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Abstract Rapid and sensitive nucleic acid detection is vital in disease diagnosis and therapeutic assessment. Herein, we propose a co-freezing localized CRISPR-Cas12a (CL-Cas12a) strategy for sensitive nucleic acid detection. The CL-Cas12a was obtained through a 15-minute co-freezing process, allowing the Cas12a/crRNA complex and hairpin reporter confined on the AuNPs surface with high load efficiency, for rapid sensing of nucleic acid with superior performance to other localized Cas12a strategies. This CL-Cas12a based platform could quantitatively detect targets down to 98 aM in 30 min with excellent specificity. Furthermore, the CL-Cas12a successful applied to detect human papillomavirus infection and human lung cancer-associated single-nucleotide mutations. We also achieved powerful signal amplification for imaging Survivin mRNA in living cells. These findings highlight the potential of CL-Cas12a as an effective tool for nucleic acid diagnostics and disease monitoring. Graphical abstract

Abstract Colorectal cancer (CRC) is a complex malignancy, influenced not only by cancer cells but also by the tumor microenvironment (TME). Within the TME, emerging evidence highlights the presence and functional roles of diverse microbial entities, referred to as intratumoral microbiota. The distribution of these microbiota exhibits significant heterogeneity and engages in dynamic interactions with tumor cells, forming a unique ecosystem. Certain bacterial strains distinctly influence the TME of CRC, affecting the characteristics and progression of the tumor. This review summarizes the intricate roles of intratumoral microbiota within CRC's TME, emphasizing their importance in the disease's development and progression, and discuss the opportunities and challenges in the field.

Abstract Bacterial extracellular vesicles (BEVs) are nano‐size particles secreted by bacteria that carry various bioactive components. These vesicles are thought to provide a new window into the mechanisms by which bacteria affect their hosts, but their fundamental proprieties within human remain poorly understood. Here, we developed a single‐vesicle analytical platform that enabled BEV detection in complex biological samples of host. Using this platform, we found the presence of BEVs in the host circulation and they were mainly derived from gut microbes. We showed that the levels of circulating BEVs in humans significantly increased with aging due to an age‐related increase in intestinal permeability. Significantly different levels of BEVs in blood were also found in patients with colorectal cancer and colitis. Together, our study provides new insights into circulating BEV biology and reveals their potential as a new class of biomarkers.

Abstract The profiling of small extracellular vesicle-associated microRNAs (sEV-miRNAs) plays a vital role in cancer diagnosis and monitoring. However, detecting sEV-miRNAs with low expression in clinical samples remains challenging. Herein, we propose a novel electrochemical biosensor using localized DNA tetrahedron-assisted catalytic hairpin assembly (LDT-CHA) for sEV-miRNA determination. The LDT-CHA contained localized DNA tetrahedrons with CHA substrates, leveraging an efficient localized reaction to enable sensitive and rapid sEV-miRNA measurement. Based on the LDT-CHA, the proposed platform can quantitatively detect sEV-miRNA down to 25 aM in 30 min with outstanding specificity. For accurate diagnosis of gastric cancer patients, a combination of LDT-CHA and a panel of four sEV-miRNAs (sEV-miR-1246, sEV-miR-21, sEV-miR-183-5P, and sEV-miR-142-5P) was employed in a gastric cancer cohort. Compared with diagnosis with single sEV-miRNA, the proposed platform demonstrated a higher accuracy of 88.3% for early gastric tumor diagnoses with higher efficiency (AUC: 0.883) and great potential for treatment monitoring. Thus, this study provides a promising method for the bioanalysis and determination of the clinical applications of LDT-CHA. Graphical Abstract

Abstract Cytopathological examination plays a crucial role in cancer diagnosis as it reflects the cellular pathology of cancer. However, this process traditionally relies on the visual examination by cytopathologists. Recent advancements in computer and digital imaging technologies have enabled the application of artificial intelligence (AI)‐based models to identify tumor cells in images, thereby assisting cytopathologists in achieving enhanced performance. AI‐based models can improve the accuracy and reproducibility of image evaluation and streamline clinical workflows. Moreover, AI‐based models can analyze a diverse range of sample types, including peripheral blood, urine, ascites, and bone marrow. AI‐based cytopathological recognition can help clinicians screen and diagnose cancer, predict prognosis and recurrence of cancers, such as leukemia, cervical cancer, urothelial carcinoma, and gastric cancer. Additionally, AI‐based models can predict the types of mutations in leukemia. A growing number of studies emphasize the potential of computational image analysis and deep learning‐based AI to build novel diagnostic tools that are conducive to the biomedical field. This review describes the recent developments in AI‐based cytopathological recognition and offers a perspective on how AI tools of cytopathology can help improve cancer diagnosis and prognosis prediction. Future developments in AI model applications can further contribute to the improvement of human health.

Abstract Recently, the interest in extracellular vesicles released by bacteria has rapidly increased. Bacterial extracellular vesicles (BEVs) have been involved in bacteria‐bacteria and bacteria‐host interactions, which strengthen health or bring about various pathologies. However, BEV separation, characterization, and functional studies require the establishment of guidelines and further optimization in order to stimulate the development of science in BEV research and a following successful transformation into clinical applications. This position paper is authored by the Microbial Vesicles Task Force of the Chinese Society for Extracellular Vesicles (CSEV) composed of experienced medical laboratory specialists, microbiologists, virologists, biologists and material biologists who are actively engaged in BEV research. Herein, we present a concise description of BEV research and discover challenges and critical gaps in current BEV‐based analyses for clinical applications. Finally, we also offer suggestions and considerations to improve experimental reproducibility and interoperability in BEV research to promote progress in the field.

Abstract Preeclampsia (PE) is a multisystem disorder with high maternal morbidity and mortality rates. Currently, no practical therapeutic approach is available to prevent PE progression, except for early delivery. Gut dysbiosis is associated with PE development. Previous data showed that the abundance of Akkermansia muciniphila (Am) was lower in patients with PE than in normotensive pregnant women. Here, in this study, decreased abundance of Am was observed in a PE mouse model. Also, we found that administration with Am could significantly attenuate systolic blood pressure, promote foetal growth and improve the placental pathology in mice with PE. Moreover, Am‐derived extracellular vesicles (AmEVs) were transferred from the gastrointestinal (GI) tract to the placenta and mitigated pre‐eclamptic symptoms in PE mice. These beneficial effects of AmEVs were mediated by enhanced trophoblast invasion of the spiral artery (SpA) and SpA remodelling through activation of the epidermal growth factor receptor (EGFR)–phosphatidylinositol‐3‐kinase (PI3K)–protein kinase B (AKT) signalling pathway. Collectively, our findings revealed the potential benefit of using AmEVs for PE treatment and highlighted important host–microbiota interactions.

Abstract Detection and imaging of α‐L‐fucosidase (AFU) is of great value to understand its roles in hepatocellular carcinoma (HCC) and tumor early diagnosis, but ideal assays are still lacking. Herein, a near‐infrared (NIR) fluorescent biosensor (α‐Fuc‐DCM) was elaborately designed and synthesized for rapid and ratiometric detection of AFU activity in cells and HCC tumor mouse models. In the presence of AFU, this biosensor shows an enhancement in NIR emission in a ratiometric manner, which significantly improves the detection accuracy with the limit of detection as low as 4.8 mU/mL. Taking advantage of these merits, the activity of AFU in lysosomes could be visualized using ratiometric and NIR dual modality in living cells. Furthermore, its remarkable application for monitoring of endogenous AFU activity in HCC tumor‐bearing mouse model is also demonstrated with bright fluorescence signal, which indicated that the biosensor could clearly monitor the liver tumor in the early stage. Importantly, the α‐Fuc‐DCM probe can be utilized to detect the AFU in serum from HCC patients. This strategy offers a promising biosensor system for early diagnosis of HCC and studying the roles of AFU in cancers.

Abstract Subarachnoid hemorrhage (SAH) is a severe subtype of stroke caused by the rupturing of blood vessels in the brain. The ability to accurately assess the degree of bleeding in an SAH model is crucial for understanding the brain‐damage mechanisms and developing therapeutic strategies. However, current methods are unable to monitor microbleeding owing to their limited sensitivities. Herein, a new bleeding assessment system using a bioprobe TTVP with aggregation‐induced emission (AIE) characteristics is demonstrated. TTVP is a water‐soluble, small‐molecule probe that specifically interacts with blood. Taking advantage of its AIE characteristics, cell membranes affinity, and albumin‐targeting ability, TTVP fluoresces in bleeding areas and detects the presence of blood with a high signal‐to‐noise (S/N) ratio. The degree of SAH bleeding in an endovascular perforation model is clearly evaluated based on the intensity of the fluorescence observed in the brain, which enables the ultrasensitive detection of mirco‐bleeding in the SAH model in a manner that outperforms the current imaging strategies. This method serves as a promising tool for the sensitive analysis of the degree of bleeding in SAHs and other hemorrhagic diseases.

Abstract 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 (PO43−‐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.

Abstract Background Acute respiratory infections (ARI) cause considerable morbidity and mortality worldwide, especially in children. Unfortunately, there are limited multi-center data on common viral respiratory infections in south China. Methods A total of 4403 nasal swabs were collected from children in 10 cities in Guangdong, China in 2019. Seven respiratory viruses, influenza A virus (IFA), influenza B virus (IFB), respiratory syncytial virus (RSV), adenoviruses (ADV) and parainfluenza virus types 1–3 (PIV1, PIV2 and PIV3), were detected by direct immunofluorescence antibody assay. The personal information and clinical characteristics were recorded and analyzed. Results The results showed that at least one virus was detected in 1099 (24.96 %) samples. The detection rates of RSV, IFA, ADV, PIV3, PIV1 and PIV2 were 7.13 % (314/4403), 5.31 % (234/4403), 4.02 % (177/4403), 3.04 % (134/4403), 1.70 % (75/4403) and 1.16 % (51/4403), respectively. The detection rate of RSV was highest in 0–6-month-old children at 18.18 % (106/583), while the detection rate of IFA was highest in 12–18-year-old children at 20.48 % (17/83). The total detection rates in winter and spring were 35.67 % (219/614) and 34.56 % (403/1166), higher than those in summer, 17.41 % (284/1631), and autumn, 19.46 % (193/992). Conclusions RSV and IFA were the main respiratory viruses in children. With increasing age the detection rate of RSV decreased in children, but the trends for the detection rates of IFA and IFB were the opposite. This study provided the viral etiology and epidemiology of pediatric patients with ARI in Guangdong, China.

Abstract Background Circulating tumor cells (CTCs) has been demonstrated as a promising liquid biopsy marker for breast cancer (BC). However, the intra-patient heterogeneity of CTCs remains a challenge to clinical application. We aim at profiling aggressive CTCs subpopulation in BC utilizing the distinctive metabolic reprogramming which is a hallmark of metastatic tumor cells. Methods Oncomine, TCGA and Kaplan–Meier plotter databases were utilized to analyze expression and survival relevance of the previously screened metastasis-promoting metabolic markers (PGK1/G6PD) in BC patients. CTCs detection and metabolic classification were performed through micro-filtration and multiple RNA in situ hybridization using CD45 and PGK1/G6PD probes. Blood samples were collected from 64 BC patients before treatment for CTCs analysis. Patient characteristics were recorded to evaluate clinical applications of CTCs metabolic subtypes, as well as morphological EMT subtypes classified by epithelial (EpCAM/CKs) and mesenchymal (Vimentin/Twist) markers. Results PGK1 and G6PD expressions were up-regulated in invasive BC tissues compared with normal mammary tissues. Increased tissue expressions of PGK1 or G6PD indicated shortened overall and relapse-free survival of BC patients (P 0 patients. Increased GM+CTCs number and positive rate were correlated with tumor metastasis and progression (P

Summary: Long-term plasma membrane (PM) tracking is important for studying the membrane function and diagnosis of membrane-related diseases. However, current PM probes can easily diffuse into the cytoplasm, which is undesirable. Here, we report an amphipathic aggregation-induced emission (AIE)-active far-red fluorescent probe, named DENPB, for long-term PM imaging. In live cancer cells, DENPB can be turned on after insertion into the PM and shows long-term membrane retention. It can be used for PM tracking for 6 h without obvious internalization. Interestingly, data show that it selectively stained the PM of live cancer cells (e.g., HeLa, HepG2, MDA-MB-231, MCF-7) but showed no signal in live non-cancer cells (e.g., RAW 264.7, NIH 3T3, HFF). It can also distinguish paraformaldehyde-fixed cancer cells from non-cancer cells by showing different signal patterns. This work provides a rational design strategy for a long-term PM-retention probe and a useful tool for cancer cell-specific staining that may greatly contribute to cancer diagnosis.

Arterial remodeling is a major pathological consequence of hypertension, which is recognized as the most common chronic non-communicable disease. However, the detailed mechanism of how arterial remodeling is induced by hypertension has not yet been fully elucidated. Evaluating the transcriptional changes in arterial tissue in response to elevated blood pressure at an early stage may provide new insights and identify novel therapeutic candidates in preventing arterial remodeling. Here, we used the ascending aorta of the transverse aortic constriction (TAC) model to induce arterial remodeling in C57BL/6 male mice. Age-matched mice were subjected to sham surgery as controls. The TAC model was only considered successful if the mice conformed to the criteria (RC/LC blood flow velocity with 5–10-fold change) 1 week after the surgery. Two weeks after surgery, the ascending aorta developed severe remodeling in TAC mice as compared to the sham group. High throughput sequencing was then applied to identify differentially expressed (DE) transcripts. In silicon analysis were then performed to systematically network transcriptional changes. A total of 1,019 mRNAs were significantly changed between TAC and the sham group at the transcriptional level. GO (Gene Ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) analysis revealed that stress/stimulus/immune-related biological processes played a crucial role during arterial remodeling. Our data provide a comprehensive understanding of global gene expression changes in the TAC model, which suggests that targeting inflammation and vascular smooth cell transformation are potential therapeutic strategies to interfere with the aortic remodeling at an early stage in the development of hypertension.

Abstract Noninvasive prenatal detection of monogenic diseases based on cell‐free DNA is hampered by challenges in obtaining a sufficient fraction and adequate quality of fetal DNA. Analyzing rare trophoblastic cells from Papanicolaou smears carrying the entire fetal genome provides an alternative method for noninvasive detection of monogenic diseases. However, intracellular labeling for identification of target cells can affect the quality of DNA in varying degrees. Here, a new approach is developed for nondestructive identification of rare fetal cells from abundant maternal cells based on endoplasmic reticulum staining and linear discriminant analysis (ER‐LDA). Compared with traditional methods, ER‐LDA has little effect on cell quality, allowing trophoblastic cells to be analyzed on the single‐cell level. Using ER‐LDA, high‐purity of trophoblastic cells can be identified and isolated at single cell resolution from 60 pregnancies between 4 and 38 weeks of gestation. Pathogenic variants, including –SEA/ deletion mutation and point mutations, in 11 fetuses at risk for α‐ or β‐thalassemia can be accurately detected by this test. The detection platform can also be extended to analyze the mutational profiles of other monogenic diseases. This simple, low‐cost, and noninvasive test can provide valuable fetal cells for fetal genotyping and holds promise for prenatal detection of monogenic diseases.

Abstract Liquid biopsies of cancer via single‐cell molecular profiling of circulating tumor cells (CTCs) are hampered by the lack of ideal CTC markers. In this study, it is reported that TPN, a bioprobe with aggregation‐induced emission (AIE) activity is capable of distinguishing various tumor cells from blood leukocytes based on the difference in cell mitochondria. TPN is a cell‐permeant live‐cell stain that has little effect on cell viability and integrity, enabling single‐cell DNA/RNA analysis with improved efficiency compared with traditional antibody‐based methods. Using TPN labeling, CTCs and CTC cluster are detected in the blood from patients with lung or liver cancer. The capability of TPN to identify rare tumor cells in the malignant pleural effusion samples is also demonstrated. Furthermore, RNA sequencing of single lung CTC identified by TPN is successfully performed. The findings presented here provide an antibody‐free, low‐cost, and nondisruptive approach for detection and genomic characterization of viable tumor cells based on a mitochondria‐targeting AIE luminogen. It might serve as a new tool for monitoring of genomics dynamic of tumor and unraveling the mechanisms of tumor metastasis.

Abstract Background Circulating tumor cells (CTCs), an advantageous target of liquid biopsy, is an important biomarker for the prognosis and monitoring of cancer. Currently, detection techniques for CTCs are mainly based on the physical and/or epithelial characteristics of tumor cells. However, biofunctional activity markers that can indicate the high metastatic capacity of CTCs are lacking. Methods Functional microarray, quantitative real-time polymerase chain reaction, and Western blot were used on five prostate cancer cell lines with different metastatic capacities to identify the metastasis-related metabolic genes. The identified genes were detected in the CTCs of 64 clinical samples using the RNA in situ hybridization. A multi-criteria weighted model was used to determine the optimal metabolic markers for the CTCs test. Based on five fluorescent signals targeting DAPI, CD45, metabolic, epithelial (EpCAM/CKs), and mesenchymal (Vimentin/Twist) markers, the filtration-enriched CTCs were classified as GM+CTCs/GM−CTCs (metabolic types) or E-CTCs/H-CTCs/M-CTCs (EMT types). Correlation analysis and ROC curve were conducted on 54 prostate cancer samples to evaluate the clinical significance of CTCs subtypes. Results Eight metastasis-related metabolic genes were identified, including HK2, PDP2, G6PD, PGK1, PHKA1, PYGL, PDK1, and PKM2. Among them, PGK1 and G6PD were determined as optimal glucose metabolic (GM) markers for CTCs. GM+CTCs (marked by PGK1/G6PD) were detectable in 64.8% (35/54) of prostate cancer patients, accounting for 46.5% (134/288) of total CTCs. An increased GM+CTCs level was associated with advanced tumor stage and metastasis (P

Exosomes, membrane vesicles of 40–100 nm in diameter, are derived from endosomes in various cells. The bioactive molecules specifically packed into exosomes can be horizontally transferred into recipient cells changing their biological properties, by which tumour cells continuously modify their surrounding microenvironment and distant target cells favouring cancer metastasis. It has been suspected for a long time that exosomes participate in the whole process of tumour metastasis. Although there is much unknown and many controversies in the role of cancer exosome, the major contribution of tumour-associated exosomes to different steps of cancer metastasis are demonstrated in this review. Mainly because these exosomes are easily accessible and capable of representing their parental cells, exosomes draw much attention as a promising biomarker for tumour screening, diagnosis and prognosis. Currently, researchers have found numerous biomarkers in exosomes with great potential to be utilized in personalized medicine. In this article, we summarize the roles of biomarkers, which are validated by clinical samples. Even though many conundrums remain, such as exosome extraction, large multicentre validation of biomarkers and data interpretation, exosomes are certain to be used in clinical practice in the near future as the field rapidly expands.

Since glucose biosensors are one of the most popular and widely used point-of-care testing devices, a novel electrochemical enzyme-linked immunosorbent assay (ELISA) for protein biomarkers has been developed based on a glucose detection strategy. In this study, α-fetoprotein (AFP) was used as the target protein. An electrochemical ELISA system was constructed using anti-AFP antibodies immobilized on microwell plates as the capture antibody (Ab1) and multi-label bioconjugates as signal tracer. The bioconjugates were synthesized by attaching glucoamylase and the secondary anti-AFP antibodies (Ab2) to gold nanoparticles (AuNPs). After formation of the sandwich complex, the Ab2-glucoamylase-AuNPs conjugates converted starch into glucose in the presence of AFP. The concentration of AFP can be calculated based on the linear relation between AFP and glucose, the concentration of which can be detected by the glucose biosensor. When the AFP concentration ranged from 0.05 to 100 ng/mL, a linear calibration plot (i (µA) = 13.62033 − 2.86252 logCAFP (ng/mL), r = 0.99886) with a detection limit of 0.02 ng/mL was obtained under optimal conditions. The electrochemical ELISA developed in this work shows acceptable stability and reproducibility, and the assay for AFP spiked in human serum also shows good recovery (97.0%–104%). This new method could be applied for detecting any protein biomarker with the corresponding antibodies.

Acquired immune deficiency syndrome (AIDS) is a severe communicable immune deficiency disease caused by the human immune deficiency virus (HIV). The analysis laboratory diagnosis of HIV infection is a crucial aspect of controlling AIDS. The p24 antigen, the HIV-1 capsid protein, is of considerable diagnostic interest because it is detectable several days earlier than host-generated HIV antibodies following HIV exposure. We present herein a new sandwich HIV p24 immunosensor based on directly electroplating an electrode surface with gold nanoparticles using chronoamperometry, which greatly increased the conductivity and reversibility of the electrode. Under optimum conditions, the electrochemical signal showed a linear relationship with the concentration of p24, ranging from 0.01 ng/mL to 100 ng/mL (R > 0.99), and the detection limit was 0.008 ng/mL. Compared with ELISA, this method increased the sensitivity by more than two orders of magnitude (the sensitivity of ELISA for p24 is about 1 ng/mL). This immunosensor may be broadly applied to clinical samples, being distinguished by its ease of use, mild reaction conditions, guaranteed reproducibility, and good anti-interference ability.