articles+ search results

DNA, Oligonucleotide Array Sequence Analysis, Equipment Design, Biosensing Techniques, Kinetics, Electronics, Semiconductors, Nanotechnology, Miniaturization, Lab-On-A-Chip Devices, Enzyme Assays, CMOS chip, biosensor, molecular electronics, single-molecule detection, single-molecule sequencing, Bioengineering, Biotechnology, Genetics, and Generic health relevance

For nearly 50 years, the vision of using single molecules in circuits has been seen as providing the ultimate miniaturization of electronic chips. An advanced example of such a molecular electronics chip is presented here, with the important distinction that the molecular circuit elements play the role of general-purpose single-molecule sensors. The device consists of a semiconductor chip with a scalable array architecture. Each array element contains a synthetic molecular wire assembled to span nanoelectrodes in a current monitoring circuit. A central conjugation site is used to attach a single probe molecule that defines the target of the sensor. The chip digitizes the resulting picoamp-scale current-versus-time readout from each sensor element of the array at a rate of 1,000 frames per second. This provides detailed electrical signatures of the single-molecule interactions between the probe and targets present in a solution-phase test sample. This platform is used to measure the interaction kinetics of single molecules, without the use of labels, in a massively parallel fashion. To demonstrate broad applicability, examples are shown for probe molecule binding, including DNA oligos, aptamers, antibodies, and antigens, and the activity of enzymes relevant to diagnostics and sequencing, including a CRISPR/Cas enzyme binding a target DNA, and a DNA polymerase enzyme incorporating nucleotides as it copies a DNA template. All of these applications are accomplished with high sensitivity and resolution, on a manufacturable, scalable, all-electronic semiconductor chip device, thereby bringing the power of modern chips to these diverse areas of biosensing.

Acetobacter, Tetracycline, Anti-Bacterial Agents, Bacteriological Techniques, Drug Resistance, Bacterial, Lab-On-A-Chip Devices, active matter, colonization resistance, microbiome stability, microfluidics, phase separation, active, , matter, and Infectious Diseases

Bacteria are efficient colonizers of a wide range of secluded microhabitats, such as soil pores, skin follicles, or intestinal crypts. How the structural diversity of these habitats modulates microbial self-organization remains poorly understood, in part because of the difficulty to precisely manipulate the physical structure of microbial environments. Using a microfluidic device to grow bacteria in crypt-like incubation chambers of systematically varied lengths, we show that small variations in the physical structure of the microhabitat can drastically alter bacterial colonization success and resistance against invaders. Small crypts are uncolonizable; intermediately sized crypts can stably support dilute populations, while beyond a second critical length scale, populations phase separate into a dilute region and a jammed region. The jammed state is characterized by extreme colonization resistance, even if the resident strain is suppressed by an antibiotic. Combined with a flexible biophysical model, we demonstrate that colonization resistance and associated priority effects can be explained by a crowding-induced phase transition, which results from a competition between proliferation and density-dependent cell leakage. The emerging sensitivity to scale underscores the need to control for scale in microbial ecology experiments. Systematic flow-adjustable length-scale variations may serve as a promising strategy to elucidate further scale-sensitive tipping points and to rationally modulate the stability and resilience of microbial colonizers.

RADIOCHEMISTRY, RADIOPHARMACEUTICALS, CHEMICAL purification, RADIOACTIVE tracers, and NUCLEAR medicine

New platforms are enabling radiochemistry to be carried out in tiny, microliter-scale volumes, and this capability has enormous benefits for the production of radiopharmaceuticals. These droplet-based technologies can achieve comparable or better yields compared to conventional methods, but with vastly reduced reagent consumption, shorter synthesis time, higher molar activity (even for low activity batches), faster purification, and ultra-compact system size. We review here the state of the art of this emerging direction, summarize the radiotracers and prosthetic groups that have been synthesized in droplet format, describe recent achievements in scaling up activity levels, and discuss advantages and limitations and the future outlook of these innovative devices. [ABSTRACT FROM AUTHOR]

Retina, Organoids, Bioreactors, Cell Differentiation, Lab-On-A-Chip Devices, Eye Disease and Disorders of Vision, Biotechnology, Bioengineering, Neurosciences, Eye, Chemical Sciences, Engineering, and Analytical Chemistry

Retinal degeneration is a leading cause of vision impairment and blindness worldwide and medical care for advanced disease does not exist. Stem cell-derived retinal organoids (RtOgs) became an emerging tool for tissue replacement therapy. However, existing RtOg production methods are highly heterogeneous. Controlled and predictable methodology and tools are needed to standardize RtOg production and maintenance. In this study, we designed a shear stress-free micro-millifluidic bioreactor for nearly labor-free retinal organoid maintenance. We used a stereolithography (SLA) 3D printer to fabricate a mold from which Polydimethylsiloxane (PDMS) was cast. We optimized the chip design using in silico simulations and in vitro evaluation to optimize mass transfer efficiency and concentration uniformity in each culture chamber. We successfully cultured RtOgs at three different differentiation stages (day 41, 88, and 128) on an optimized bioreactor chip for more than 1 month. We used different quantitative and qualitative techniques to fully characterize the RtOgs produced by static dish culture and bioreactor culture methods. By analyzing the results from phase contrast microscopy, single-cell RNA sequencing (scRNA seq), quantitative polymerase chain reaction (qPCR), immunohistology, and electron microscopy, we found that bioreactor-cultured RtOgs developed cell types and morphology comparable to static cultured ones and exhibited similar retinal genes expression levels. We also evaluated the metabolic activity of RtOgs in both groups using fluorescence lifetime imaging (FLIM), and found that the outer surface region of bioreactor cultured RtOgs had a comparable free/bound NADH ratio and overall lower long lifetime species (LLS) ratio than static cultured RtOgs during imaging. To summarize, we validated an automated micro-millifluidic device with significantly reduced shear stress to produce RtOgs of comparable quality to those maintained in conventional static culture.

Caco-2 Cells, Glycocalyx, Humans, Neuraminic Acids, Alkaloids, Glycosyltransferases, Fucose, Glycoproteins, Polysaccharides, Enzyme Inhibitors, Chromatography, Liquid, Microfluidic Analytical Techniques, Proteomics, Molecular Structure, Structure-Activity Relationship, Glycosylation, Mass Spectrometry, Glycomics, Lab-On-A-Chip Devices, A549 Cells, N-glycan, glycosyltransferase, and mass spectrometry

Glycomic profiling methods were used to determine the effect of metabolic inhibitors on glycan production. These inhibitors are commonly used to alter the cell surface glycosylation. However, structural analysis of the released glycans has been limited. In this research, the cell membranes were enriched and the glycans were released to obtain the N-glycans of the glycocalyx. Glycomic analysis using liquid chromatography-mass spectrometry (LC-MS) with a PGC chip column was used to profile the structures in the cell membrane. Glycans of untreated cells were compared to glycans of cells treated with inhibitors, including kifunensine, which inhibits the formation of complex- and hybrid-type structures, 2,4,7,8,9-Penta-O-acetyl-N-acetyl-3-fluoro-b-d-neuraminic acid methyl ester for sialylated glycans, 2-deoxy-2-fluorofucose, and 6-alkynyl fucose for fucosylated glycans. Kifunensine was the most effective, converting nearly 95% of glycans to high mannose types. The compound 6-alkynyl fucose inhibited some fucosylation but also incorporated into the glycan structure. Proteomic analysis of the enriched membrane for the four inhibitors showed only small changes in the proteome accompanied by large changes in the N-glycome for Caco-2. Future works may use these inhibitors to study the cellular behavior associated with the alteration of glycosylation in various biological systems, e.g., viral and bacterial infection, drug binding, and cell-cell interactions.

Trophoblasts, Humans, Placenta Accreta, Diagnosis, Differential, Cohort Studies, Reproducibility of Results, ROC Curve, Cell Aggregation, Pregnancy, Nanostructures, Adult, Middle Aged, Female, Placenta Previa, Lab-On-A-Chip Devices, Maternal Serum Screening Tests, and Biomarkers

Placenta accreta spectrum (PAS) is a high-risk obstetrical condition associated with significant morbidity and mortality. Current clinical screening modalities for PAS are not always conclusive. Here, we report a nanostructure-embedded microchip that efficiently enriches both single and clustered circulating trophoblasts (cTBs) from maternal blood for detecting PAS. We discover a uniquely high prevalence of cTB-clusters in PAS and subsequently optimize the device to preserve the intactness of these clusters. Our feasibility study on the enumeration of cTBs and cTB-clusters from 168 pregnant women demonstrates excellent diagnostic performance for distinguishing PAS from non-PAS. A logistic regression model is constructed using a training cohort and then cross-validated and tested using an independent cohort. The combined cTB assay achieves an Area Under ROC Curve of 0.942 (throughout gestation) and 0.924 (early gestation) for distinguishing PAS from non-PAS. Our assay holds the potential to improve current diagnostic modalities for the early detection of PAS.

Liver, Hepatocytes, Humans, Liver Diseases, Alcoholic, Ethanol, Gene Expression Profiling, Polyploidy, Models, Biological, Lab-On-A-Chip Devices, ALD, ASH, NASH, alcohol, bile canaliculi, digital pathology, fatty liver, liver disease, organ-on-chip, steatosis, Substance Abuse, Alcoholism, Alcohol Use and Health, Digestive Diseases, Liver Disease, Chronic Liver Disease and Cirrhosis, 2.1 Biological and endogenous factors, Oral and gastrointestinal, Biochemistry and Cell Biology, and Medical Physiology

Alcohol-associated liver disease (ALD) is a global health issue and leads to progressive liver injury, comorbidities, and increased mortality. Human-relevant preclinical models of ALD are urgently needed. Here, we leverage a triculture human Liver-Chip with biomimetic hepatic sinusoids and bile canaliculi to model ALD employing human-relevant blood alcohol concentrations (BACs) and multimodal profiling of clinically relevant endpoints. Our Liver-Chip recapitulates established ALD markers in response to 48 h of exposure to ethanol, including lipid accumulation and oxidative stress, in a concentration-dependent manner and supports the study of secondary insults, such as high blood endotoxin levels. We show that remodeling of the bile canalicular network can provide an in vitro quantitative readout of alcoholic liver toxicity. In summary, we report the development of a human ALD Liver-Chip as a powerful platform for modeling alcohol-induced liver injury with the potential for direct translation to clinical research and evaluation of patient-specific responses.

Transducers, Acoustics, Ultrasonics, Electric Power Supplies, Lab-On-A-Chip Devices, Analytical Chemistry, Chemical Sciences, and Engineering

Acoustofluidics has promised to enable lab-on-a-chip and point-of-care devices in ways difficult to achieve using other methods. Piezoelectric ultrasonic transducers-as small as the chips they actuate-provide rapid fluid and suspended object transport. Acoustofluidic lab-on-chip devices offer a vast range of benefits in early disease identification and noninvasive drug delivery. However, their potential has long been undermined by the need for benchtop or rack-mount electronics. The piezoelectric ultrasonic transducers within require these equipment and thus acoustofluidic device implementation in a bedside setting has been limited. Here we detail a general process to enable the reader to produce battery or mains-powered microcircuits ideal for driving 1-300 MHz acoustic devices. We include the general design strategy for the circuit, the blocks that collectively define it, and suitable, specific choices for components to produce these blocks. We furthermore illustrate how to incorporate automated resonance finding and tracking, sensing and feedback, and built-in adjustability to accommodate devices' vastly different operating frequencies and powers in a single driver, including examples of fluid and particle manipulation typical of the needs in our discipline. With this in hand, the many groups active in lab-on-a-chip acoustofluidics can now finally deliver on the promise of handheld, point-of-care technologies.

Humans, Carcinoma, Hepatocellular, Liver Neoplasms, Liver Cirrhosis, Disease Progression, Dimethylpolysiloxanes, RNA, Messenger, Diagnosis, Differential, Neoplasm Staging, Microfluidic Analytical Techniques, Case-Control Studies, ROC Curve, Reverse Transcriptase Polymerase Chain Reaction, Nanostructures, Computer Simulation, Aged, Middle Aged, Female, Male, Nanowires, Early Detection of Cancer, Lab-On-A-Chip Devices, Hep G2 Cells, Click Chemistry, Extracellular Vesicles, Biomarkers, Tumor, Liquid Biopsy, and Computational Chemistry

We report a covalent chemistry-based hepatocellular carcinoma (HCC)-specific extracellular vesicle (EV) purification system for early detection of HCC by performing digital scoring on the purified EVs. Earlier detection of HCC creates more opportunities for curative therapeutic interventions. EVs are present in circulation at relatively early stages of disease, providing potential opportunities for HCC early detection. We develop an HCC EV purification system (i.e., EV Click Chips) by synergistically integrating covalent chemistry-mediated EV capture/release, multimarker antibody cocktails, nanostructured substrates, and microfluidic chaotic mixers. We then explore the translational potential of EV Click Chips using 158 plasma samples of HCC patients and control cohorts. The purified HCC EVs are subjected to reverse-transcription droplet digital PCR for quantification of 10 HCC-specific mRNA markers and computation of digital scoring. The HCC EV-derived molecular signatures exhibit great potential for noninvasive early detection of HCC from at-risk cirrhotic patients with an area under receiver operator characteristic curve of 0.93 (95% CI, 0.86 to 1.00; sensitivity = 94.4%, specificity = 88.5%).

Polyketide Synthases, Microfluidic Analytical Techniques, Soil Microbiology, Genomic Library, Plasmids, Biosynthetic Pathways, Metagenomics, Lab-On-A-Chip Devices, Workflow, Developmental Biology, Environmental Sciences, Biological Sciences, and Information and Computing Sciences

Microbial biosynthetic gene clusters are a valuable source of bioactive molecules. However, because they typically represent a small fraction of genomic material in most metagenomic samples, it remains challenging to deeply sequence them. We present an approach to isolate and sequence gene clusters in metagenomic samples using microfluidic automated plasmid library enrichment. Our approach provides deep coverage of the target gene cluster, facilitating reassembly. We demonstrate the approach by isolating and sequencing type I polyketide synthase gene clusters from an Antarctic soil metagenome. Our method promotes the discovery of functional-related genes and biosynthetic pathways.

The skin is the first line of defense of our body, and it is composed of the epidermis and dermis with diverse immune cells. Various in vitro models have been investigated to recapitulate the immunological functions of the skin and to model inflammatory skin diseases. The simplest model is a two-dimensional (2D) co-culture system, which helps understand the direct and indirect cellto- cell interactions between immune and structural cells; however, it has limitations when observing three-dimensional (3D) interactions or reproducing skin barriers. Conversely, 3D skin constructs can mimic the human skin characteristics in terms of epidermal and dermal structures, barrier functions, cell migration, and cell-to-cell interaction in the 3D space. Recently, as the importance of neuro-immune-cutaneous interactions in the inflammatory response is emerging, 3D skin constructs containing both immune cells and neurons are being developed. A microfluidic culture device called "skin-on-a-chip," which simulates the structures and functions of the human skin with perfusion, was also developed to mimic immune cell migration through the vascular system. This review summarizes the in vitro skin models with immune components, focusing on two highly prevalent chronic inflammatory skin diseases: atopic dermatitis and psoriasis. The development of these models will be valuable in studying the pathophysiology of skin diseases and evaluating the efficacy and toxicity of new drugs. [ABSTRACT FROM AUTHOR]

Humans, Enterobacteriaceae, Anti-Bacterial Agents, Microfluidic Analytical Techniques, Polymerase Chain Reaction, Particle Size, Surface Properties, Methicillin-Resistant Staphylococcus aureus, Lab-On-A-Chip Devices, Vancomycin-Resistant Enterococci, Hematology, Emerging Infectious Diseases, Vaccine Related, Sepsis, Prevention, Antimicrobial Resistance, Biotechnology, Infectious Diseases, Detection, screening and diagnosis, 4.1 Discovery and preclinical testing of markers and technologies, Infection, Good Health and Well Being, Chemical Sciences, Engineering, and Analytical Chemistry

Sepsis due to antimicrobial resistant pathogens is a major health problem worldwide. The inability to rapidly detect and thus treat bacteria with appropriate agents in the early stages of infections leads to excess morbidity, mortality, and healthcare costs. Here we report a rapid diagnostic platform that integrates a novel one-step blood droplet digital PCR assay and a high throughput 3D particle counter system with potential to perform bacterial identification and antibiotic susceptibility profiling directly from whole blood specimens, without requiring culture and sample processing steps. Using CTX-M-9 family ESBLs as a model system, we demonstrated that our technology can simultaneously achieve unprecedented high sensitivity (10 CFU per ml) and rapid sample-to-answer assay time (one hour). In head-to-head studies, by contrast, real time PCR and BioRad ddPCR only exhibited a limit of detection of 1000 CFU per ml and 50-100 CFU per ml, respectively. In a blinded test inoculating clinical isolates into whole blood, we demonstrated 100% sensitivity and specificity in identifying pathogens carrying a particular resistance gene. We further demonstrated that our technology can be broadly applicable for targeted detection of a wide range of antibiotic resistant genes found in both Gram-positive (vanA, nuc, and mecA) and Gram-negative bacteria, including ESBLs (blaCTX-M-1 and blaCTX-M-2 families) and CREs (blaOXA-48 and blaKPC), as well as bacterial speciation (E. coli and Klebsiella spp.) and pan-bacterial detection, without requiring blood culture or sample processing. Our rapid diagnostic technology holds great potential in directing early, appropriate therapy and improved antibiotic stewardship in combating bloodstream infections and antibiotic resistance.