Mutation detection of urinary cell-free DNA via catch-and-release isolation on nanowires for liquid biopsy.

Cell-free DNA (cfDNA) and extracellular vesicles (EVs) are molecular biomarkers in liquid biopsies that can be applied for cancer detection, which are known to carry information on the necessary conditions for oncogenesis and cancer cell-specific activities after oncogenesis, respectively. Analyses for both cfDNA and EVs from the same body fluid can provide insights into screening and identifying the molecular subtypes of cancer; however, a major bottleneck is the lack of efficient and standardized techniques for the isolation of cfDNA and EVs from clinical specimens. Here, we achieved catch-and-release isolation by hydrogen bond-mediated binding of cfDNA in urine to zinc oxide (ZnO) nanowires, which also capture EVs by surface charge, and subsequently we identified genetic mutations in urinary cfDNA. The binding strength of hydrogen bonds between single-crystal ZnO nanowires and DNA was found to be equal to or larger than that of conventional hydrophobic interactions, suggesting the possibility of isolating trace amounts of cfDNA. Our results demonstrated that nanowire-based cancer screening assay can screen cancer and can identify the molecular subtypes of cancer in urine from brain tumor patients through EV analysis and cfDNA mutation analysis. We anticipate our method to be a starting point for more sophisticated diagnostic models of cancer screening and identification.

Tailoring ZnO nanowire crystallinity and morphology for label-free capturing of extracellular vesicles.

Zinc oxide (ZnO) nanowires have shown their potential in isolation of cancer-related biomolecules such as extracellular vesicles (EVs), RNAs, and DNAs for early diagnosis and therapeutic development of diseases. Since the function of inorganic nanowires changes depending on their morphology, previous studies have established strategies to control the morphology and have demonstrated attainment of improved properties for gas and organic compound detection, and for dye-sensitized solar cells and photoelectric conversion performance. Nevertheless, crystallinity and morphology of ZnO nanowires for capturing EVs, an important biomarker of cancer, have not yet been discussed. Here, we fabricated ZnO nanowires with different crystallinities and morphologies using an ammonia-assisted hydrothermal method, and we comprehensively analyzed the crystalline nature and oriented growth of the synthesized nanowires by X-ray diffraction and selected area electron diffraction using high resolution transmission electron microscopy. In evaluating the performance of label-free EV capture in a microfluidic device platform, we found both the crystallinity and morphology of ZnO nanowires affected EV capture efficiency. In particular, the zinc blende phase was identified as important for crystallinity, while increasing the nanowire density in the array was important for morphology to improve EV capture performance. These results highlighted that the key physicochemical properties of the ZnO nanowires were related to the EV capture performance.

Molecular profiling of extracellular vesicles via charge-based capture using oxide nanowire microfluidics.

Extracellular vesicles (EVs) have shown promising features as biomarkers for early cancer diagnoses. The outer layer of cancer cell-derived EVs consists of organotropic metastasis-induced membrane proteins and specifically enriched proteoglycans, and these molecular compositions determine EV surface charge. Although many efforts have been devoted to investigating the correlation between EV subsets obtained through density-, size-, and immunoaffinity-based captures and expressed membrane proteins, understanding the correlation between EV subsets obtained through surface charge-based capture and expressed membrane proteins is lacking. Here, we propose a methodology to profile membrane proteins of EV subsets obtained through surface charge-based capture. Nanowire-induced charge-based capture of EVs and in-situ profiling of EV membrane proteins are the two key methodology points. The oxide nanowires allowed EVs to be obtained through surface charge-based capture due to the diverse isoelectric points of the oxides and the large surface-to-volume ratios of the nanowire structures. And, with the ZnO nanowire device, whose use does not require any purification and concentration processes, we demonstrated the correlation between negatively-charged EV subsets and expressed membrane proteins derived from each cell. Furthermore, we determined that a colon cancer related membrane protein was overexpressed on negatively charged surface EVs derived from colon cancer cells.

Annealed ZnO/Al2O3 Core-Shell Nanowire as a Platform to Capture RNA in Blood Plasma.

RNA analytical platforms gained extensive attention recently for RNA-based molecular analysis. However, the major challenge for analyzing RNAs is their low concentration in blood plasma samples, hindering the use of RNAs for diagnostics. Platforms that can enrich RNAs are essential to enhance molecular detection. Here, we developed the annealed ZnO/Al2O3 core-shell nanowire device as a platform to capture RNAs. We showed that the annealed ZnO/Al2O3 core-shell nanowire could capture RNAs with high efficiency compared to that of other circulating nucleic acids, including genomic DNA (gDNA) and cell-free DNA (cfDNA). Moreover, the nanowire was considered to be biocompatible with blood plasma samples due to the crystalline structure of the Al2O3 shell which serves as a protective layer to prevent nanowire degradation. Our developed device has the potential to be a platform for RNA-based extraction and detection.

Rapid Discrimination of Extracellular Vesicles by Shape Distribution Analysis.

A rapid and simple cancer detection method independent of cancer type is an important technology for cancer diagnosis. Although the expression profiles of biological molecules contained in cancer cell-derived extracellular vesicles (EVs) are considered candidates for discrimination indexes to identify any cancerous cells in the body, it takes a certain amount of time to examine these expression profiles. Here, we report the shape distributions of EVs suspended in a solution and the potential of these distributions as a discrimination index to discriminate cancer cells. Distribution analysis is achieved by low-aspect-ratio nanopore devices that enable us to rapidly analyze EV shapes individually in solution, and the present results reveal a dependence of EV shape distribution on the type of cells (cultured liver, breast, and colorectal cancer cells and cultured normal breast cells) secreting EVs. The findings in this study provide realizability and experimental basis for a simple method to discriminate several types of cancerous cells based on rapid analyses of EV shape distributions.

A Method to Analyze Urinary Extracellular Vesicles.

Extracellular vesicles (EVs) play an important role in cell-to-cell communication by carrying molecular messages that reflect physiological and pathological conditions of the parent cells. EVs have been identified in all body fluids; and among them, urine stands out as a sample that is easy and inexpensive to obtain and can be collected over time to monitor changes. Various protocols have been established to study urinary extracellular vesicles (UEVs) and they have shown great potential as a biomarker source for clinical applications, not only for urological, but also non-urological diseases. Due to the high variability and low reproducibility of pre-analytical and analytical methods for UEVs, establishing a standardized protocol remains a challenge in the field of diagnosis. Here, we review UEV studies and present the techniques that are most commonly used, those that have been applied as new developments, and those that have the most potential for future applications. The workflow procedures from the sampling step to the qualitative and quantitative analysis steps are summarized along with advantages and disadvantages of the methodologies, in order to give consideration for choosing the most promising and suitable method to analyze human UEVs.

Alterations of mineralized matrix by lead exposure in osteoblast (MC3T3-E1) culture.

The present study investigated the effect of lead (Pb) on bone ultrastructure and chemistry using an in vitro bone model. MC3T3-E1 preosteoblasts were differentiated and treated with lead acetate at 0.4, 2, 10, and 50 µM. No abnormalities in either cell growth or bone nodule formation were observed with the treated dose of lead acetate. However, Pb treatments could significantly increase Pb accumulation in differentiated osteoblast cultures and upregulate expression of Divalent metal transporter 1 (Dmt1) in a dose dependent manner. Pb treatments also altered the expression of osteogenic genes, including secreted phosphoprotein 1, osteocalcin, type I collagen, and osteoprotegerin. Moreover, in mineralized osteoblast cultures, Pb was found to be mainly deposited as Pb salts and oxides, respectively. Ultrastructure analysis revealed Pb localizing with calcium and phosphorus in the mineralized matrix. In mineralizing osteoblast cells, Pb was found in the intracellular calcified vesicles which is one of the bone mineralization mechanisms. Pb was also present in mineral deposits with various shapes and sizes, such as small and large globular or needle-like mineral deposits representing early to mature stages of mineral deposits. Furthermore, Pb was found more in the globular deposits than the needle shaped mineral crystals. Taken together, our observations revealed how Pb incorporates into bone tissue, and showed a close association with bone apatite.