CRISPR-Cas and catalytic hairpin assembly technology for target-initiated amplification detection of pancreatic cancer specific tsRNAs.

Transfer RNA-derived small RNAs (tsRNAs) tRF-LeuCAG-002 (ts3011a RNA) is a novel class of non-coding RNAs biomarker for pancreatic cancer (PC). Reverse transcription polymerase chain reaction (RT-qPCR) has been unfit for community hospitals that are short of specialized equipment or laboratory setups. It has not been reported whether isothermal technology can be used for detection, because the tsRNAs have rich modifications and secondary structures compared with other non-coding RNAs. Herein, we have employed a catalytic hairpin assembly (CHA) circuit and clustered regularly interspaced short palindromic repeats (CRISPR) to develop an isothermal and target-initiated amplification method for detecting ts3011a RNA. In the proposed assay, the presence of target tsRNA triggers the CHA circuit that transforms new DNA duplexes to activate collateral cleavage activity of CRISPR-associated proteins (CRISPR-Cas) 12a, achieving cascade signal amplification. This method showed a low detection limit of 88 aM at 37 °C within 2 h. Moreover, it was demonstrated for the first time that, this method is less likely to produce aerosol contamination than RT-qPCR by simulating aerosol leakage experiments. This method has good consistency with RT-qPCR in the detection of serum samples and showed great potential for PC-specific tsRNAs point-of-care testing (POCT).

MicroRNAs in extracellular vesicles: Sorting mechanisms, diagnostic value, isolation, and detection technology.

MicroRNAs (miRNAs) are a class of short, single-stranded, noncoding RNAs, with a length of about 18-22 nucleotides. Extracellular vesicles (EVs) are derived from cells and play a vital role in the development of diseases and can be used as biomarkers for liquid biopsy, as they are the carriers of miRNA. Existing studies have found that most of the functions of miRNA are mainly realized through intercellular transmission of EVs, which can protect and sort miRNAs. Meanwhile, detection sensitivity and specificity of EV-derived miRNA are higher than those of conventional serum biomarkers. In recent years, EVs have been expected to become a new marker for liquid biopsy. This review summarizes recent progress in several aspects of EVs, including sorting mechanisms, diagnostic value, and technology for isolation of EVs and detection of EV-derived miRNAs. In addition, the study reviews challenges and future research avenues in the field of EVs, providing a basis for the application of EV-derived miRNAs as a disease marker to be used in clinical diagnosis and even for the development of point-of-care testing (POCT) platforms.

Highly efficient and automated isolation technology for extracellular vesicles microRNA.

MicroRNA (miRNA) in extracellular vesicles (EVs) has great potential to be a promising marker in liquid biopsy. However, the present EV isolation methods, such as ultracentrifugation, have complicated and long-time operation, which impedes research on EV miRNA. The downstream complex miRNA extraction process will also significantly increase the detection cycle and loss. We first established a simple automated technique to efficiently extract target miRNAs in EVs from plasma based on Fe3O4@TiO2 beads with high affinity and capture efficiency. We combined a heat-lysis method for quick and simple EV miRNA extraction and detection. The results indicated that our method has more RNA yield than TRIzol or a commercial kit and could complete EV enrichment and miRNA extraction in 30 min. Through the detection of miRNA-21, healthy people and lung cancer patients were distinguished, which verified the possibility of the application in clinical detection. The automated isolation technology for EV miRNA has good repeatability and high throughput, with great application potential in clinical diagnosis.

A Washing-Free and Easy-to-Operate Fluorescent Biosensor for Highly Efficient Detection of Breast Cancer-Derived Exosomes.

Traditional detection methods for protein tumor markers in the early screening of breast cancer are restricted by complicated operation procedures and unstable reproducibility. As one of alternative emerging tumor markers, exosomes play an important role in diagnosing and treating cancers at the early stage due to traceability of their origins and great involvement in occurrence and development of cancers. Herein, a washing-free and efficient fluorescent biosensor has been proposed to realize simple and straightforward analysis of breast cancer cell-derived exosomes based on high affinity aptamers and G quadruplex-hemin (G4-hemin). The whole reaction process can be completed by several simple steps, which realizes washing-free and labor-saving. With simplified operation procedures and high repeatability, the linear detection range for this developed fluorescent biosensing strategy to breast cancer cell-derived exosomes is from 2.5 × 105 to 1.00 × 107 particles/ml, and the limit of detection is down to 0.54 × 105 particles/ml.

Rapid Capturing and Chemiluminescent Sensing of Programmed Death Ligand-1 Expressing Extracellular Vesicles.

Cancer specific extracellular vesicles (EVs) are of significant clinical relevance, for instance programmed death ligand-1 (PD-L1) expressing EVs (PD-L1@EVs) have been shown to be ideal biomarker for non-invasive diagnosis of cancer and can predate the response of cancer patients to anti-PD-1/PD-L-1 immunotherapy. The development of sensitive and straightforward methods for detecting PD-L1@EVs can be a vital tool for non-invasive diagnosis of cancer. Most of the contemporary methods for EVs detection have limitations such as involvement of long and EV's loss prone isolation methods prior to detection or they have employed expensive antibodies and instruments to accomplish detection. Therefore, we designed an ultracentrifugation-free and antibody-free sensing assay for PD-L1@EV by integrating Titanium oxide (TiO2) coated magnetic beads (Fe3O4@TiO2) rapid capturing of EVs from undiluted serum with aptamers specificity and chemiluminescence (CL) sensitivity. To accomplish this we used Fe3O4@TiO2 beads to rapidly capture EVs from the undiluted patient serum and added biotin labelled PD-L1 aptamer to specifically recognize PD-L1@EVs. Later, added streptavidin-modified Alkaline phosphates (ALP) taking advantage of biotin-streptavidin strong binding. Addition of CDP-star, a chemiluminescent substrate of ALP, initiates the chemiluminiscense that was recorded using spectrophotometer. The sensing assay showed high sensitivity with limit of detection (LOD) as low as 2.584×105 EVs/mL and a wider linear correlation of CL intensity (a.u.) with the concentration of PD-L1@EVs from 105 to 108 EVs/mL. To examine the clinical utility of sensing assay we used undiluted serum samples from lung cancer patients and healthy individuals and successfully discern between healthy individuals and lung cancer patients. We are optimistic that the sensing assay can ameliorate our ability to be able to diagnose lung cancer non-invasively and can be helpful to predate the patient's response to anti-PD-1/PD-L1 immunotherapy.

Combined Detection of Exosome Concentration and Tumor Markers in Gastric Cancer.

It has been an established fact that exosomes act as a mediator in tumor microenvironment as well as participate actively in intercellular communication between cancer cells. Exosomes carry a variety of molecular cargoes that prevent cyclic degradation and represent the cells of their origin. In this study, the difference in expression levels of exosomes was measured for diagnosis of gastric cancer. We isolated exosomes from plasma by size-selective method. The morphology of the exosomes was characterized by transmission electron microscopy, and the particle size and concentration of the exosomes were detected by NanoSight's Nanoparticle Tracking Analysis. Results indicated that the expression level of exosomes in gastric cancer patients was higher than that in healthy individuals. The specificity and sensitivity were 65.2% and 73.1%, respectively. Currently, clinical tumor markers for gastric cancer detection mainly included Carbohydrate antigen 72-4 (CA72-4), Alpha-fetoprotein, Carbohydrate antigen 125, Carbohydrate antigen 19-9 (CA19-9), Carcinoembryonic Antigen, Carbohydrate antigen 242. When we combined positive rate for combined gastric cancer biomarkers, results showed that exosomes concentration +CA19-9 and exosomes concentration +CA72-4 in the two-combined test can provide enough positive rate. Therefore, it can be concluded that for gastric cancer, the concentration of exosomes may be regarded as a diagnostic indicator, eventually.

Recent advances in single extracellular vesicle detection methods.

Extracellular vesicles (EVs) are secreted by a variety of cells. They are known for their pertinent role in intercellular communication, and participation in different pathological processes, making them ideal candidate for utilization as a biomarker for diagnosis and treatment of diseases. In contemporary years, the concept of a well-established liquid biopsy technology, and detection and utilization of EVs as a biomarkers have received unprecedented attention. Many rapid and precise EVs detection methods have been proposed, however, majority of them detect EVs in a bulk. As the prevalent heterogeneity of single extracellular vesicle (SEV) plays an important role in the analysis of disease progression, therefore, to prevent information loss, increased attention has been paid to SEV detection with remarkable successes. Technologies like fluorescence labeling, micro imaging and microfluidic chip were successfully employed for EVs detection at SEV level. This review summarizes the recent advances in SEV detection methods, their potential targets, applications as well as concludes future prospects for developing new SEV detection strategies.

Progress in exosome associated tumor markers and their detection methods.

Exosomes are secreted by cells and are widely present in body fluids. Exosomes contain various molecular constituents of their cells of origin such as proteins, mRNA, miRNAs, DNA, lipid and glycans which are very similar as the content in tumor cells. These contents play an important role in various stages of tumor development, and make the tumor-derived exosome as a hot and emerging biomarker for various cancers diagnosis and management in non-invasive manner. The present problems of exosome isolation and detection hinder the application of exosomes. With the development of exosome isolation and detection technology, the contents of exosomes can be exploited for early cancer diagnosis. This review summarizes the recent progress on exosome-associated tumor biomarkers and some new technologies for exosome isolation and detection. Furthermore, we have also discussed the future development direction in exosome analysis methods.