Extracellular vesicles (EV) have been emerging as potential biomarkers for disease monitoring. In particular, tumor‐derived EV (TDE) are known to carry oncogenic miRNA, so they can be used for ...diagnosis of early cancer by analyzing the expression levels of EV‐miRNA circulating in the blood. Here, using our novel microfluidic device, we rapidly and selectively isolate cancerous EV expressing breast cancer‐derived surface markers CD49f and EpCAM within 2 minutes. Based on seven candidates of miRNA nominated from The Cancer Genome Atlas (TCGA) database, the expression levels of miRNA in TDE were validated in a total of 82 individuals, including 62 breast cancer patients and 20 healthy controls. Among seven candidates, four miRNAs (miR‐9, miR‐16, miR‐21, and miR‐429) from the EV were highly elevated in early‐stage breast cancer patients compared with healthy donors. The combination of significant miRNAs from specific EV has high sensitivities of 0.90, 0.86, 0.88, and 0.84 of the area under the receiver operating characteristic curve (AUC) in each subtype (luminal A, luminal B, HER‐2, and triple‐negative) of early‐stage breast cancer. Our results suggest that the combination of four miRNA signatures of specific EV could serve as a sensitive and specific biomarker and enable early diagnosis of breast cancer using liquid biopsy.
A new combination of miRNA from cancer‐specific extracellular vesicles was proposed for the highly sensitive and specific diagnosis of early‐stage breast cancer in association with a microfluidic‐based immuno‐isolation technique. These findings suggest that miRNA in EV could be used as important biomarkers for the early detection of breast cancer and identification of cancer subtype.
Extracellular vesicles (EVs) are recognized as promising biomarkers for several diseases. However, their conventional isolation methods have several drawbacks, such as poor yields, low purity, and ...time-consuming operations. Therefore, a simple, low-cost, and rapid microfluidic platform has been extensively developed to meet the requirement in biomedical applications. Herein, a modular microfluidic platform is demonstrated to isolate and enrich EVs directly from plasma, in a combination of continuous capture and purification of EVs. The EVs were selectively captured by target-specific antibody-coated beads in a horseshoe-shaped orifice micromixer (HOMM) chip within 2 min. A fish-trap-shaped microfilter unit was subsequently used to elute and purify the affinity-induced captured EVs from the microbeads. The ability of the modular chip to capture, enrich, and release EVs was demonstrated in 5 min (100 μL sample) at high throughput (100 μL min
). The two chips can be modularized or individually operated, depending on the clinical applications such as diagnostics and therapeutics. For the diagnostic applications, the EVs on microbeads can be directly subjected to the molecular analysis whereas the pure EVs should be released from the microbeads for the therapeutic treatments. This study reveals that the fabricated modular chip can be appropriately employed as a platform for EV-related research tools.
•The high-throughput microfluidic chip for enrichment and photothermal lysis of bacteria was introduced.•Concanavalin A was used as binding molecules to bind bacteria to the magnetic particles.•The ...microfluidic chip consists of vortex mixing, magnetic enrichment and photothermal lysis channels.•Bacteria in real food samples were detected by through the pretreatment of the microfluidic device.
We have developed a high-throughput pretreatment microfluidic chip for enrichment of microorganisms in food using magnetic particles and extracting DNAs using photothermal effects of magnetic particles. Magnetic particles modified with Concanavalin A can capture a variety of pathogens in the sample. As magnetic particles and bacteria injected into the microfluidic chip at a high flow rate, they bound actively in the mixing channel. After passing through the mixing channel, the combined bacteria and magnetic particles complexes were captured and enriched by magnetic force at the chambers which rectangular neodymium magnets were assembled in the form of dozens of arrays. After the magnet arrays were removed, the elution buffer was injected at a slightly lower flow rate and the eluted particles were captured in a small lysis chamber. The laser with a wavelength of 532 nm was irradiated at the lysis chamber to dissolve the captured bacteria as strong heat generated by the photothermal effects of the magnetic particles. Finally, the extracted DNAs were detected by real-time PCR.
The outbreak of the COVID-19 pandemic has led to millions of fatalities worldwide. For preventing epidemic transmission, rapid and accurate virus detection methods to early identify infected people ...are urgently needed in the current situation. Therefore, an electrochemical biosensor based on the trans-cleavage activity of CRISPR/Cas13a was developed in this study for rapid, sensitive, and nucleic-acid-amplification-free detection of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Herein, a redox probe conjugated with ssRNA is immobilized on the electrode surface modified with a nanocomposite (NC) and gold nanoflower (AuNF) for enhancing the sensing performance. The SARS-CoV-2 RNA is captured by the Cas13a–crRNA complex, which triggers the RNase function of Cas13a. The enzymatically activated Cas13a–crRNA complex is subsequently introduced to the reRNA-conjugated electrochemical sensor, and consequently cleaves the reRNA. A change in current occurs due to the release of the redox molecule labeled on the reRNA, which is trans-cleaved from the Cas13a–crRNA complex. The biosensor can detect as low as 4.4 × 10−2 fg/mL and 8.1 × 10−2 fg/mL of ORF and S genes, respectively, over a wide dynamic range (1.0 × 10−1 to 1.0 × 105 fg/mL). Moreover, the biosensor was evaluated by measuring SARS-CoV-2 RNA spiked in artificial saliva. The recovery of the developed sensor was found to be in an agreeable range of 96.54–101.21%. The designed biosensor lays the groundwork for pre-amplification-free detection of ultra-low concentrations of SARS-CoV-2 RNA and on-site and rapid diagnostic testing for COVID-19.
•An electrochemical sensor applied with Cas13a trans-cleavage activity is presented for the detection of SARS-CoV-2 RNA.•The high sensitivity of the biosensor was achieved without nucleic acid amplification.•The biosensor could detect as low as 4.4 × 10-2 fg/mL and 8.1 × 10-2 fg/mL of ORF and S genes, respectively.•The recovery of ORF and S genes in the salivary matrix were observed to be 109.4 to 111.3% and 96.5% to 101.2%, respectively.
The severe acute respiratory syndrome (SARS-CoV-2) outbreak triggered global concern and emphasized the importance of virus monitoring. During a seasonal influenza A outbreak, relatively low ...concentrations of 103–104 viral genome copies are available per 1 m3 of air, which makes detection and monitoring very challenging because the limit of detection of most polymerase chain reaction (PCR) devices is approximately 103 viral genome copies/mL. In response to the urgent need for the rapid detection of airborne coronaviruses and influenza viruses, an electrostatic aerosol-to-hydrosol (ATH) sampler was combined with a concanavalin A (ConA)-coated high-throughput microfluidic chip. The samples were then used for PCR detection. The results revealed that the enrichment capacity of the ATH sampler was 30,000-fold for both HCoV-229E and H1N1 influenza virus, whereas the enrichment capacities provided by the ConA-coated microfluidic chip were 8-fold and 16-fold for HCoV-229E and H1N1 virus, respectively. Thus, the total enrichment capacities of our combined ATH sampler and ConA-coated microfluidic chip were 2.4 × 105-fold and 4.8 × 105-fold for HCoV-229E and H1N1 virus, respectively. This methodology significantly improves PCR detection by providing a higher concentration of viable samples.
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•Continuous and simultaneous enrichment of sampled airborne viruses is realized via concanavalin A-coated microfluidic chip.•Microfluidic chip design provides surface aera enough for sampled airborne viruses to be attached.•The integrated sampling and enrichment system provides high concentration of viable samples for PCR detection.
Over the past few decades, circulating tumor cells (CTCs) have been studied as a means of overcoming cancer. However, the rarity and heterogeneity of CTCs have been the most significant hurdles in ...CTC research. Many techniques for CTC isolation have been developed and can be classified into positive enrichment (i.e., specifically isolating target cells using cell size, surface protein expression, and so on) and negative enrichment (i.e., specifically eluting non-target cells). Positive enrichment methods lead to high purity, but could be biased by their selection criteria, while the negative enrichment methods have relatively low purity, but can isolate heterogeneous CTCs. To compensate for the known disadvantages of the positive and negative enrichments, in this study we introduced a two-stage microfluidic chip. The first stage involves a microfluidic magnetic activated cell sorting (μ-MACS) chip to elute white blood cells (WBCs). The second stage involves a geometrically activated surface interaction (GASI) chip for the selective isolation of CTCs. We observed up to 763-fold enrichment in cancer cells spiked into 5 mL of blood sample using the μ-MACS chip at 400 μL/min flow rate. Cancer cells were successfully separated with separation efficiencies ranging from 10.19% to 22.91% based on their EpCAM or HER2 surface protein expression using the GASI chip at a 100 μL/min flow rate. Our two-stage microfluidic chips not only isolated CTCs from blood cells, but also classified heterogeneous CTCs based on their characteristics. Therefore, our chips can contribute to research on CTC heterogeneity of CTCs, and, by extension, personalized cancer treatment.
Much research has been performed over the past several decades in an attempt to conquer cancer. Tissue biopsy is the conventional method for gathering biological materials to analyze cancer and has ...contributed greatly to the understanding of cancer. However, this method is limited because it is time-consuming (requires tissue sectioning, staining, and pathological analysis), costly, provides scarce starting materials for multiple tests, and is painful. A liquid biopsy, which analyzes cancer-derived materials from various body fluids using a minimally invasive procedure, is more practical for real-time monitoring of disease progression than tissue biopsy. Biomarkers analyzable through liquid biopsy include circulating tumor cells (CTCs), exosomes, circulating cell-free DNA (cfDNA), miRNA, and proteins. Research on CTCs has been actively conducted because CTCs provide information on the whole cell, unlike the other biomarkers mentioned above. However, owing to the rarity and heterogeneity of CTCs, CTC research faces many critical concerns. Although exosomes and cfDNA have some technical challenges, they are being highlighted as new target materials. That is because they also have genetic information on cancers. Even though the number of exosomes and cfDNA from early stage cancer patients are similar to healthy individuals, they are present in high concentrations after metastasis. In this article, we review several technologies for material analyses of cancer, discuss the critical concerns based on hands-on experience, and describe future directions for cancer screening, detection, and diagnostics.
Circulating tumor cells (CTCs) have attracted a great deal of attention, as they can be exploited to investigate metastasis. The molecular and cellular characteristics of these cells are little ...understood because they are rare and difficult to isolate. Many methods of isolation have centered on affinity-based positive enrichment (i.e., capturing target cells and eluting nontarget cells) using epithelial cell adhesion molecule (EpCAM) antibodies. It is known, however, that not all CTCs express the EpCAM antigen because they are heterogeneous by nature. In addition, negative enrichment (i.e., capturing nontarget cells and eluting target cells) has advantages over positive enrichment in isolating CTCs since the former can collect the target cells in an intact form. In this paper, we introduce a geometrically activated surface interaction (GASI) chip with an asymmetric herringbone structure designed to generate enhanced mixing flows, increasing the surface interaction between the nontarget cells and the channel surface. CD45 antibodies were immobilized inside the channel to capture leukocytes and release CTCs to the outlet. Blood samples from breast, lung, and gastric cancer patients were analyzed. The number of isolated CTCs varied from 1 to 51 in 1 mL of blood. Because our device does not require any labeling processes (e.g., EpCAM antibodies), intact and heterogeneous CTCs can be isolated regardless of EpCAM expression.
Over the past two decades, circulating tumor cells (CTCs) have been widely recognized for their importance in clinical trials. While most enrichment methods for these cells have been conducted ...through the batch process due to their rarity in blood and the need for large sample volumes, the batch process leads to unavoidable cell loss. Given the heterogenetic features of CTCs, this cell loss may limit the validity of research that relies on the isolation of CTCs; such research includes cancer prognosis, diagnosis of minimal residual diseases, assessment of tumor sensitivity to anticancer drugs, and the personalization of anticancer therapies. Recent advances in microfluidic approaches have made it possible to enrich CTCs with a small degree of cell loss. In this review, we highlight several microfluidic-based positive and negative enrichment methods that are the subject of considerable research interest (e.g. EpCAM-dependent assay and EpCAM-independent assay) and suggest a microfluidic-based single cell analysis platform for the down-stream analysis of CTCs. We also discuss critical concerns and future directions for research.
Plastic waste is a pernicious environmental pollutant that threatens ecosystems and human health by releasing contaminants including di(2-ethylhexyl) phthalate (DEHP) and bisphenol A (BPA). ...Therefore, a machine-learning (ML)-powered electrochemical aptasensor was developed in this study for simultaneously detecting DEHP and BPA in river waters, particularly to minimize the electrochemical signal errors caused by varying pH levels. The aptasensor leverages a straightforward and effective surface modification strategy featuring gold nanoflowers to achieve low detection limits for DEHP and BPA (0.58 and 0.59 pg/mL, respectively), excellent specificity, and stability. The least-squares boosting (LSBoost) algorithm was introduced to reliably monitor the targets regardless of pH; it employs a layer that adjusts the number of multi-indexes and the parallel learning structure of an ensemble model to accurately predict concentrations by preventing overfitting and enhancing the learning effect. The ML-powered aptasensor successfully detected targets in 12 river sites with diverse pH values, exhibiting higher accuracy and reliability. To our knowledge, the platform proposed in this study is the first attempt to utilize ML for the simultaneous assessment of DEHP and BPA. This breakthrough allows for comprehensive investigations into the effects of contamination originating from diverse plastics by eliminating external interferent-caused influences.
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