Sensitive electrochemical biosensor for rapid detection of sEV-miRNA based turbo-like localized catalytic hairpin assembly.

Small extracellular vesicle-associated microRNAs (sEV-miRNAs) have emerged as critical biomarkers for cancer diagnosis, yet the rapid detection of these low-abundance molecules in clinical samples remains a formidable challenge. Herein, a simple turbo-like localized catalytic hairpin assembly (TL-CHA) was proposed for sEV-miR-1246 measurement. This electrochemical sensor achieves dual localization through the ingeniously use of AuNPs and DNA nanowires, which provides rich sites for CHA cascade amplification, significantly enhancing the effective reaction and amplify the detection response. Leveraging this innovative design, this biosensor demonstrated the ability to detect sEV-miRNA at concentrations as low as 5.24 aM in a time frame of 30 min. The precision of the measurements was validated through reverse transcription quantitative polymerase chain reaction. Furthermore, the sensor was used for analyzing plasma samples from gastric cancer patients yielded AUC values of 0.973 for all stages and 0.945 for early stages. This demonstrates the sensor's robust performance in both the staging diagnosis and early screening of gastric cancer. Therefore, this platform has great potential for the clinical cancer diagnosis.

Controlled-release hydrogel loaded with magnesium-based nanoflowers synergize immunomodulation and cartilage regeneration in tendon-bone healing.

Tendon-bone interface injuries pose a significant challenge in tissue regeneration, necessitating innovative approaches. Hydrogels with integrated supportive features and controlled release of therapeutic agents have emerged as promising candidates for the treatment of such injuries. In this study, we aimed to develop a temperature-sensitive composite hydrogel capable of providing sustained release of magnesium ions (Mg2+). We synthesized magnesium-Procyanidin coordinated metal polyphenol nanoparticles (Mg-PC) through a self-assembly process and integrated them into a two-component hydrogel. The hydrogel was composed of dopamine-modified hyaluronic acid (Dop-HA) and F127. To ensure controlled release and mitigate the "burst release" effect of Mg2+, we covalently crosslinked the Mg-PC nanoparticles through coordination bonds with the catechol moiety within the hydrogel. This crosslinking strategy extended the release window of Mg2+ concentrations for up to 56 days. The resulting hydrogel (Mg-PC@Dop-HA/F127) exhibited favorable properties, including injectability, thermosensitivity and shape adaptability, making it suitable for injection and adaptation to irregularly shaped supraspinatus implantation sites. Furthermore, the hydrogel sustained the release of Mg2+ and Procyanidins, which attracted mesenchymal stem and progenitor cells, alleviated inflammation, and promoted macrophage polarization towards the M2 phenotype. Additionally, it enhanced collagen synthesis and mineralization, facilitating the repair of the tendon-bone interface. By incorporating multilevel metal phenolic networks (MPN) to control ion release, these hybridized hydrogels can be customized for various biomedical applications.

Turmeric-Derived Nanoparticles Functionalized Aerogel Regulates Multicellular Networks to Promote Diabetic Wound Healing.

Regulation of excessive inflammation and impaired cell proliferation is crucial for healing diabetic wounds. Although plant-to-mammalian regulation offers effective approaches for chronic wound management, the development of a potent plant-based therapeutic presents challenges. This study aims to validate the efficacy of turmeric-derived nanoparticles (TDNPs) loaded with natural bioactive compounds. TDNPs can alleviate oxidative stress, promote fibroblast proliferation and migration, and reprogram macrophage polarization. Restoration of the fibroblast-macrophage communication network by TDNPs stimulates cellular regeneration, in turn enhancing diabetic wound healing. To address diabetic wound management, TDNPs are loaded in an ultralight-weight, high swelling ratio, breathable aerogel (AG) constructed with cellulose nanofibers and sodium alginate backbones to obtain TDNPs@AG (TAG). TAG features wound shape-customized accessibility, water-adaptable tissue adhesiveness, and capacity for sustained release of TDNPs, exhibiting outstanding performance in facilitating in vivo diabetic wound healing. This study highlights the potential of TDNPs in regenerative medicine and their applicability as a promising solution for wound healing in clinical settings.

Isolation and Enrichment of Extracellular Vesicles with Double-Positive Membrane Protein for Subsequent Biological Studies.

The isolation and enrichment of specific extracellular vesicle (EV) subpopulations are essential in the context of precision medicine. However, the current methods predominantly rely on a single-positive marker and are susceptible to interference from soluble proteins or impurities. This limitation represents a significant obstacle to the widespread application of EVs in biological research. Herein, a novel approach that utilizes proximity ligation assay (PLA) and DNA-RNA hybridization are proposed to facilitate the binding of two proteins on the EV membrane in advance enabling the isolation and enrichment of intact EVs with double-positive membrane proteins followed by using functionalized magnetic beads for capture and enzymatic cleavage for isolated EVs release. The isolated subpopulations of EVs can be further utilized for cellular uptake studies, high-throughput small RNA sequencing, and breast cancer diagnosis. Hence, developing and implementing a specialized system for isolating and enriching a specific subpopulation of EVs can enhance basic and clinical research in this field.

Biomimetic cryogel promotes the repair of osteoporotic bone defects through altering the ROS niche via down-regulating the ROMO1.

Osteoporosis is a systemic bone disease that is prone to fractures due to decreased bone density and bone quality, and delayed union or nonunion often occurs in osteoporotic fractures. Therefore, it is particularly important to develop tissue engineering materials to promote osteoporotic fracture healing. In this study, a series of biomimetic cryogels prepared from the decellularized extracellular matrix (dECM), methacrylate gelatin (GelMA), and carboxymethyl chitosan (CMCS) via unidirectional freezing, photo- and genipin crosslinking were applied for the regeneration of osteoporotic fractures. Specifically, dECM extracted from normal or osteoporotic rats was applied for the preparation of the cryogels, named as GC-Normal dECM or GC-OVX dECM, respectively. It was verified that the GC-Normal dECM demonstrated superior performance in promoting the proliferation of BMSCs isolated from osteoporotic rats (OVX-BMSCs), and the differentiation of OVX-BMSCs into osteoblasts both in vitro and in vivo. RNA sequencing and further verifications confirmed that GC-Normal dECM cryogel could scavenge the intracellular reactive oxygen species (ROS) in OVX-BMSCs to accelerate the regeneration of osteoporotic fracture by down-regulating the reactive oxygen species modulator 1 (Romo1). The results indicated that by regulating the ROS niche of OVX-BMSCs, biomimetic the GC-Normal dECM cryogel was expected to be a clinical candidate for repairing osteoporotic bone defects.

Design strategies and advanced applications of primer exchange reactions in biosensing: A review.

Early disease diagnosis relies on the sensitive detection and imaging of biomarkers. Signal amplification is one of the most commonly used methods to improve detection sensitivity. Primer exchange reaction (PER) is a novel signal amplification technique that has garnered attention because of its simple and sensitive features. The classical PER involves a single catalytic hairpin, which enables the attachment of custom sequences to the primer chain, generating a long repeat sequence that can bind numerous signaling molecules and achieve powerful signal amplification. Currently, numerous PER-based signal amplification strategies are available that can improve detection sensitivity and promote the development of the signal amplification field. This review focuses on the mechanism of typical PER, the diversification of PER, and PER-based biosensors for various targets. Finally, the challenges and prospects of PER development are discussed.

A sensitive and rapid electrochemical biosensor for sEV-miRNA detection based on domino-type localized catalytic hairpin assembly.

Small extracellular-vesicule-associated microRNA (sEV-miRNA) is an important biomarker for cancer diagnosis. However, rapid and sensitive detection of low-abundance sEV-miRNA in clinical samples is challenging. Herein, a simple electrochemical biosensor that uses a DNA nanowire to localize catalytic hairpin assembly (CHA), also called domino-type localized catalytic hairpin assembly (DT-LCHA), has been proposed for sEV-miRNA1246 detection. The DT-LCHA offers triple amplification, (i). CHA system was localized in DNA nanowire, which shorten the distance between hairpin substrate, inducing the high collision efficiency of H1 and H2 and domino effect. Then, larger numbers of CHAs were triggered, capture probe bind DT-LCHA by exposed c sites. (ii) The DNA nanowire can load large number of electroactive substance RuHex as amplified electrochemical signal tags. (iii) multiple DT-LCHA was carried by the DNA nanowire, only one CHA was triggered, the DNA nanowire was trapped by the capture probe, which greatly improve the detection sensitivity, especially when the target concentration is extremely low. Owing to the triple signal amplification in this strategy, sEV-miRNA at a concentration of as low as 24.55 aM can be detected in 20 min with good specificity. The accuracy of the measurements was also confirmed using reverse transcription quantitative polymerase chain reaction. Furthermore, the platform showed good performance in discriminating healthy donors from patients with early gastric cancer (area under the curve [AUC]: 0.96) and was equally able to discriminate between benign gastric tumors and early cancers (AUC: 0.77). Thus, the platform has substantial potential in biosensing and clinical diagnosis.

Immunogenic radiation therapy for enhanced anti-tumor immunity via core-shell nanocomposite-mediated multiple strategies.

Background: Due to the immunosuppressive tumor microenvironment (TME), radiation therapy (RT)-mediated immune response is far from satisfactory. How to improve the efficacy of immunogenic RT by priming strong immunogenic cell death (ICD) is an interesting and urgent challenge. Methods: A polyacrylic acid-coated core-shell UiO@Mn3O4 (denoted as UMP) nanocomposite is constructed for immunogenic RT via multiple strategies. Results: Reshaping the TME via Mn3O4-mediated integration of O2 production, GSH depletion, ROS generation and cell cycle arrest, accompanied by Hf-based UiO-mediated radiation absorption, eventually amplifies UMP-mediated RT to induce intense ICD. With the potent ICD induction and reprogrammed tumor-associated macrophages, this synergetic strategy can promote dendritic cells maturation and CD8+ T cells infiltration, and potentiate anti-tumor immunity against primary, distant, and metastatic tumors. Conclusion: This work is expected to shed light on the immunosuppressive TME-reshaping via multiple strategies to reinforce the immunogenic RT outcome and facilitate the development of effective cancer nanomedicine.

Engineered Enzyme-Loaded Erythrocyte Vesicles Precisely Deprive Tumoral Nutrients to Induce Synergistic Near-Infrared-II Photothermal Therapy and Immune Activation.

Starvation therapy has been considered a promising strategy in cancer treatment for altering the tumor microenvironment (TME) and causing a cascade of therapeutic effects. However, it is still highly challenging to establish a therapeutic strategy for precisely and potently depriving tumoral nutrition. In this study, a glucose oxidase (GOx) and thrombin-incorporated erythrocyte vesicle (EV) with cyclic (Arg-Gly-Asp) (cRGD) peptide modification, denoted as EV@RGT, were synthesized for precisely depriving tumoral nutrition and sequentially inducing second near-infrared region (NIR-II) photothermal therapy (PTT) and immune activation. The EV@RGT could specifically accumulate at the tumor site and release the enzymes at the acidic TME. The combination of GOx and thrombin exhausts tumoral glucose and blocks the nutrition supply at the same time, resulting in severe energy deficiency and reactive oxygen species (ROS) enrichment within tumor cells. Subsequently, the abundant clotted erythrocytes in tumor vessels present outstanding localized NIR-II PTT for cancer eradication owing to the hemoglobin. Furthermore, the abundant ROS generated by enhanced starvation therapy repolarizes resident macrophages into the antitumor M1 phenotype via a DNA damage-induced STING/NF-κB pathway, ultimately contributing to tumor elimination. Consequently, the engineered EV@RGT demonstrates powerful antitumor efficiency based on precise nutrition deprivation, sequential NIR-II PTT, and immune activation effect. This work provides an effective strategy for the antitumor application of enzyme-based reinforced starvation therapy.

NIR-II Responsive Nanohybrids Incorporating Thermosensitive Hydrogel as Sprayable Dressing for Multidrug-Resistant-Bacteria Infected Wound Management.

Developing an effective dressing against bacterial infection and synchronously addressing wound complications, such as bleeding, long-term inflammation, and reinfection, are highly desirable in clinical practice. In this work, a second near-infrared (NIR-II) responsive nanohybrid consisting of imipenem encapsulated liposome with gold-shell and lipopolysaccharide (LPS)-targeting aptamer, namely ILGA, is constructed for bacteria elimination. Benefiting from the delicate structure, ILGA exhibits strong affinity and a reliable photothermal/antibiotic therapeutic effect toward multidrug-resistant Pseudomonas aeruginosa (MDR-PA). Furthermore, by incorporating ILGA with a thermosensitive hydrogel poly(lactic-co-glycolic acid)-polyethylene glycol-poly(lactic-co-glycolic acid) (PLGA-PEG-PLGA), a sprayable dressing ILGA@Gel was prepared, which enables a quick on-demand gelation (10 s) for wound hemostasis and offers excellent photothermal/antibiotic efficacy to sterilize the infected wound. Additionally, ILGA@Gel provides satisfactory wound-healing environments by reeducating wound-associated macrophages for inflammation alleviation and forming a gel layer to block exogenous bacterial reinfection. This biomimetic hydrogel reveals excellent bacteria eradication and wound recovery effectiveness, demonstrating its promising potential for managing complicated infected wounds.

Selective Photothermal Therapy Based on Lipopolysaccharide Aptamer Functionalized MoS2 Nanosheet-Coated Gold Nanorods for Multidrug-Resistant Pseudomonas aeruginosa Infection.

Chronic wounds infected by multidrug-resistant gram-negative bacteria have evolved resistance to traditional antibiotic therapy, posing a threat to global public health in recent years. Herein, a selective therapeutic nanorod (MoS2 -AuNRs-apt) based on molybdenum disulfide (MoS2 ) nanosheets coated gold nanorods (AuNRs) targeting lipopolysaccharide (LPS) is presented. AuNRs have excellent photothermal conversion efficiency in 808 nm laser-guided photothermal therapy (PTT), and the MoS2 nanosheets coating significantly enhances the biocompatibility of AuNRs. Furthermore, the conjugation of the nanorods with aptamer permits active targeting of LPS on the surface of gram-negative bacteria and a specific anti-inflammatory ability in the multidrug-resistant Pseudomonas aeruginosa (MRPA)-infected wound murine model. It is concluded that the antimicrobial effect of these nanorods is considerably more significant than non-targeted PTT. Moreover, they can precisely overcome MRPA bacteria by physical damage and effectively reduce excess M1 inflammatory macrophages to accelerate the healing of infected wounds. Overall, this molecular therapeutic strategy displays great potential as a prospective antimicrobial treatment for MRPA infections.

Quantification of placental extracellular vesicles in different pregnancy status via single particle analysis method.

BACKGROUND: The nano-sized, lipid bilayer-delimited placental extracellular vesicles (PEVs) released by the placenta are now regarded as important mediators involved in various physiological and pathological processes of pregnant women. The number and contents of PEVs are significantly altered in preeclampsia and are considered as potential biomarkers. However, the distribution pattern of PEVs in the maternal circulation in different pregnancy status is still unclear for the limitation of the traditional method with low sensitivity. METHODS: In this work, we recruited 561 pregnant women with different pregnancy status and investigated the distribution pattern of PEVs in the maternal circulation based on a single extracellular vesicle analysis method and placental alkaline phosphatase (PLAP), a placenta-specific marker. RESULTS: The concentration of PEVs in pregnant women increased with the progression of gestational age, while the ratio of PEVs decreased to about 10% in the third trimester. Surprisingly, the PLAP+ EVs also presented in the plasma of non-pregnant women and normal male about 5%. The change in the ratio of PEVs can reflect the pregnancy status and also had a better diagnostic value in severe preeclampsia (AUC = 0.7811). CONCLUSIONS: Our study not only reveals the distribution pattern of PEVs, but also identifies the diagnostic potential of PEVs as biomarkers.

Rapid and efficient isolation platform for plasma extracellular vesicles: EV-FISHER.

Extracellular vesicles (EVs) have found diverse applications in clinical theranostics. However, the current techniques to isolate plasma EVs suffer from burdensome procedures and limited yield. Herein, we report a rapid and efficient EV isolation platform, namely, EV-FISHER, constructed from the metal-organic framework featuring cleavable lipid probes (PO4 3- -spacer-DNA-cholesterol, PSDC). The EV-FISHER baits EVs from plasma by cholesterol and separates them with an ordinary centrifuge. The captured EVs could be released and collected upon subsequent cleavage of PSDC by deoxyribonuclease I. We conclude that EV-FISHER dramatically outperforms the ultracentrifugation (UC) in terms of time (∼40 min vs. 240 min), isolation efficiency (74.2% vs. 18.1%), and isolation requirement (12,800 g vs. 135,000 g). In addition to the stable performance in plasma, EV-FISHER also exhibited excellent compatibility with downstream single-EV flow cytometry, enabling the identification of glypican-1 (GPC-1) EVs for early diagnosis, clinical stages differentiation, and therapeutic efficacy evaluation in breast cancer cohorts. This work portrays an efficient strategy to isolate EVs from complicated biological fluids with promising potential to facilitate EVs-based theranostics.

Microenvironment-driven sequential ferroptosis, photodynamic therapy, and chemotherapy for targeted breast cancer therapy by a cancer-cell-membrane-coated nanoscale metal-organic framework.

Designing and developing nanomedicine based on the tumor microenvironment (TME) for effective cancer treatment is highly desirable. In this work, polyvinyl pyrrolidone (PVP) dispersed nanoscale metal-organic framework (NMOF) of Fe-TCPP (TCPP = tetrakis (4-carboxyphenyl) porphyrin) loaded with hypoxia-activable prodrug tirapazamine (TPZ) and coated by the cancer cell membrane (CM) is constructed (the formed nanocomposite denoted as PFTT@CM). Due to the functionalization with the homologous cancer cell membrane, PFTT@CM is camouflaged to evade the immune clearance and preferentially accumulates at the tumor site. Once internalized by cancer cells, PFTT@CM is activated by the TME through redox reaction and Fenton reaction between Fe3+ in nano-platform and endogenous glutathione (GSH) and hydrogen peroxide (H2O2) to promote GSH exhausting as well as •OH and O2 production, which triggers ferroptosis and dramatically enhances photodynamic therapy (PDT) efficacy. Subsequently, the PDT process mediated by TCPP and light would consume oxygen and aggravate tumor hypoxia to further activate the prodrug TPZ for cancer chemotherapy. As a consequence, the TME-driven PFTT@CM nano-platform not only demonstrated its TME modulation ability but also showed a sequential synergistic therapy, which eventually inhibited the cancer cell proliferation. This multimodal nano-platform is expected to shed light on the design of TME-activatable reaction to reinforce the synergistic therapeutic outcome and facilitate the development of effective cancer nanomedicine.

Multiplexed analysis of small extracellular vesicle-derived mRNAs by droplet digital PCR and machine learning improves breast cancer diagnosis.

Breast cancer has become the leading cause of global cancer incidence and a serious threat to women's health. Accurate diagnosis and early treatment are of great importance to prognosis. Although clinically used diagnostic approaches can be used for cancer screening, accurate diagnosis of breast cancer is still a critical unmet need. Here, we report a 4-plex droplet digital PCR technology for simultaneous detection of four small extracellular vesicle (sEV)-derived mRNAs (PGR, ESR1, ERBB2 and GAPDH) in combination with machine learning (ML) algorithms to improve breast cancer diagnosis. We evaluate the diagnsotic results with and without the assistance of the ML models. The results indicate that ML-assisted analysis exhibits higher diagnostic performance even using a single marker for breast cancer diagnosis, and demonstrate improved diagnostic performance under the best combination of biomarkers and suitable ML diagnostic model. Therefore, multiple sEV-derived mRNAs analysis coupled with ML not only provides the best combination of markers for breast cancer diagnosis, but also significantly improves the diagnostic efficiency of breast cancer.

EDTA-K2 Improves the Detection Sensitivity of SARS-CoV-2 IgM and IgG Antibodies by Chelating Colloidal Gold in the Immunochromatographic Assay.

OBJECTIVE: The coronavirus disease (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is now rapidly spreading globally. Serological tests are an important method to assist in the diagnosis of COVID-19, used for epidemiological investigations. In this study, we aimed to investigate the impact of different types of vacuum collection tubes on the detection of SARS-CoV-2 IgM and IgG antibodies, using the colloidal gold immunochromatographic assay (GICA). PATIENTS AND METHODS: A total of 112 patients with COVID-19 and 200 healthy control subjects with no infection were enrolled in this study. Their serum and plasma were collected into four different types of vacuum blood collection tubes. SARS-CoV-2 IgM and IgG specific antibodies in the plasma and serum were then detected by GICA and chemiluminescence assay (CA), respectively. In addition, the particle sizes of different colloidal gold solutions in the presence of different anticoagulants and coagulants were evaluated by both laser diffraction (Malvern) and confocal laser microscope, respectively. RESULTS: Our results revealed that anticoagulated plasma with EDTA-K2 improved the positive detection rate of SARS-CoV-2 IgM antibodies. Furthermore, our results shown that the detection results by GICA and CA were highly consistent, especially, the results of EDTA-K2 anticoagulated plasma detected by GICA was more consistent with CA results. We confirmed that EDTA-K2 could improve the detection sensitivity of SARS-CoV-2 IgG antibodies by chelating excessive colloidal gold compared with sodium citrate or lithium heparin, these methodologies did not appear to cause false positives. Colloidal gold particles could be chelated and aggregated by EDTA-K2, but not by sodium citrate, lithium heparin and coagulants. CONCLUSION: GICA is widely used to detect antibodies for the advantages of convenient, fast, low cost, suitable for screening large sample and require minimal equipment. In this study, we found that EDTA-K2 amplified the positive antibody signal by chelating colloidal gold and improved the detection sensitivity of SARS-CoV-2 IgM and IgG antibodies when using the GICA. Therefore, we suggested that EDTA-K2 anticoagulated plasma was more suitable for the detection of SARS-CoV-2 antibodies.

Facile and recyclable dopamine sensing by a label-free terbium(III) metal-organic framework.

Herein, we present a facile strategy for dopamine (DA) sensing by a water-stable MOF of {[Tb(Cmdcp)(H2O)3]2(NO3)2·5H2O}n (1, H3CmdcpBr = N-carboxymethyl-(3,5-dicarboxyl)pyridinium bromide). Without any post-modification, MOF 1 functions as an effective fluorescent sensor for the label-free detection of DA with the detection limit of 0.41 µM (S/N = 3). Under the optimum condition of 80 °C, pH 9 for 80 min in Tris-HCl with natural ambient oxygen, DA polymerizes to give polydopamine (pDA), which adheres to the surface of MOF 1 and quenched its green luminescence thoroughly. The sensing process is visible to naked eyes under 365 nm UV light irradiation due to the partial overlap of its excitation spectrum with the absorption spectrum of pDA. The sensing process is not interfered by coexisting of bio-related organic substances, such as glucose (Glu), 5-hydroxytryptamine (5-HT), homocysteine (Hcy), ascorbic acid (AA), uric acid (UA), cysteine (Cys), glutathione (GSH), as well as the presence of metal ions, including Zn2+, Ca2+, Mg2+, Ni2+ and Co2+. The sensing process is also adaptable in biological fluids of serum and urine with satisfactory recoveries ranging from 96.14% to 104.32%.

Selective and recyclable tandem sensing of PO43- and Al3+ by a water-stable terbium-based metal-organic framework.

Herein, a luminescent water-stable terbium-based metal-organic framework (MOF) {[Tb(Cmdcp)(H2O)3]2(NO3)2·5H2O}n (1, H3CmdcpBr = N-carboxymethyl-(3,5-dicarboxyl)pyridinium bromide) has been synthesized and used for the recyclable sensing of PO43- and Al3+ in tandem. MOF 1 acts as a fluorescent sensor for PO43- by the luminescence "turn-off" mechanism with high selectivity over other anions, such as F-, Cl-, Br-, I-, NO3-, H2PO4-, HSO4-, HCO3-, HSO3-, SO42-, CO32- and HPO42-. The formed PO43-@1 complex further acts as the Al3+ sensor with the luminescence "turn-on" mechanism, also with high selectivity over diverse inorganic cations of Fe2+, Mn2+, Co2+, Ni2+, Hg2+, Na+, K+, Li+, Ag+, Mg2+, Ca2+, Cd2+, Pb2+, Cu2+, and Zn2+. The detection process for both PO43- and Al3+ can be directly observed with naked eyes under the UV light at 365 nm. The detection limits for PO43- and Al3+ are 1.1 µM and 6.6 µM, respectively. Such a sensing cycle is further transferable to urine and serum samples with a satisfactory near-quantitative recovery, highlighting its good potential in biologically relevant applications.

The future of Extracellular Vesicles as Theranostics - an ISEV meeting report.

The utilization of extracellular vesicles (EVs) in clinical theranostics has rapidly advanced in the past decade. In November 2018, the International Society for Extracellular Vesicles (ISEV) held a workshop on "EVs in Clinical Theranostic". Here, we report the conclusions of roundtable discussions on the current advancement in the analysis technologies and we provide some guidelines to researchers in the field to consider the use of EVs in clinical application. The main challenges and the requirements for EV separation and characterization strategies, quality control and clinical investigation were discussed to promote the application of EVs in future clinical studies.

Facile fluorescent aptasensor using aggregation-induced emission luminogens for exosomal proteins profiling towards liquid biopsy.

Surface protein patterns of tumor-derived exosomes could be promising noninvasive diagnostic biomarkers for liquid biopsy. However, a convenient and cost-effective platform for exosomal protein profiling is still lacking. Herein, a facile fluorescent aptasensor is developed to assess exosomal tumor-associated proteins, combining aptamers, aggregation-induced emission luminogens (AIEgens), and graphene oxide (GO) as recognition elements, fluorescent dye, and the quencher, respectively. Specifically, numberous TPE-TAs could bind one aptamer and form aggregates rapidly, resulting in an amplified fluorescence signal. In the absence of tumor-derived exosomes, GO absorbs the TPE-TAs/aptamer complex, allowing fluorescence quenching. When the target exosomes are introduced, the aptamer preferentially binds with its target. Thus the TPE-TAs/aptamer complexes detach from GO surface, followed by the appearance of a "turn-on" fluorescent signal. Under the optimized conditions, the linear range of target exosomes is estimated to be 4.07 × 105 to 1.83 × 107 particles/µL (0.68-30.4 pM) with a detection limit of 3.43 × 105 particles/µL (0.57 pM). This strategy demonstrated great performance in differentiating prostate cancer from healthy individuals (AUC: 0.9790). Furthermore, by profiling three tumor-associated protein markers including epidermal growth factor receptor (EGFR), epithelial cell adhesion molecule (EpCAM), and human epidermal growth factor receptor 2 (HER2) on exosomes in a breast tumor cohort, this sensing platform diagnoses breast tumors with high efficiency (AUC: 0.9845) and exhibits a high sensitivity of 97.37% for distinguishing malignant breast cancers, where the stage I cases were detected with 92.31% sensitivity. Therefore, this aptasensor provides a promising strategy to profile tumor-derived exosomal proteins for early diagnosis in liquid biopsy.