Capillary force-driven reverse-Tesla valve structure for microfluidic bioassays.

Biological assays involve the lysis of biological particles, enzyme reactions, and gene amplification, and require a certain amount of time for completion. Microfluidic chips are regarded as powerful devices for biological assays and in vitro diagnostics; however, they cannot achieve a high mixing efficiency, particularly in some time-consuming biological reactions. Herein, we introduce a microfluidic reverse-Tesla (reTesla) valve structure in which the fluid is affected by vortices and branch flow convergence, resulting in flow retardation and a high degree of mixing. The reTesla is passively operated by a microfluidic capillary force without any pumping facility. Compared with our previously developed micromixers, this innovative pumpless microfluidic chip exhibited high performance, with a mixing efficiency of more than 93%. The versatility of our reTesla chip will play a pivotal role in the study of various biological and chemical reactions.

Efficient separation of large particles and giant cancer cells using an isosceles trapezoidal spiral microchannel.

Polyploid giant cancer cells (PGCCs) contribute to the genetic heterogeneity and evolutionary dynamics of tumors. Their size, however, complicates their isolation from mainstream tumor cell populations. Standard techniques like fluorescence-activated cell sorting (FACS) rely on fluorescent labeling, introducing potential challenges in subsequent PGCC analyses. In response, we developed the Isosceles Trapezoidal Spiral Microchannel (ITSµC), a microfluidic device optimizing the Dean drag force (FD) and exploiting uniform vortices for enhanced separation. Numerical simulations highlighted ITSµC's advantage in producing robust FD compared to rectangular and standard trapezoidal channels. Empirical results confirmed its ability to segregate larger polystyrene (PS) particles (avg. diameter: 50 µm) toward the inner wall, while directing smaller ones (avg. diameter: 23 µm) outward. Utilizing ITSµC, we efficiently isolated PGCCs from doxorubicin-resistant triple-negative breast cancer (DOXR-TNBC) and patient-derived cancer (PDC) cells, achieving outstanding purity, yield, and viability rates (all greater than 90%). This precision was accomplished without fluorescent markers, and the versatility of ITSµC suggests its potential in differentiating a wide range of heterogeneous cell populations.

Analysis of the Setomimycin Biosynthetic Gene Cluster from Streptomyces nojiriensis JCM3382 and Evaluation of Its α-Glucosidase Inhibitory Activity Using Molecular Docking and Molecular Dynamics Simulations.

The formation of atroposelective biaryl compounds in plants and fungi is well understood; however, polyketide aglycone synthesis and dimerization in bacteria remain unclear. Thus, the biosynthetic gene cluster (BGC) responsible for antibacterial setomimycin production from Streptomyces nojiriensis JCM3382 was examined in comparison with the BGCs of spectomycin, julichromes, lincolnenins, and huanglongmycin. The setomimycin BGC includes post-polyketide synthase (PKS) assembly/cycling enzymes StmD (C-9 ketoreductase), StmE (aromatase), and StmF (thioesterase) as key components. The heterodimeric TcmI-like cyclases StmH and StmK are proposed to aid in forming the setomimycin monomer. In addition, StmI (P-450) is predicted to catalyze the biaryl coupling of two monomeric setomimycin units, with StmM (ferredoxin) specific to the setomimycin BGC. The roles of StmL and StmN, part of the nuclear transport factor 2 (NTF-2)-like protein family and unique to setomimycin BGCs, could particularly interest biochemists and combinatorial biologists. α-Glucosidase, a key enzyme in type 2 diabetes, hydrolyzes carbohydrates into glucose, thereby elevating blood glucose levels. This study aimed to assess the α-glucosidase inhibitory activity of EtOAc extracts of JCM 3382 and setomimycin. The JCM 3382 EtOAc extract and setomimycin exhibited greater potency than the standard inhibitor, acarbose, with IC50 values of 285.14 ± 2.04 µg/mL and 231.26 ± 0.41 µM, respectively. Molecular docking demonstrated two hydrogen bonds with maltase-glucoamylase chain A residues Thr205 and Lys480 (binding energy = -6.8 kcal·mol-1), two π-π interactions with Trp406 and Phe450, and one π-cation interaction with Asp542. Residue-energy analysis highlighted Trp406 and Phe450 as key in setomimycin's binding to maltase-glucoamylase. These findings suggest that setomimycin is a promising candidate for further enzymological research and potential antidiabetic therapy.

1-Acetyl-ß-Carboline from a Jeju Gotjawal Strain Lentzea sp. JNUCC 0626 and Its Melanogenic Stimulating Activity in B16F10 Melanoma Cells.

The genus Lentzea is a prolific source of bioactive and structurally diverse secondary metabolites. We isolated a novel strain, Lentzea sp. JNUCC 0626, from Hwasun Gotjawal on Jeju Island, Korea. Based on 16S rRNA partial gene sequence analysis, strain JNUCC 0626 is closely related to Lentzea isolaginshaensis NX62 (99.41% similarity), Lentzea pudingi DHS C021 (99.31%), and Lentzea cavernae SYSU K10001 (99.26%). From the fermentation broth of JNUCC 0626, we isolated 1-acetyl-ß-carboline, whose structure was established using IR, HR-ESI-MS, and 1D- and 2D-NMR techniques. 1-acetyl-ß-carboline was found to activate melanogenesis in mouse B16F10 cells without cytotoxicity at concentrations up to 50 µM. At this concentration, the compound increased melanin content by 27.44% and tyrosinase activity by 240.64% compared to the control, by upregulating key melanogenic enzymes, including tyrosinase, TRP-1, TRP-2, and microphthalmia-associated transcription factor (MITF), a central regulator of melanogenesis. In addition, 1-acetyl-ß-carboline significantly inhibited ERK phosphorylation, reducing it by 20.79% at a concentration of 12.5 µM and by 25.63% at 25 µM. This inhibition supports the hypothesis that 1-acetyl-ß-carboline enhances melanin synthesis by upregulating MITF and melanogenic enzymes via the ERK signaling pathway. This study aimed to isolate and identify 1-acetyl-ß-carboline from a novel strain of Lentzea sp. JNUCC 0626, discovered in Gotjawal, Jeju Island, and to evaluate its effect on melanin production in B16F10 melanoma cells. Skin irritation tests on 32 subjects confirmed its safety for topical use, and the findings suggest that 1-acetyl-ß-carboline, which enhances melanogenesis without cytotoxicity, holds promise as a therapeutic agent for hypopigmentation-related conditions or as a cosmetic ingredient.

A modular microfluidic platform for serial enrichment and harvest of pure extracellular vesicles.

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-1). 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.

Coil spring-powered pump with inertial microfluidic chip for size-based isolation and enrichment of biological cells.

Microfluidic chips have been widely used for in vitro diagnostics using pretreatment of biological samples; however, biologists and clinical researchers have difficulties using them in resource-limited settings. Sample injection systems for microfluidic chips are bulky, expensive, electricity-powered, and complex. A coiled spring-powered device, which can be used to isolate variously sized cells with high efficiency continuously and passively, was developed for portable, low-cost, electricity-free, and simple sample injection. The flow driving power was provided by releasing the compression spring in the mechanical syringe driver with a one-click action. In general, a syringe pump generates a stable passive flow rate. However, the syringe pumps are large in size and expensive because they have many functions such as infusion/withdrawal flow injection and the use of syringes of various sizes, allowing them to be applied in a variety of applications performed in the laboratory. In addition, it is not suitable for portable devices because of the considerable amount of electric power required. To overcome these drawbacks, we developed a device prototype that sorts different-sized particles and separates rare tumor cells or blood cells from blood with high efficiency. The performance of the coiled spring-powered device was evaluated and found to be comparable with that of syringe pump-powered devices. In situations where trained personnel cannot handle microfluidic chips for isolating circulating biomarkers (CTCs, WBCs, or plasma) from blood samples, the coiled spring-powered device can provide diagnostic tools, especially in resource-limited countries.

Multi-miRNA panel of tumor-derived extracellular vesicles as promising diagnostic biomarkers of early-stage breast cancer.

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.

Automatically Controlled Microfluidic System for Continuous Separation of Rare Bacteria from Blood.

Bloodstream infection by microorganisms is a major public health concern worldwide. Millions of people per year suffer from microbial infections, and current blood culture-based diagnostic methods are time-consuming because of the low concentration of infectious microorganisms in the bloodstream. In this study, we introduce an efficient automated microfluidic system for the continuous isolation of rare infectious bacteria (Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa) from blood. Bacteria received a balanced force between a fluidic drag force and a periodically controlled dielectrophoretic (DEP) force from tilted electrodes to minimize cell adhesion to the electrodes, which prevented the loss of rare infectious bacteria. Target bacteria were efficiently segregated from the undesired blood cells to ensure that only the bacteria received the DEP force under the hypotonic condition, while the blood cells received no DEP force and exited the channel via a laminar flow. Thus, the bacteria were successfully extracted from the blood with a high recovery yield of 91.3%, and the limit of the bacteria concentration for isolation was 100 cfu/ml. We also developed an automated system that performed every step from blood-sample loading to application of electricity to the microfluidic chip for bacteria separation. It reduced the standard deviation of the bacteria recovery yield from 6.16 to 2.77 compared with the conventional batch process, providing stable bacteria-extraction performance and minimizing errors and bacteria loss caused by user mistakes. © 2019 International Society for Advancement of Cytometry.

Isolation and enrichment of circulating biomarkers for cancer screening, detection, and diagnostics.

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.

Enhanced enrichment of collected airborne coronavirus and influenza virus samples via a ConA-coated microfluidic chip for PCR detection.

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.

High-resolution spiral microfluidic channel integrated electrochemical device for isolation and detection of extracellular vesicles without lipoprotein contamination.

Recent studies have indicated significant correlation between the concentration of immune checkpoint markers borne by extracellular vesicles (EVs) and the efficacy of immunotherapy. This study introduces a high-resolution spiral microfluidic channel-integrated electrochemical device (HiMEc), which is designed to isolate and detect EVs carrying the immune checkpoint markers programmed death ligand 1 (PD-L1) and programmed death protein 1 (PD-1), devoid of plasma-abundant lipoprotein contamination. Antigen-antibody reactions were applied to immobilize the lipoproteins on bead surfaces within the plasma, establishing a size differential with EVs. A plasma sample was then introduced into the spiral microfluidic channel, which facilitated the acquisition of nanometer-sized EVs and the elimination of micrometer-sized lipoprotein-bead complexes, along with the isolation and quantification of EVs using HiMEc. PD-L1 and PD-1 expression on EVs was evaluated in 30 plasma samples (10 from healthy donors, 20 from lung cancer patients) using HiMEc and compared to the results obtained from standard tissue-based PD-L1 testing, noting that HiMEc could be utilized to select further potential candidates. The obtained results are expected to contribute positively to the clinical assessment of potential immunotherapy beneficiaries.

Negative enrichment of circulating tumor cells using a geometrically activated surface interaction chip.

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.

Microfluidic devices for the isolation of circulating rare cells: a focus on affinity-based, dielectrophoresis, and hydrophoresis.

Circulating rare cells have attracted interest because they can be good indicators of various types of diseases. For example, enumeration of circulating tumor cells is used for cancer diagnosis and prognosis, while DNA analysis or enumeration of nucleated red blood cells is useful for prenatal diagnosis or hypoxic anemia, and that of circulating stem cells to diagnose cancer metastasis. Isolation of these cells and their downstream analyses can provide significant information such as the origin and characteristics of a disease. Novel approaches based on microfluidics have many advantages, including the continuous process and integration with other components for analysis. For these reasons, a variety of microfluidic devices have been developed to isolate and characterize rare cells. In this article, we review several microfluidic devices, with a focus on affinity-based isolation (e.g. antigen-antibody reaction) and label-free separation (DEP and hydrophoresis).

Microfluidic sorting of fluorescently activated cells depending on gene expression level.

During the last few years, fluorescence activated cell sorter has played an important role in a variety of biological investigations as well as clinical diagnostics. However, the conventional fluorescence activated cell sorter has several limitations, such as large size, large sample volumes required for operation, and high cost. In this paper, we present a novel microfluidic device that can separate cells based on various fluorescent protein expression levels. Our system consists of three major parts: focusing, detection, and separation. The operating principles are briefly as follows: first fluorescent cells were delivered into the microfluidic chip and focused in the center of channel by sheath flow. Subsequently, the cells were excited by a 532 nm laser at 30 µW and concurrently detected by a photomultiplier tube. Based on their fluorescence intensities, the cells were separated into three outlets by a dielectrophoretic force. Using this system, we successfully separated the genetically modified cells at 0.1 µL/min (sample flow rate) to sheath flow rate at 1:5, 5 Vpp voltage, and 800 kHz frequency. The separation efficiency was measured as high as 94.7%. In conclusion, we found that our system has the capability of separating genetically modified cells with various fluorescent intensities and help study biology and medicine in a molecular level.

An all-in-one platform to deplete pathogenic bacteria for rapid and safe enrichment of plant-derived extracellular vesicles.

Plant-derived extracellular vesicles (PDEVs) have exhibited several advantages, such as high biocompatibility, improvement of skin conditions, and the prevention of skin aging. However, traditional methods of extraction for plant substances, such as heating under reflux or solvent extraction, are complicated, time-consuming, and low in purity. Accordingly, a simple and efficient platform is necessary for purely isolating natural substances from plants. In this study, we report a newly designed platform for removing impurities to purify PDEVs. The proposed platform comprises three parts: (i) inflow of samples, (ii) depletion of impurities, and (iii) collection of PDEVs. The platform is designed to flow from top to bottom using gravity without the need for electric components. The platform allows the delimitation of impurities, such as the pathogenic bacteria in PDEVs, by capturing magnetic beads coated with Concanavalin A (Con A). We validate the practicality of our platform using extracellular vesicles derived from liquorice (LdEVs). Notably, the LdEVs purified using the Con A-coated magnetic beads provide better cell uptake and wound recovery than the commercialized extract LdEVs. This highlights the therapeutic potential of fresh LdEVs purified using our platform, particularly in preventing skin aging. The findings of this study hold significant practical implications for the cosmeceutical and therapeutic field, providing a promising approach for the extraction and purification of natural substances from plants to harness their benefits effectively.

Modularized dynamic cell culture platform for efficient production of extracellular vesicles and sequential analysis.

Extracellular vesicles (EVs) are nanometer-sized particles naturally secreted by cells for intercellular communication that encapsulate bioactive cargo, such as proteins and RNA, with a lipid bilayer. Tumor cell-derived EVs (tdEVs) are particularly promising biomarkers for cancer research because their contents reflect the cell of origin. In most studies, tdEVs have been obtained from cancer cells cultured under static conditions, thus lacking the ability to recapitulate the microenvironment of cells in vivo. Recent developments in perfusable cell culture systems have allowed oxygen and a nutrient gradient to mimic the physiological and cellular microenvironment. However, as these systems are perfused by circulating the culture medium within the unified structure, independently harvesting cells and EVs at each time point for analysis presents a limitation. In this study, a modularized cell culture system is designed for the perfusion and real-time collection of EVs. The system consists of three detachable chambers, one each for fresh medium, cell culture, and EV collection. The fresh medium flows from the medium chamber to the culture chamber at a flow rate controlled by the hydraulic pressure injected with a syringe pump. When the culture medium containing EVs exceeds a certain volume within the chamber, it overflows into the collection chamber to harvest EVs. The compact and modularized chambers are highly interoperable with conventional cell culture modalities used in the laboratory, thus enabling various EV-based assays.

E2E-BPF microscope: extended depth-of-field microscopy using learning-based implementation of binary phase filter and image deconvolution.

Several image-based biomedical diagnoses require high-resolution imaging capabilities at large spatial scales. However, conventional microscopes exhibit an inherent trade-off between depth-of-field (DoF) and spatial resolution, and thus require objects to be refocused at each lateral location, which is time consuming. Here, we present a computational imaging platform, termed E2E-BPF microscope, which enables large-area, high-resolution imaging of large-scale objects without serial refocusing. This method involves a physics-incorporated, deep-learned design of binary phase filter (BPF) and jointly optimized deconvolution neural network, which altogether produces high-resolution, high-contrast images over extended depth ranges. We demonstrate the method through numerical simulations and experiments with fluorescently labeled beads, cells and tissue section, and present high-resolution imaging capability over a 15.5-fold larger DoF than the conventional microscope. Our method provides highly effective and scalable strategy for DoF-extended optical imaging system, and is expected to find numerous applications in rapid image-based diagnosis, optical vision, and metrology.

Electrochemical detection and analysis of tumor-derived extracellular vesicles to evaluate malignancy of pancreatic cystic neoplasm using integrated microfluidic device.

Tumor-derived extracellular vesicles (tdEVs) are one of the most promising biomarkers for liquid biopsy-based cancer diagnostics, owing to the expression of specific membrane proteins of their cellular origin. The investigation of epithelial-to-mesenchymal transition (EMT) in cancer using tdEVs is an alternative way of evaluating the risk of malignancy transformation. An ultra-sensitive selection and detection methodology is an essential step in developing a tdEVs-based cancer diagnostic device. In this study, we developed an indium-tin-oxide (ITO) sensor integrated microfluidic device consisting of two main parts: 1) a multi-orifice flow-fractionation (MOFF) channel for extraction of pure EVs by removing blood cellular debris, and 2) an ITO sensor coupled with a geometrically activated surface interaction (GASI) channel for enrichment and quantification of tdEV. The microfluidic channel and the ITO sensors are assembled with a 3D printed magnetic housing to prevent sample leakage and to easily attach/detach the sensors to/from the microfluidic channel. The tdEVs were successfully captured on the specific antibody modified ITO surfaces in the integrated microfluidic channel. The integrated sensors showed an excellent linear response between 103 and 109 tdEVs/mL. Simultaneous evaluation of the epithelial and mesenchymal markers on the tdEV surfaces successfully revealed the EMT index of the corresponding pancreatic cancer cells. Our ITO sensor integrated microfluidic device showed excellent detection in the clinically relevant tdEVs-concentration range for patients with pancreatic cystic neoplasms. Hence, this system is expected to open a new avenue for liquid biopsy-based cancer prognostics and diagnostics.

Electrochemical biosensor for nucleic acid amplification-free and sensitive detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA via CRISPR/Cas13a trans-cleavage reaction.

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.

Microfluidic recapitulation of circulating tumor cell-neutrophil clusters via double spiral channel-induced deterministic encapsulation.

Circulating tumor cell (CTC)-neutrophil clusters are highly potent precursors of cancer metastasis. However, their rarity in patients' blood has restricted research thus far, and moreover, studies on in vitro methods for mimicking cell clusters have generally neglected in vivo conditions. Here, we introduce an inertial-force-assisted droplet microfluidic chip that allows the recapitulation of CTC-neutrophil clusters in terms of physical as well as biochemical features. The deterministic encapsulation of cells via double spiral channels facilitates the pairing of neutrophils and cancer cells with ratios of interest (from 1 : 1 to 1 : 3). The encapsulated cells are spontaneously associated to form clusters, achieving the physical emulation of CTC-neutrophil clusters. Furthermore, the molecular signatures of CTC-neutrophil clusters (e.g., their E-cadherin, VCAM-1, and mRNA expressions) were well defined. Our novel microfluidic platform for exploring CTC-neutrophil clusters can therefore play a promising role in cancer-metastasis studies.