Avoiding Pre-Isolation Step in Exosome Analysis: Direct Isolation and Sensitive Detection of Exosomes Using Gold-Loaded Nanoporous Ferric Oxide Nanozymes.

Most of the current exosome-analysis strategies are time-consuming and largely dependent on commercial extraction kit-based preisolation step, which requires extensive sample manipulations, costly isolation kits, reagents, tedious procedures, and sophisticated equipment and is prone to bias/artifacts. Herein we introduce a simple method for direct isolation and subsequent detection of a specific population of exosomes using an engineered superparamagnetic material with multifunctional properties, namely, gold-loaded ferric oxide nanocubes (Au-NPFe2O3NC). In this method, the Au-NPFe2O3NC were initially functionalized with a generic tetraspanin (exosomes-associated) antibody (i.e., CD63) and dispersed in sample fluids where they work as "dispersible nanocarriers" to capture the bulk population of exosomes. After magnetic collection and purification, Au-NPFe2O3NC-bound exosomes were transferred to the tissue-specific, antibody-modified, screen-printed electrode. As a proof of principle, we used a specific placental marker, placenta alkaline phosphatase (PLAP), to detect exosomes secreted from placental cells. The peroxidase-like activity of Au-NPFe2O3NC was then used to accomplish an enzyme-linked immunosorbent assay (ELISA)-based sensing protocol for naked-eye observation along with UV-visible and electrochemical detection of PLAP-specific exosomes present in placental cell-conditioned media. We demonstrated excellent agreement in analytical performance for the detection of placental cell-derived exosomes (i.e., linear dynamic range, 103-107 exosomes/mL; limit of detection, 103 exosomes/mL; relative standard deviation (%RSD) of <5.5% for n = 3) using with and without commercial "total exosome isolation kit"-based preisolation step. We envisage that this highly sensitive, rapid, and inexpensive assay could be useful in quantifying specific populations of exosomes for various clinical applications, focusing on pregnancy complications.

An amplification-free electrochemical detection of exosomal miRNA-21 in serum samples.

Recent evidence suggests that small non-coding RNAs such as microRNA (miRNA) encapsulated in exosomes represent an important mechanism of communication between the cells. Exosomal miRNAs play an important role in carcinogenesis via enhancing the cell to cell communication and targeting the cell growth molecular pathways which in turn facilitate metastasis in cancers. Despite progressive advances, the current methods for the exosomal miRNA detection mostly rely on labor-intensive sequencing approaches which are often prone to amplification bias and require costly and bulky equipment. Herein, we report an electrochemical approach for the detection of cancer-derived exosomal miRNAs in human serum samples by selectively isolating the target miRNA using magnetic beads pre-functionalized with capture probes and then directly adsorbing the targets onto a gold electrode surface. The level of adsorbed miRNA is detected electrochemically in the presence of an [Fe(CN)6]4-/3- redox system. This method enabled an excellent detection sensitivity of 1.0 pM with a relative standard deviation (%RSD) of <5.5% in cancer cells and serum samples (n = 8) collected from patients with colorectal adenocarcinoma (CRC). We believe that our approach could be useful in clinical settings for the quantification of exosomal miRNA in cancer patients.

Biological Functions and Current Advances in Isolation and Detection Strategies for Exosome Nanovesicles.

Exosomes are nanoscale (≈30-150 nm) extracellular vesicles of endocytic origin that are shed by most types of cells and circulate in bodily fluids. Exosomes carry a specific composition of proteins, lipids, RNA, and DNA and can work as cargo to transfer this information to recipient cells. Recent studies on exosomes have shown that they play an important role in various biological processes, such as intercellular signaling, coagulation, inflammation, and cellular homeostasis. These functional roles are attributed to their ability to transfer RNA, proteins, enzymes, and lipids, thereby affecting the physiological and pathological conditions in various diseases, including cancer and neurodegenerative, infectious, and autoimmune diseases (e.g., cancer initiation, progression, and metastasis). Due to these unique characteristics, exosomes are considered promising biomarkers for the diagnosis and prognosis of various diseases via noninvasive or minimally invasive procedures. Over the last decade, a plethora of methodologies have been developed for analyzing disease-specific exosomes using optical and nonoptical tools. Here, the major biological functions, significance, and potential role of exosomes as biomarkers and therapeutics are discussed. Furthermore, an overview of the most commonly used techniques for exosome analysis, highlighting the major technical challenges and limitations of existing techniques, is presented.

Quantum dot-based sensitive detection of disease specific exosome in serum.

Tumor-derived exosomes have emerged as promising cancer biomarkers due to their unique composition and functions. Herein, we report a stripping voltammetric immunoassay for the electrochemical detection of disease-specific exosomes using quantum dots as signal amplifiers. The assay involves three subsequent steps where bulk exosome populations are initially magnetically captured on magnetic beads by a generic tetraspanin antibody (e.g., CD9 or CD63) followed by the identification of disease-specific exosomes using cancer-related. Here, we used CdSe quantum dot (CdSeQD) functionalised-biotinylated HER-2 and FAM134B antibodies as breast and colon cancer markers. After magnetic washing and purification steps, acid dissolution of CdSeQDs and subsequent anodic stripping voltammetric quantification of Cd2+ were carried out at the bare glassy carbon working electrode. This method enabled sensitive detection of 100 exosomes per µL with a relative standard deviation (%RSD) of <5.5% in cancer cell lines and a small cohort of serum samples (n = 9) collected from patients with colorectal adenocarcinoma. We believe that our approach could potentially represent an effective bioassay for the quantification of disease-specific exosomes in clinical samples.

An Electromagnetically Actuated Double-Sided Cell-Stretching Device for Mechanobiology Research.

Cellular response to mechanical stimuli is an integral part of cell homeostasis. The interaction of the extracellular matrix with the mechanical stress plays an important role in cytoskeleton organisation and cell alignment. Insights from the response can be utilised to develop cell culture methods that achieve predefined cell patterns, which are critical for tissue remodelling and cell therapy. We report the working principle, design, simulation, and characterisation of a novel electromagnetic cell stretching platform based on the double-sided axial stretching approach. The device is capable of introducing a cyclic and static strain pattern on a cell culture. The platform was tested with fibroblasts. The experimental results are consistent with the previously reported cytoskeleton reorganisation and cell reorientation induced by strain. Our observations suggest that the cell orientation is highly influenced by external mechanical cues. Cells reorganise their cytoskeletons to avoid external strain and to maintain intact extracellular matrix arrangements.