extracellular vesicles. Tumor therapy is a major part of the therapy section that illustrates the role of engineered extracellular vesicles in synergetic therapy such as phototherapy, theragnostic, and delivery of genetic materials. In addition, extracellular vesicles have shown their potential in the treatment of retinal disorders, neurodegenerative disease, tuberculosis, osteoporosis, inflammatory bowel disease, vaccine production, and wound healing.

Engineered extracellular vesicles can deliver cargo to the specific cells, elicit an immune response and could be used for the development of the vaccines in the future. However, the progress is at the initial stage. Overall, this review will provide a comprehensive understanding and could serve as a reference for researchers in the clinical translation of engineered extracellular vesicles in different biomedical fields.

This review focusses on the significance of fluorescent, phosphorescent labelling and tracking of extracellular vesicles (EVs) for unravelling their biology, pathophysiology, and potential diagnostic and therapeutic uses. Various labeling strategies, such as lipid membrane, surface protein, luminal, nucleic acid, radionuclide, quantum dot labels, and metal complex-based stains, are evaluated for visualizing and characterizing EVs. Direct labelling with fluorescent lipophilic dyes is simple but generally lacks specificity, while surface protein labelling offers selectivity but may affect EV-cell interactions. Luminal and nucleic acid labelling strategies have their own advantages and challenges. Each labelling approach has strengths and weaknesses, which require a suitable probe and technique based on research goals, but new tetranuclear polypyridylruthenium(II) complexes as phosphorescent probes have strong phosphorescence, selective staining, and stability. Future research should prioritize the design of novel fluorescent probes and labelling platforms that can significantly enhance the efficiency, accuracy, and specificity of EV labeling, while preserving their composition and functionality. It is crucial to reduce false positive signals and explore the potential of multimodal imaging techniques to gain comprehensive insights into EVs.

Exosomes, a category of extracellular vesicle (EV), are phospholipid bilayer structures ranging from 30 to 150 nm, produced by various organisms through the endosomal pathway. Recent studies have established the utilization of exosomes as nanocarriers for drug distribution across various therapeutic areas including cancer, acute liver injury, neuroprotection, oxidative stress, inflammation, etc. The importance of plant-derived exosomes and exosome vesicles derived from mammalian cells or milk, loaded with potent plant bioactives for various therapeutic indications are discussed along with insights into future perspectives. Moreover, this review provides a detailed understanding of exosome biogenesis, their composition, classification, stability of different types of exosomes, and different routes of administration along with the standard techniques used for isolating, purifying, and characterizing exosomes.

As one of the deadliest gynaecological cancers, ovarian cancer has been on the list. With lesser-known symptoms and lack of an accurate detection method, it is still difficult to catch it early. In terms of both the diagnosis and outlook for cancer, liquid biopsy has come a long way with significant advancements. Exosomes, extracellular components commonly shed by cancerous cells, are nucleic acid-rich particles floating in almost all body fluids and hold enormous promise, leading to minimallyinvasive molecular diagnostics. They have been shown as potential biomarkers in liquid biopsy, being implicated in tumour growth and metastasis. In order to address the drawbacks of ovarian cancer tumor heterogeneity, a liquid biopsy-based approach is being investigated by detecting cell-free nucleic acids, particularly non-coding RNAs, having the advantage of being less invasive and more prominent in nature. microRNAs are known to actively contribute to cancer development and their existence inside exosomes has also been made quite apparent which can be leveraged to diagnose and treat the disease. Extraction of miRNAs and exosomes is an arduous execution, and while other approaches have been investigated, none have produced results that are as encouraging due to limits in time commitment, yield, and, most significantly, damage to the exosomal structure resulting discrepancies in miRNA-based expression profiling for disease diagnosis. We have briefly outlined and reviewed the difficulties with exosome isolation techniques and the need for their standardization. The several widely used procedures and their drawbacks in terms of the exosomal purity they may produce have also been outlined.

The accurate quantification of cancer-derived exosomes, which are emerging as promising noninvasive biomarkers for liquid biopsies in the early diagnosis of cancer, is becoming increasingly imperative. In our work, we developed a magnetically controlled photothermal, colorimetric, and fluorescence trimode aptasensor for human gastric cancer cell (SGC-7901)-derived exosomes. This sensor relied on CP/Mn-PBA DSNBs nanocomposites, created by decorating copper peroxide (CP) nanodots on polyethyleneimine-modified manganese-containing Prussian blue analogues double-shelled nanoboxes (PEI-Mn-PBA DSNBs). Through self-assembly, we attached CD63 aptamer-labeled CP/Mn-PBA DSNBs (Apt-CP/Mn-PBA DSNBs) to complementary DNA-labeled magnetic beads (cDNA-MB). During exosome incubation, these aptamers preferentially formed complexes with exosomes, and we efficiently removed the released CP/Mn-PBA DSNBs by using magnetic separation. The CP/Mn-PBA DSNBs exhibited high photoreactivity and photothermal conversion efficiency under near-infrared (NIR) light, leading to temperature variations under 808 nm irradiation, correlating with different exosome concentrations. Additionally, colorimetric detection was achieved by monitoring the color change in a 3,3',5,5'-tetramethylbenzidine (TMB) system, facilitated by PEI modification, NIR-enhanced peroxidase-like activity of CP/Mn-PBA DSNBs and their capacity to generate Cu2+ and H2O2 under acidic conditions. Moreover, in the presence of Cu2+ and ascorbic acid (AA), DNA sequences could form dsDNA-templated copper nanoparticles (CuNPs), which emitted strong fluorescence at around 575 nm. Increasing exosome concentrations correlated with decreases in temperature, absorbance, and fluorescence intensity. This trimode biosensor demonstrated satisfactory ability in differentiating gastric cancer patients from healthy individuals using human serum samples.

Extracellular vesicles (EVs) secreted by various cells offer great potential for use in the diagnosis and treatment of disease. EVs are heterogeneous membranous vesicles. Exosomes are a subtype of EVs, 40-150 nm spherical vesicles with a lipid layer derived from endosomes. Exosomes, which are involved in signal transduction and maintain homeostasis, are released from almost all cells, tissues, and body fluids. Although several methods exist to isolate and characterize EVs and exosomes, each technique has significant drawbacks and limitations that prevent progress in the field. New approaches in the biology of EVs show great potential for isolating and characterizing EVs, which will help us better understand their biological function. The strengths and limitations of conventional strategies and novel methods (microfluidic) for EV isolation are outlined in this review. We also present various exosome isolation techniques and kits that are commercially available and assess the global market demand for exosome assays.

Colorectal carcinoma (CRC) is the third most common cancer in terms of diagnosis and the second in terms of mortality. Recent studies have shown that various proteins, extracellular vesicles (i.e., exosomes), specific genetic variants, gene transcripts, cell-free DNA (cfDNA), circulating tumor DNA (ctDNA), microRNAs (miRNAs), long non-coding RNAs (lncRNAs), and altered epigenetic patterns, can be used to detect, and assess the prognosis of CRC. Over the last decade, a plethora of conventional methodologies (e.g., polymerase chain reaction [PCR], direct sequencing, enzyme-linked immunosorbent assay [ELISA], microarray, in situ hybridization) as well as advanced analytical methodologies (e.g., microfluidics, electrochemical biosensors, surface-enhanced Raman spectroscopy [SERS]) have been developed for analyzing genetic and epigenetic biomarkers using both optical and non-optical tools. Despite these methodologies, no gold standard detection method has yet been implemented that can analyze CRC with high specificity and sensitivity in an inexpensive, simple, and time-efficient manner. Moreover, until now, no study has critically reviewed the advantages and limitations of these methodologies. Here, an overview of the most used genetic and epigenetic biomarkers for CRC and their detection methods are discussed. Furthermore, a summary of the major biological, technical, and clinical challenges and advantages/limitations of existing techniques is also presented.

Recently, studies have revealed that human herpesvirus 4 (HHV-4), also known as the Epstein-Barr virus, might be associated with the severity of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Compared to SARS-CoV-2 infection alone, patients coinfected with SARS-CoV-2 and HHV-4 had higher risks of fever, inflammation, and even death, thus, confirming that HHV-4/SARS-CoV-2 coinfection in patients could benefit from clinical investigation. Although several intelligent devices can simultaneously discern multiple genes related to SARS-CoV-2, most operate via label-based detection, which restricts them from directly measuring the product. In this study, we developed a device that can replicate and detect SARS-CoV-2 and HHV-4 DNA. This device can conduct a duplex polymerase chain reaction (PCR) in a microfluidic channel and detect replicates in a non-labeled manner through a plasmonic-based sensor. Compared to traditional instruments, this device can reduce the required PCR time by 55% while yielding a similar amount of amplicon. Moreover, our device's limit of detection (LOD) reached 100 fg/mL, while prior non-labeled sensors for SARS-CoV-2 detection were in the range of ng/mL to pg/mL. Furthermore, the device can detect desired genes by extracting cells artificially infected with HHV-4/SARS-CoV-2. We expect that this device will be able to help verify HHV-4/SARS-CoV-2 coinfected patients and assist in the evaluation of practical treatment approaches.

Cancer is the most challenging global health crisis. In the recent times, studies on extracellular vesicles (EVs) are adding a new chapter to cancer research and reports on EVs explores cancer in a new dimension. Exosomes are a group of subpopulations of EVs. It originates from the endosomes and carries biologically active molecules to the neighboring cells which in turn transforms the recipient cell activity. In general, it plays a role in cellular communication. The correlation between exosomes and cancer is fascinating. Tumor-derived exosomes (TEXs) play a dynamic role in cancer progression and are associated with uncontrolled cell growth, angiogenesis, immune suppression, and metastasis. Its molecular cargo is an excellent source of cancer biomarkers. Several advanced molecular profiling approaches assist in exploring the TEXs in depth. This paves the way for a strong foundation for identifying and detecting more specific and efficient biomarkers. TEXs are also gaining importance in scientific society for its role in cancer therapy and several clinical trials based on TEXs is a proof of its significance. In this review, we have highlighted the role of TEXs in mediating immune cell reprogramming, cancer development, metastasis, EMT, organ-specific metastasis, and its clinical significance in cancer theranostics. TEXs profiling is an effective method to understand the complications associated with cancer leading to good health and well-being of the individual and society as a whole.

Extracellular vesicles (EVs) are extensively dispersed lipid bilayer membrane vesicles involved in the delivery and transportation of molecular payloads to certain cell types to facilitate intercellular interactions. Their significant roles in physiological and pathological processes make EVs outstanding biomarkers for disease diagnosis and treatment monitoring as well as ideal candidates for drug delivery. Nevertheless, differences in the biogenesis processes among EV subpopulations have led to a diversity of biophysical characteristics and molecular cargos. Additionally, the prevalent heterogeneity of EVs has been found to substantially hamper the sensitivity and accuracy of disease diagnosis and therapeutic monitoring, thus impeding the advancement of clinical applications. In recent years, the evolution of single EV (SEV) analysis has enabled an in-depth comprehension of the physical properties, molecular composition, and biological roles of EVs at the individual vesicle level. This review examines the sample acquisition tactics prior to SEV analysis, i.e., EV isolation techniques, and outlines the current state-of-the-art label-free and label-based technologies for SEV identification. Furthermore, the challenges and prospects of biomedical applications based on SEV analysis are systematically discussed.

Blood-circulating exosomes as a disease biomarker have great potential in clinical applications as they contain molecular information about their parental cells. However, label-free characterization of exosomes is challenging due to their small size. Without labeling, exosomes are virtually indistinguishable from other entities of similar size. Over recent years, several techniques have been developed to overcome the existing challenges. This paper demonstrates a new label-free approach based on dynamic PlAsmonic NanO-apeRture lAbel-free iMAging (D-PANORAMA), a bright-field technique implemented on arrayed gold nanodisks on invisible substrates (AGNIS). PANORAMA provides high surface sensitivity and has been shown to count single 25 nm polystyrene beads (PSB) previously. Herein, we show that using the dynamic imaging mode, D-PANORAMA can yield 3-dimensional, sub-diffraction limited localization of individual 25 nm beads. Furthermore, we demonstrate D-PANORAMA's capability to size, count, and localize the 3-dimensional, sub-diffraction limited position of individual exosomes as they bind to the AGNIS surface. We emphasize the importance of both the in-plane and out-of-plane localization, which exploit the synergy of 2-dimensional imaging and the intensity contrast.

Colorectal cancer (CRC) remains an enormous challenge to human health worldwide. Unfortunately, the mechanism underlying CRC progression is not well understood. Mounting evidence has confirmed that exosomes play a vital role in CRC progression, which has attracted extensive attention among researchers. In addition to acting as messengers between CRC cells, exosomes also participate in the CRC immunomodulatory process and reshape immune function. As stable message carriers and liquid biopsy option under development, exosomes are promising biomarkers in the diagnosis or treatment of CRC. In this review we have described and analyzed the biogenesis and release of exosomes and current research on the role of exosomes in immune regulation and metastasis of CRC. Moreover, we have discussed candidate exosomal molecules as potential biomarkers to diagnose CRC, predict CRC progression, or determine CRC chemoresistance, and described the significance of exosomes in the immunotherapy of CRC. This review provides insight to further understand the role of exosomes in CRC progression and identify valuable biomarkers that facilitate the clinical management of CRC patients.

Exosomes are nanosized extracellular vesicles secreted by nearly all viable cells following the fusing of multivesicular bodies and the plasma membrane and discharged into the encircling bodily fluids. Exosomes can transport cell-specific components from the source cell to the target cell. Given the enormous potential of exosomes as non-invasive diagnostic biomarkers and therapeutic nanovehicles. Lately, accumulated evidence has demonstrated that exosomes serve an important role in prognosis, diagnosis, and even treatment strategies. While several reviews have collective information on the biomedical application of exosomes, a comprehensive review incorporating updated and improved methodologies for beneficial applications of such vesicles in cancer theranostics is indispensable. In the current review, we first provided a comprehensive review of the introduction of exosomes, featuring their discovery, separation, characterization, function, biogenesis, secretion. The implications of exosomes as promising nanovehicles for drug and gene delivery, application of exosome inhibitors in the management of cancers, completed and ongoing clinical trials on the biological relevance of exosomes are then discussed in detail. As the field of exosome research grows, a better understanding of the subcellular parts and mechanisms involved in exosome secretion and targeting of specific cells will help figure out what their exact physiological functions are in the body.

Surface-Enhanced Raman Scattering (SERS) technology, as a powerful tool to identify molecular species by collecting molecular spectral signals at the single-molecule level, has achieved substantial progresses in the fields of environmental science, medical diagnosis, food safety, and biological analysis. As deepening research is delved into SERS sensing, more and more high-performance or multifunctional SERS substrate materials emerge, which are expected to push Raman sensing into more application fields. Especially in the field of biological analysis, intrinsic and extrinsic SERS sensing schemes have been widely used and explored due to their fast, sensitive and reliable advantages. Herein, recent developments of SERS substrates and their applications in biomolecular detection (SARS-CoV-2 virus, tumor etc.), biological imaging and pesticide detection are summarized. The SERS concepts (including its basic theory and sensing mechanism) and the important strategies (extending from nanomaterials with tunable shapes and nanostructures to surface bio-functionalization by modifying affinity groups or specific biomolecules) for improving SERS biosensing performance are comprehensively discussed. For data analysis and identification, the applications of machine learning methods and software acquisition sources in SERS biosensing and diagnosing are discussed in detail. In conclusion, the challenges and perspectives of SERS biosensing in the future are presented.

Extracellular vesicles (EVs), acting as important mediators of intercellular communication, play an essential role in physiological processes, which have unique potential in the medical field. However, the heterogeneity of EVs limits their development for disease diagnosis and therapy, making the EV subpopulation analysis extremely valuable. In this article, a simple microfluidic approach was presented for the on-chip specific isolation and detection of two phenotypes of EVs (Annexin V⁺ EGFR⁺ EVs and Annexin V^- EGFR⁺ EVs) based on different biomolecule-modified magnetic nanospheres and a fluorescence labeling technique. Combined with the control of the magnetic field in the microzone and fluid flow, it was easy to form two separate functional regions in the chip to capture different EV subpopulations. This method was successfully applied to the tests of clinical saliva samples in 75 oral squamous cell carcinoma (OSCC) patients and 10 healthy people. The results showed that the total level of EGFR⁺ EVs was much higher in OSCC patients that in healthy people. Meantime, the ratio of Annexin V⁺ EGFR⁺ EVs to Annexin V^- EGFR⁺ EVs was found to be negatively correlated with tumor T stage of OSCC patients with a statistical difference, which suggested the ratio as a clinical index for monitoring the progression of OSCC in real time based on a noninvasive method. The approach provided a novel idea for evaluating the tumor T stage of OSCC and a powerful tool for clinical application.

Surface Plasmon Resonance Imaging (SPRI) is a robust technique for visualizing refractive index changes, which enables researchers to observe interactions between nanoscale objects in an imaging manner. In the past period, scholars have been attracted by the Prism-Coupled and Non-prism Coupled configurations of SPRI and have published numerous experimental results. This review describes the principle of SPRI and discusses recent developments in Prism-Coupled and Non-prism Coupled SPRI techniques in detail, respectively. And then, major advances in biological applications of SPRI are reviewed, including four sub-fields (cells, viruses, bacteria, exosomes, and biomolecules). The purpose is to briefly summarize the recent advances of SPRI and provide an outlook on the development of SPRI in various fields.

Liquid biopsy for the analysis of circulating cancer biomarkers (CBs) is a major advancement toward the early detection of cancer. In comparison to tissue biopsy techniques, liquid biopsy is relatively painless, offering multiple sampling opportunities across easily accessible bodily fluids such as blood, urine, and saliva. Liquid biopsy is also relatively inexpensive and simple, avoiding the requirement for specialized laboratory equipment or trained medical staff. Major advances in the field of liquid biopsy are attributed largely to developments in nanotechnology and microfabrication that enables the creation of highly precise chip-based platforms. These devices can overcome detection limitations of an individual biomarker by detecting multiple markers simultaneously on the same chip, or by featuring integrated and combined target separation techniques. In this review, the major advances in the field of portable and semi-portable micro, nano, and multiplexed platforms for CB detection for the early diagnosis of cancer are highlighted. A comparative discussion is also provided, noting merits and drawbacks of the platforms, especially in terms of portability. Finally, key challenges toward device portability and possible solutions, as well as discussing the future direction of the field are highlighted.

Exosomes derived from cancer cells have been recognized as a promising biomarker for minimally invasive liquid biopsy. Herein, a novel sandwich-type biosensor was fabricated for highly sensitive detection of exosomes. Amino-functionalized Fe₃O₄ nanoparticles were synthesized as a sensing interface with a large surface area and rapid enrichment capacity, while two-dimensional MXene nanosheets were used as signal amplifiers with excellent electrical properties. Specifically, CD63 aptamer attached Fe₃O₄ nanoprobes capture the target exosomes. MXene nanosheets modified with epithelial cell adhesion molecule (EpCAM) aptamer were tethered on the electrode surface to enhance the quantification of exosomes captured with the detection of remaining protein sites. With such a design, the proposed biosensor showed a wide linear range from 10² particles μL^-1 to 10⁷ particles μL^-1 for sensing 4T1 exosomes, with a low detection limit of 43 particles μL^-1. In addition, this sensing platform can determine four different tumor cell types (4T1, Hela, HepG2, and A549) using surface proteins corresponding to aptamers 1 and 2 (CD63 and EpCAM) and showcases good specificity in serum samples. These preliminary results demonstrate the feasibility of establishing a sensitive, accurate, and inexpensive electrochemical sensor for detecting exosome concentrations and species. Moreover, they provide a significant reference for exosome applications in clinical settings, such as liquid biopsy and early cancer diagnosis.

Extracellular vesicles (EVs) are nanoscale membrane vesicles, which contain a wide variety of cargo such as proteins, miRNAs, and lipids. A growing body of evidence suggests that EVs are promising biomarkers for disease diagnosis and therapeutic strategies. Although the excellent clinical value, their use in personalized healthcare practice is not yet feasible due to their highly heterogeneous nature. Taking the difficulty of isolation and the small size of EVs into account, the characterization of EVs at a single-particle level is both imperative and challenging. In a bid to address this critical point, more research has been directed into a microfluidic platform because of its inherent advantages in sensitivity, specificity, and throughput. This review discusses the biogenesis and heterogeneity of EVs and takes a broad view of state-of-the-art advances in microfluidics-based EV research, including not only EV separation, but also the single EV characterization of biophysical detection and biochemical analysis. To highlight the advantages of microfluidic techniques, conventional technologies are included for comparison. The current status of artificial intelligence (AI) for single EV characterization is then presented. Furthermore, the challenges and prospects of microfluidics and its combination with AI applications in single EV characterization are also discussed. In the foreseeable future, recent breakthroughs in microfluidic platforms are expected to pave the way for single EV analysis and improve applications for precision medicine.

Extracellular vesicles (EVs) have emerged as a novel resource of biomarkers for cancer and certain other diseases. Probing EVs in body fluids has become of major interest in the past decade in the development of a new-generation liquid biopsy for cancer diagnosis and monitoring. However, sensitive and specific molecular detection and analysis are challenging, due to the small size of EVs, low amount of antigens on individual EVs, and the complex biofluid matrix. Nanomaterials have been widely used in the technological development of protein and nucleic acid-based EV detection and analysis, owing to the unique structure and functional properties of materials at the nanometer scale. In this review, we summarize various nanomaterial-based analytical technologies for molecular EV detection and analysis. We discuss these technologies based on the major types of nanomaterials, including plasmonic, fluorescent, magnetic, organic, carbon-based, and certain other nanostructures. For each type of nanomaterial, functional properties are briefly described, followed by the applications of the nanomaterials for EV biomarker detection, profiling, and analysis in terms of detection mechanisms.

Single vesicle molecular profiling has the potential to transform cancer detection and monitoring by precisely probing cancer-associated extracellular vesicles (EVs) in the presence of normal EVs in body fluids, but it is challenging due to the small EV size, low abundance of antigens on individual vesicles, and a complex biological matrix. Here, we report a facile dual imaging single vesicle technology (DISVT) for surface protein profiling of individual EVs and quantification of target-specific EV subtypes based on direct molecular capture of EVs from diluted biofluids, dual EV-protein fluorescence-light scattering imaging, and fast image analysis using Bash scripts, Python, and ImageJ. Plasmonic gold nanoparticles (AuNPs) were used to label and detect targeted surface protein markers on individual EVs with dark-field light scattering imaging at the single particle level. Monte Carlo calculations estimated that the AuNPs could detect EVs down to 40 nm in diameter. Using the DISVT, we profiled surface protein markers of interest across individual EVs derived from several breast cancer cell lines, which reflected the parental cells. Studies with plasma EVs from healthy donors and breast cancer patients revealed that the DISVT, but not the traditional bulk enzyme-linked immunosorbent assay, detected human epidermal growth factor receptor 2 (HER2)-positive breast cancer at an early stage. The DISVT also precisely differentiated HER2-positive breast cancer from HER2-negative breast cancer. We additionally showed that the amount of tumor-associated EVs was tripled in locally advanced patients compared to that in early-stage patients. These studies suggest that single EV surface protein profiling with DISVT can provide a facile and high-sensitivity method for early cancer detection and quantitative monitoring.

Research into extracellular vesicles (EVs) has grown significantly over the last few decades with EVs being widely regarded as a source of biomarkers for human health and disease with massive clinical potential. Secreted by every cell type in the body, EVs report on the internal cellular conditions across all tissue types. Their presence in readily accessible biofluids makes the potential of EV biosensing highly attractive as a noninvasive diagnostic platform via liquid biopsies. However, their small size (50-250 nm), inherent heterogeneity, and the complexity of the native biofluids introduce challenges for effective characterization, thus, limiting their clinical utility. This has led to a surge in the development of various novel EV biosensing techniques, with capabilities beyond those of conventional methods that have been directly transferred from cell biology. In this review, key detection principles used for EV biosensing are summarized, with a focus on some of the most recent and fundamental developments in the field over the last 5 years. This article is categorized under: Diagnostic Tools > Biosensing Diagnostic Tools > In Vitro Nanoparticle-Based Sensing.

Extracellular vesicles (EVs) are double-layered lipid membrane vesicles released by cells. Currently, EVs are attracting a lot of attention in the biological and medical fields due to their role as natural carriers of proteins, lipids, and nucleic acids. Thus, they can transport useful genomic information from their parental cell through body fluids, promoting cell-to-cell communication even between different organs. Due to their functionality as cargo carriers and their protein expression, they can play an important role as possible diagnostic and prognostic biomarkers in various types of diseases, e.g., cancers, neurodegenerative, and autoimmune diseases. Today, given the invaluable importance of EVs, there are some pivotal challenges to overcome in terms of their isolation. Conventional methods have some limitations: they are influenced by the starting sample, might present low throughput and low purity, and sometimes a lack of reproducibility, being operator dependent. During the past few years, several microfluidic approaches have been proposed to address these issues. In this review, we summarize the most important microfluidic-based devices for EV isolation, highlighting their advantages and disadvantages compared to existing technology, as well as the current state of the art from the perspective of the use of these devices in clinical applications.

Vascular endothelial growth factor receptor 2 (VEGF-R2) is a marker of angiogenesis and metastasis of cancer. Two biosensors for the determination of VEGF-R2 in plasma have been developed. One of them is based on a pure gold chip, and the other on a silver/gold bimetallic chip; both have the receptor, monoclonal rabbit antibody specific for human VEGF-R2, attached to the chip via a cysteamine linker. The biosensor with the gold chip exhibits linearity of the analytical signal between 0.03 and 2 ng/mL, a precision of 1.4% and recovery between 99% and 102%. The biosensor with the bimetallic chip exhibits linearity between 0.03 and 1 ng/mL, a precision of 2.2% and recovery between 99% and 103%. Both biosensors tolerate a 1:100 excess of VEGF, VEGF-R1 and VEGF-R3. Both biosensors were validated by parallel determination of VEGF-R2 in 27 different plasma samples using the ELISA immunosensor assay, with very good agreement of the results. Thermodynamic parameters of the interaction of VEGF-R2 with the antibody were determined by QCM (Quartz Crystal Microbalance) and SPRi (Surface Plasmon Resonance imaging) measurements.

Exosome analysis is a promising tool for clinical and biological research applications. However, detection and biomarker quantification of exosomes is technically challenging because they are small and highly heterogeneous. Here, we report an optical approach for imaging exosomes and quantifying their protein markers without labels using plasmonic scattering microscopy (PSM). PSM can provide improved spatial resolution and distortion-free image compared to conventional surface plasmon resonance (SPR) microscopy, with the signal-to-noise ratio similar to objective coupled surface plasmon resonance (SPR) microscopy, and millimeter-scale field of view as a prism-coupled SPR system, thus allowing exosome size distribution analysis with high throughput. In addition, PSM retains the high specificity and surface sensitivity of the SPR sensors and thus allows selection of exosomes from extracellular vesicles with antibody-modified sensor surfaces and in situ analyzing binding kinetics between antibody and the surface protein biomarkers on the captured exosomes. Finally, the PSM can be easily constructed on a popular prism-coupled SPR system with commercially available components. Thus, it may provide an economical and powerful tool for clinical exosome analysis and exploration of fundamental issues such as exosome biomarker binding properties.

The development of a CMOS manufactured THz sensing platform could enable the integration of state-of-the-art sensing principles with the mixed signal electronics ecosystem in small footprint, low-cost devices. To this aim, in this work we demonstrate a label-free protein sensing platform using highly doped germanium plasmonic antennas realized on Si and SOI substrates and operating in the THz range of the electromagnetic spectrum. The antenna response to different concentrations of BSA shows in both cases a linear response with saturation above 20 mg/mL. Ge antennas on SOI substrates feature a two-fold sensitivity as compared to conventional Si substrates, reaching a value of 6 GHz/(mg/mL), which is four-fold what reported using metal-based metamaterials. We believe that this result could pave the way to a low-cost lab-on-a-chip biosensing platform.

Exosomes are lipid-bilayer enclosed vesicles with diameters of 30-150 nm, which play a pivotal role in cell communication by transporting their cargoes such as proteins, lipids, and genetic materials. In recent years, exosomes have been under intense investigation, as they show great promise in numerous areas, especially as bio-markers in liquid biopsies. However, due to the high heterogeneity and the nano size of exosomes, the separation of exosomes is not easy. This review will deliver an outline of the conventional methods and the microfluidic-based technologies for exosome separation. Particular attention is devoted to microfluidic devices, highlighting the efficiency of exosome isolation by these methods. Additionally, this review will introduce advances made in the integrated microfluidics technologies that enable the separation and analysis of exosomes.

Liquid biopsy has remarkably facilitated clinical diagnosis and surveillance of cancer via employing a non-invasive way to detect cancer-derived components, such as circulating tumor DNA and circulating tumor cells from biological fluid samples. The cancer-derived exosomes, which are nano-sized vesicles secreted by cancer cells have been investigated in liquid biopsy as their important roles in intracellular communication and disease development have been revealed. Given the challenges posed by the complicated humoral microenvironment, which contains a variety of different cells and macromolecular substances in addition to the exosomes, it has attracted a large amount of attention to effectively isolate exosomes from collected samples. In this review, the authors aim to analyze classic strategies for separation of cancer-derived exosomes, giving an extensive discussion of advantages and limitations of these methods. Furthermore, the innovative multi-strategy methods to realize efficient isolation of cancer-derived exosomes in practical applications are also presented. Additionally, the possible development trends of exosome separation in to the future is discussed in this review.

Current methods for the early diagnosis of cancer can be invasive and costly. In recent years, exosomes have been recognized as potential biomarkers for cancer diagnostics. The common methods for quantitative detection of exosomes, such as nanoparticle tracking analysis (NTA) and flow cytometry, rely on large-scale instruments and complex operation, with results not specific for cancer. Herein, we present a tri-channel electrochemical immunobiosensor for enzyme-free and label-free detecting carcino-embryonic antigen (CEA), neuron-specific enolase (NSE), and cytokeratin 19 fragments (Cyfra21-1) from exosomes for specific early diagnosis of lung cancer. The electrochemical immunobiosensor showed good selectivity and stability. Under optimum experimental conditions, the linear ranges were from 10^-3 to 10 ng/mL for CEA, 10^-4 to 10² ng/mL for NSE, and 10^-3 to 10² ng/mL for Cyfra21-1, and a detection limit down to 10^-4 ng/mL was achieved. Furthermore, we performed exosome analysis in three kinds of lung cancer. The results showed a distinct expression level of exosomal markers in different types. These works provide insight into a promising alternative for the quantification of exosomal markers in specific diseases in the following clinical bioassays.