Transcriptomic profiling analysis of the effect of palmitic acid on 3D spheroids of ß-like cells derived from induced pluripotent stem cells.

Type 2 diabetes (T2D) is posing a serious public health concern with a considerable impact on human life and health expenditures worldwide. The disease develops when insulin plasma level is insufficient for coping insulin resistance, caused by the decline of pancreatic ß-cell function and mass. In ß-cells, the lipotoxicity exerted by saturated free fatty acids in particular palmitate (PA), which is chronically elevated in T2D, plays a major role in ß-cell dysfunction and mass. However, there is a lack of human relevant in vitro model to identify the underlying mechanism through which palmitate induces ß-cell failure. In this frame, we have previously developed a cutting-edge 3D spheroid model of ß-like cells derived from human induced pluripotent stem cells. In the present work, we investigated the signaling pathways modified by palmitate in ß-like cells derived spheroids. When compared to the 2D monolayer cultures, the transcriptome analysis (FDR set at  0.1) revealed that the 3D spheroids upregulated the pancreatic markers (such as GCG, IAPP genes), lipids metabolism and transporters (CD36, HMGSC2 genes), glucose transporter (SLC2A6). Then, the 3D spheroids are exposed to PA 0.5 mM for 72 h. The differential analysis demonstrated that 32 transcription factors and 135 target genes were mainly modulated (FDR set at  0.1) including the upregulation of lipid and carbohydrates metabolism (HMGSC2, LDHA, GLUT3), fibrin metabolism (FGG, FGB), apoptosis (CASP7). The pathway analysis using the 135 selected targets extracted the fibrin related biological process and wound healing in 3D PA treated conditions. An overall pathway gene set enrichment analysis, performed on the overall gene set (with pathway significance cutoff at 0.2), highlighted that PA perturbs the citrate cycle, FOXO signaling and Hippo signaling as observed in human islets studies. Additional RT-PCR confirmed induction of inflammatory (IGFBP1, IGFBP3) and cell growth (CCND1, Ki67) pathways by PA. All these changes were associated with unaffected glucose-stimulated insulin secretion (GSIS), suggesting that they precede the defect of insulin secretion and death induced by PA. Overall, we believe that our data demonstrate the potential of our spheroid 3D islet-like cells to investigate the pancreatic-like response to diabetogenic environment.

Dynamic, IPSC-derived hepatic tissue tri-culture system for the evaluation of liver physiologyin vitro.

Availability of hepatic tissue for the investigation of metabolic processes is severely limited. While primary hepatocytes or animal models are widely used in pharmacological applications, a change in methodology towards more sustainable and ethical assays is highly desirable. Stem cell derived hepatic cells are generally regarded as a viable alternative for the above model systems, if current limitations in functionality and maturation can be overcome. By combining microfluidic organ-on-a-chip technology with individually differentiated, multicellular hepatic tissue fractions, we aim to improve overall functionality of hepatocyte-like cells, as well as evaluate cellular composition and interactions with non-parenchymal cell populations towards the formation of mature liver tissue. Utilizing a multi-omic approach, we show the improved maturation profiles of hepatocyte-like cells maintained in a dynamic microenvironment compared to standard tissue culture setups without continuous perfusion. In order to evaluate the resulting tissue, we employ single cell sequencing to distinguish formed subpopulations and spatial localization. While cellular input was strictly defined based on established differentiation protocols of parenchyma, endothelial and stellate cell fractions, resulting hepatic tissue was shown to comprise a complex mixture of epithelial and non-parenchymal fractions with specific local enrichment of phenotypes along the microchannel. Following this approach, we show the importance of passive, paracrine developmental processes in tissue formation. Using such complex tissue models is a crucial first step to develop stem cell-derivedin vitrosystems that can compare functionally with currently used pharmacological and toxicological applications.

Activationless Electron Transfer of Redox-DNA in Electrochemical Nanogaps.

Our recent discovery of decreased reorganization energy in electrode-tethered redox-DNA systems prompts inquiries into the origin of this phenomenon and suggests its potential use to lower the activation energy of electrochemical reactions. Here, we show that the confinement of the DNA chain in a nanogap amplifies this effect to an extent to which it nearly abolishes the intrinsic activation energy of electron transfer. Employing electrochemical atomic force microscopy (AFM-SECM), we create sub-10 nm nanogaps between a planar electrode surface bearing end-anchored ferrocenylated DNA chains and an incoming microelectrode tip. The redox cycling of the DNA's ferrocenyl (Fc) moiety between the surface and the tip generates a measurable current at the scale of ∼10 molecules. Our experimental findings are rigorously interpreted through theoretical modeling and original molecular dynamics simulations (Q-Biol code). Several intriguing findings emerge from our investigation: (i) The electron transport resulting from DNA dynamics is many times faster than predicted by simple diffusion considerations. (ii) The current in the nanogap is solely governed by the electron transfer rate at the electrodes. (iii) This rate rapidly saturates as overpotentials applied to the nanogap electrodes increase, implying near-complete suppression of the reorganization energy for the oxidation/reduction of the Fc heads within confined DNA. Furthermore, evidence is presented that this may constitute a general, previously unforeseen, behavior of redox polymer chains in electrochemical nanogaps.

Correction: Generation of ß-like cell subtypes from differentiated human induced pluripotent stem cells in 3D spheroids.

Correction for 'Generation of ß-like cell subtypes from differentiated human induced pluripotent stem cells in 3D spheroids' by Lisa Morisseau et al., Mol. Omics, 2023, https://doi.org/10.1039/d3mo00050h.

Validation of an on-chip p16ink4a/Ki-67 dual immunostaining cervical cytology system using microfluidic device technology.

More specific screening systems for cervical cancer may become necessary as the human papillomavirus (HPV) vaccine becomes more widespread. Although p16/Ki-67 dual-staining cytology has several advantages, it requires advanced diagnostic skills. Here, we developed an automated on-chip immunostaining method using a microfluidic device. An electroactive microwell array (EMA) microfluidic device with patterned thin-film electrodes at the bottom of each microwell was used for single-cell capture by dielectrophoresis. Immunostaining and dual staining for p16/Ki-67 were performed on diagnosed liquid cytology samples using the EMA device. The numbers of p16/Ki-67 dual-stained cells captured by the EMA device were determined and compared among the cervical intraepithelial neoplasia (CIN) lesion samples. Seven normal, fifteen CIN grade 3, and seven CIN grade 2 samples were examined. The percentage of dual-positive cells was 18.6% in the CIN grade 2 samples and 23.6% in the CIN grade 3 samples. The percentages of dual-positive staining increased significantly as the severity of the cervical lesions increased. p16/Ki67 dual immunostaining using the EMA device is as sensitive as the conventional method of confirming the histopathological diagnosis of cervical samples. This system enables a quantified parallel analysis at the individual cell level.

Ceria-vesicle nanohybrid therapeutic for modulation of innate and adaptive immunity in a collagen-induced arthritis model.

Commencing with the breakdown of immune tolerance, multiple pathogenic factors, including synovial inflammation and harmful cytokines, are conjointly involved in the progression of rheumatoid arthritis. Intervening to mitigate some of these factors can bring a short-term therapeutic effect, but other unresolved factors will continue to aggravate the disease. Here we developed a ceria nanoparticle-immobilized mesenchymal stem cell nanovesicle hybrid system to address multiple factors in rheumatoid arthritis. Each component of this nanohybrid works individually and also synergistically, resulting in comprehensive treatment. Alleviation of inflammation and modulation of the tissue environment into an immunotolerant-favourable state are combined to recover the immune system by bridging innate and adaptive immunity. The therapy is shown to successfully treat and prevent rheumatoid arthritis by relieving the main symptoms and also by restoring the immune system through the induction of regulatory T cells in a mouse model of collagen-induced arthritis.

Ballistic Brownian Motion of Nanoconfined DNA.

Theoretical treatments of polymer dynamics in liquid generally start with the basic assumption that motion at the smallest scale is heavily overdamped; therefore, inertia can be neglected. We report on the Brownian motion of tethered DNA under nanoconfinement, which was analyzed by molecular dynamics simulation and nanoelectrochemistry-based single-electron shuttle experiments. Our results show a transition into the ballistic Brownian motion regime for short DNA in sub-5 nm gaps, with quality coefficients as high as 2 for double-stranded DNA, an effect mainly attributed to a drastic increase in stiffness. The possibility for DNA to enter the underdamped regime could have profound implications on our understanding of the energetics of biomolecular engines such as the replication machinery, which operates in nanocavities that are a few nanometers wide.

Generation of ß-like cell subtypes from differentiated human induced pluripotent stem cells in 3D spheroids.

Since the identification of four different pancreatic ß-cell subtypes and bi-hormomal cells playing a role in the diabetes pathogenesis, the search for in vitro models that mimics such cells heterogeneity became a key priority in experimental and clinical diabetology. We investigated the potential of human induced pluripotent stem cells to lead to the development of the different ß-cells subtypes in honeycomb microwell-based 3D spheroids. The glucose-stimulated insulin secretion confirmed the spheroids functionality. Then, we performed a single cell RNA sequencing of the spheroids. Using a knowledge-based analysis with a stringency on the pancreatic markers, we extracted the ß-cells INS+/UCN3+ subtype (11%; ß1-like cells), the INS+/ST8SIA1+/CD9- subtype (3%, ß3-like cells) and INS+/CD9+/ST8SIA1-subtype (1%; ß2-like cells) consistently with literature findings. We did not detect the INS+/ST8SIA1+/CD9+ cells (ß4-like cells). Then, we also identified four bi-hormonal cells subpopulations including δ-like cells (INS+/SST+, 6%), γ-like cells (INS+/PPY+, 3%), α-like-cells (INS+/GCG+, 6%) and ε-like-cells (INS+/GHRL+, 2%). Using data-driven clustering, we extracted four progenitors' subpopulations (with the lower level of INS gene) that included one population highly expressing inhibin genes (INHBA+/INHBB+), one population highly expressing KCNJ3+/TPH1+, one population expressing hepatocyte-like lineage markers (HNF1A+/AFP+), and one population expressing stem-like cell pancreatic progenitor markers (SOX2+/NEUROG3+). Furthermore, among the cycling population we found a large number of REST+ cells and CD9+ cells (CD9+/SPARC+/REST+). Our data confirm that our differentiation leads to large ß-cell heterogeneity, which can be used for investigating ß-cells plasticity under physiological and pathophysiological conditions.

Single-Cell Electrochemical Aptasensor Array.

Despite several demonstrations of electrochemical devices with limits of detection (LOD) of 1 cell/mL, the implementation of single-cell bioelectrochemical sensor arrays has remained elusive due to the challenges of scaling up. In this study, we show that the recently introduced nanopillar array technology combined with redox-labeled aptamers targeting epithelial cell adhesion molecule (EpCAM) is perfectly suited for such implementation. Combining nanopillar arrays with microwells determined for single cell trapping directly on the sensor surface, single target cells are successfully detected and analyzed. This first implementation of a single-cell electrochemical aptasensor array, based on Brownian-fluctuating redox species, opens new opportunities for large-scale implementation and statistical analysis of early cancer diagnosis and cancer therapy in clinical settings.

Electrochemical Shot Noise of a Redox Monolayer.

Redox monolayers are the base for a wide variety of devices including high-frequency molecular diodes or biomolecular sensors. We introduce a formalism to describe the electrochemical shot noise of such a monolayer, confirmed experimentally at room temperature in liquid. The proposed method, carried out at equilibrium, avoids parasitic capacitance, increases the sensitivity, and allows us to obtain quantitative information such as the electronic coupling (or standard electron transfer rates), its dispersion, and the number of molecules. Unlike in solid-state physics, the homogeneity in energy levels and transfer rates in the monolayer yields a Lorentzian spectrum. This first step for shot noise studies in molecular electrochemical systems opens perspectives for quantum transport studies in a liquid environment at room temperature as well as highly sensitive measurements for bioelectrochemical sensors.

Silicon chambers for enhanced incubation and imaging of microfluidic droplets.

Droplet microfluidics has become a powerful tool in life sciences, underlying digital assays, single-cell sequencing or directed evolution, and it is making foray in physical sciences as well. Imaging and incubation of droplets are crucial, yet they are encumbered by the poor optical, thermal and mechanical properties of PDMS, a material commonly used in microfluidics labs. Here we show that Si is an ideal material for droplet chambers. Si chambers pack droplets in a crystalline and immobile monolayer, are immune to evaporation or sagging, boost the number of collected photons, and tightly control the temperature field sensed by droplets. We use the mechanical and optical benefits of Si chambers to image ≈1 million of droplets from a multiplexed digital assay - with an acquisition rate similar to the best in-line methods. Lastly, we demonstrate their applicability with a demanding assay that maps the thermal dependence of Michaelis-Menten constants with an array of ≈150 000 droplets. The design of the Si chambers is streamlined to avoid complicated fabrication and improve reproducibility, which makes Si a complementary material to PDMS in the toolbox of droplet microfluidics.

Electrochemical response of surface-attached redox DNA governed by low activation energy electron transfer kinetics.

The mechanism responsible for electron transport within layers of redox DNA anchored to electrodes has been extensively studied over the last twenty years, but remains controversial. Herein, we thoroughly study the electrochemical behavior of a series of short, model, ferrocene (Fc) end-labeled dT oligonucleotides, terminally attached to gold electrodes, using high scan rate cyclic voltammetry complemented by molecular dynamics simulations. We evidence that the electrochemical response of both single-stranded and duplexed oligonucleotides is controlled by the electron transfer kinetics at the electrode, obeying Marcus theory, but with reorganization energies considerably lowered by the attachment of the ferrocene to the electrode via the DNA chain. This so far unreported effect, that we attribute to a slower relaxation of water around Fc, uniquely shapes the electrochemical response of Fc-DNA strands and, being markedly dissimilar for single-stranded and duplexed DNA, contributes to the signaling mechanism of E-DNA sensors.

First report of leaf anthracnose caused by Colletotrichum camelliae on tea plants (Camellia sinensis) in South Korea.

The tea plant (Camellia sinensis (L.) O. Kuntze) is a popular non-alcoholic beverage crop worldwide. The tea market in South Korea is projected to increase annually by 4.59% (Statista, 2022). Boseong, Hadong, and Jeju Island are the main tea-growing regions in South Korea. Anthracnose is one of the major diseases of tea plants and is responsible for substantial yield loss and poor tea quality. In 2021, anthracnose of tea (disease incidence of 30%) was observed in a garden (33°28'45.5"N 126°42'02.2"E) at Jeju Island, where the Yabukita cultivar has been cultivated. The typical symptoms consisted of round or irregularly shaped lesions with gray-white centers and purple-brown borders. Twelve morphologically similar isolates were recovered from 12 infected leaves using the single spore isolation method on solid potato dextrose agar (PDA) (Cai et al. 2009). Four representative isolates (GT6, GT7, GT8, and GT11) were identified based on morphology, molecular analysis, and pathogenicity tests. The upper side of seven-day-old colonies on PDA (incubated at 25 °C in the dark) was off-white with white aerial mycelia and gray-white with black zonation on their reverse side. Conidia were hyaline, aseptate, cylindrical, with both obtuse ends, and measuring 12.3 - 25.8 µm × 4.4 - 9.3 µm (n = 50). Appressoria were dark brown, irregularly shaped with a smooth edge, and measuring 7.3 -18.8 µm × 6.9 - 11.3 µm (n = 50). According to morphological characteristics, the fungal isolates were tentatively identified as the Colletotrichum gloeosporioides complex, including C. caelliae (Wang et al. 2016; Weir et al. 2012). The genomic DNA was extracted, and the ribosomal internal transcribed spacer (ITS), ß-tubulin-2 (TUB2) gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene, actin (ACT), calmodulin (CAL), and the Apn2-Mat1-2 intergenic spacer and partial mating type (ApMat) genes were amplified and subsequently sequenced using primer sets ITS1/ITS4, BT2a/BT2b, GDF1/GDR1, ACT-512F/ACT-783R1, CL1C/CL2C, and AM-F/AM-R, respectively (Silva et al. 2012; Weir et al. 2012). The resulting sequences were deposited in GenBank accession numbers (LC738932-LC738959). All the representative isolates were identified as C. camelliae by constructing the 50% majority rule consensus and maximum likelihood phylogenetic treebased on the combined ITS, TUB2, GAPDH, ACT, CAL, and ApMat sequences using MrBayes v. 3.2.2 and Mega X, respectively (Kumar et al., 2018; Ronquist et al. 2012). The pathogenicity of these isolates was tested on healthy leaves of 2- years-old tea seedlings (the Yabukita cultivar). Onside of unwounded or wounded leaves of seedlings were inoculated with 20 µL of conidial suspension (1 × 106 conidia or spores/ml) per spot (3-4 wounded or unwounded spots per side per leaf). Another side of the leaves received sterile distilled water and served as a control. Each treatment was replicated three times (three seedlings/isolate and four leaves per seedling) and this experiment was repeated twice. All plants were covered with plastic bags, placed in a growth chamber, and incubated at 25 °C with a 12-h photoperiod and 90% relative humidity. Typical anthracnose symptoms appeared on wounded leaves after two days of inoculation. Unwounded and controlled leaves remain asymptotic. To confirm Koch's postulates, fungal isolates were re-isolated from inoculated leaf lesions and identified as C. camelliae based on morphology and ITS sequences. Colletotrichum camelliae is a very common pathogen associated with tea anthracnose worldwide, including China (Liu et al. 2015; Wang et al. 2016).To the best of our knowledge, this is the first report of anthracnose in tea trees caused by C. camelliae in South Korea. The results of this study could help come up with better ways to keep an eye on and deal with this devastating on tea plants. Key words: Tea anthracnose, Colletotrichum camelliae, pathogenicity References Cai, L., et al. 2009. Fungal Divers. 39:183. Kumar, S., et al. 2018. Mol. Biol. Evol. 35:1547. Liu, F. et al. 2015. Persoonia. 35: 63-86. Ronquist, F. et al. 2012. Syst. Biol. 61:539-542. Silva, D. N. et al. 2012. Mycologia. 104:396-409. Statista 2022. Statista Digital Market out Look. Available at www.statista.com. Wang, Y.-C. et al. 2016. Sci. Rep. 6: 35287. Weir, B. S., et al. 2012. Stud. Mycol. 73:115.

Transcriptomic and proteomic studies suggest the establishment of advanced zonation-like profiles in human-induced pluripotent stem cell-derived liver sinusoidal endothelial cells and carboxypeptidase M-positive liver progenitor cells cocultured in a fluidic microenvironment.

AIM: Hepatic zonation is a physiological feature of the liver, known to be key in the regulation of the metabolism of nutrients and xenobiotics and the biotransformation of numerous substances. However, the reproduction of this phenomenon remains challenging in vitro as only part of the processes involved in the orchestration and maintenance of zonation are fully understood. The recent advances in organ-on-chip technologies, which allow for the integration of multicellular 3D tissues in a dynamic microenvironment, could offer solutions for the reproduction of zonation within a single culture vessel. METHODS: An in-depth analysis of zonation-related mechanisms observed during the coculture of human-induced pluripotent stem cell (hiPSC)-derived carboxypeptidase M-positive liver progenitor cells and hiPSC-derived liver sinusoidal endothelial cells within a microfluidic biochip was carried out. RESULTS: Hepatic phenotypes were confirmed in terms of albumin secretion, glycogen storage, CYP450 activity, and expression of specific endothelial markers such as PECAM1, RAB5A, and CD109. Further characterization of the patterns observed in the comparison of the transcription factor motif activities, the transcriptomic signature, and the proteomic profile expressed at the inlet and the outlet of the microfluidic biochip confirmed the presence of zonation-like phenomena within the biochips. In particular, differences related to Wnt/ß-catenin, transforming growth factor-ß, mammalian target of rapamycin, hypoxia-inducible factor-1, and AMP-activated protein kinase signaling, to the metabolism of lipids, and cellular remolding were observed. CONCLUSIONS: The present study shows the interest in combining cocultures of hiPSC-derived cellular models and microfluidic technologies for reproducing in vitro complex mechanisms such as liver zonation and further incites the use of those solutions for accurate reproduction of in vivo situations.

Enhanced podocyte differentiation and changing drug toxicity sensitivity through pressure-controlled mechanical filtration stress on a glomerulus-on-a-chip.

Podocytes, localized in the glomerulus, are a prognostic factor of proteinuria in kidney disease and are exposed to distinct physiological stimuli from basal to apical filtration flow. Research studies on drug discovery and disease modeling for glomerulopathy have developed a glomerulus-on-a-chip and studied podocyte mechanobiology to realize alternative methods to animal experiments. However, the effect of filtration stimulus on podocytes has remained unclear. Herein, we report the successful development of a user-friendly filtration culture device and system that can precisely control the filtration flow using air pressure control by incorporating a commercially available culture insert. It allows mouse podocytes to be cultured under filtration conditions for three days with a guarantee of maintaining the integrity of the podocyte layer. Using our system, this study demonstrated that podocyte damage caused by hyperfiltration resulting from glomerular hypertension, a common pathophysiology of many glomerulopathies, was successfully recapitulated and that filtration stimulus promotes the maturation of podocytes in terms of their morphology and gene expression. Furthermore, we demonstrated that filtration stimulus induced different drug responsiveness in podocytes than those seen under static conditions, and that the difference in drug responsiveness was dependent on the pharmacological mechanism. Overall, this study has revealed differentiating and pharmacodynamic properties of filtration stimulus and brings new insights into the research field of podocyte mechanobiology towards the realization of glomerulus-on-a-chip.

Semibulk RNA-seq analysis as a convenient method for measuring gene expression statuses in a local cellular environment.

When biologically interpretation of the data obtained from the single-cell RNA sequencing (scRNA-seq) analysis is attempted, additional information on the location of the single cells, behavior of the surrounding cells, and the microenvironment they generate, would be very important. We developed an inexpensive, high throughput application while preserving spatial organization, named "semibulk RNA-seq" (sbRNA-seq). We utilized a microfluidic device specifically designed for the experiments to encapsulate both a barcoded bead and a cell aggregate (a semibulk) into a single droplet. Using sbRNA-seq, we firstly analyzed mouse kidney specimens. In the mouse model, we could associate the pathological information with the gene expression information. We validated the results using spatial transcriptome analysis and found them highly consistent. When we applied the sbRNA-seq analysis to the human breast cancer specimens, we identified spatial interactions between a particular population of immune cells and that of cancer-associated fibroblast cells, which were not precisely represented solely by the single-cell analysis. Semibulk analysis may provide a convenient and versatile method, compared to a standard spatial transcriptome sequencing platform, to associate spatial information with transcriptome information.

Analysis of the transcriptome and metabolome of pancreatic spheroids derived from human induced pluripotent stem cells and matured in an organ-on-a-chip.

Functional differentiation of pancreatic like tissue from human induced pluripotent stem cells is one of the emerging strategies to achieve an in vitro pancreas model. Here, we propose a protocol to cultivate hiPSC-derived ß-like-cells coupling spheroids and microfluidic technologies to improve the pancreatic lineage maturation. The protocol led to the development of spheroids producing the C-peptide and containing cells positive to insulin and glucagon. In order to further characterize the cellular and molecular profiles, we performed full transcriptomics and metabolomics analysis. The omics analysis confirmed the activation of key transcription factors together with the upregulation of genes and the presence of metabolites involved in functional pancreatic tissue development, extracellular matrix remodeling, lipid and fatty acid metabolism, and endocrine hormone signaling. When compared to static 3D honeycomb cultures, dynamic 3D biochip cultures contributed to increase specifically the activity of the HIF transcription factor, to activate the calcium activated cation channels, to enrich the glucagon and insulin pathways and glycolysis/gluconeogenesis, and to increase the secretion of serotonin, glycerol and glycerol-3-phosphate at the metabolic levels.

An electroactive microwell array device to realize simultaneous trapping of single cancer cells and clusters.

The importance of circulating tumor cells (CTCs) as biomarkers has been greatly increased for early diagnosis and detection of cancer metastases. Along with a single form of CTCs, CTC clusters have recently attracted much attention due to their characteristics, such as suppression of apoptosis and survival from immune responses with high metastatic potential. Thus, it is highly necessary to investigate not only single cells but clustered cells at the same time to perform precise analysis of the current cancer state and develop suitable treatment. However, no cancer marker-free microfluidic devices have been realized to trap single cells and clusters at the same time in a single device yet. In this paper, we introduced a novel microfluidic device utilizing a microwell-on-electrode (MOE) array to realize simultaneous trapping of a single cell and clustered cells at a single cell/cluster level. Cell-sized microwells fabricated on interdigitated electrodes efficiently arrayed single cells with high trapping efficiency and single-cell occupancy (more than 90%) using dielectrophoresis (DEP). This high single cell trapping performance of MOE allows arraying of single clusters by trapping one of the cells that constitute a cluster. The feasibility of the MOE device for simultaneous arraying of single cancer cells and clusters was demonstrated by trapping a mixture of single cancer cells and clusters and measuring the size distribution of trapped clusters, which was almost identical with that of introduced cell population. Our work demonstrated that the developed MOE device can be one of the promising methods for trapping single cancer cells as well as clusters on a single device for cancer diagnosis and performing further analyses at a single cell/cluster level.

Direct Capture and Amplification of Small Fragmented DNAs Using Nitrogen-Mustard-Coated Microbeads.

Circulating cell-free DNA (cfDNA) has been implicated as an important biomarker and has been intensively studied for "liquid biopsy" applications in cancer diagnostics. Owing to its small fragment size and its low concentration in circulation, cfDNA extraction and purification from serum samples are complicated, and the extraction yield affects the precision of subsequent molecular diagnostic tests. Here, we report a novel approach using nitrogen-mustard-coated DNA capture beads (NMD beads) that covalently capture DNA and allow direct subsequent polymerase chain reaction (PCR) amplification from the NMD bead without elusion. The complex DNA extraction and purification processes are not required. To illustrate the diagnostic use of the NMD beads, we detected short DNA fragments (142 bp) that were spiked into fetal bovine serum (as a model serum sample). The spiked DNAs were captured directly from serum samples and detected using real-time PCR at concentrations as low as 10 fg/mL. We anticipate that this DNA capture bead technique has the potential to simplify the preanalytical processes required for cfDNA detection, which could significantly expand the diagnostic applications of liquid biopsy.