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Optics Express May 2024In this paper, a highly integrated terahertz (THz) biosensor is proposed and implemented, which pioneered the preparation of low-temperature gallium arsenide (LT-GaAs)...
In this paper, a highly integrated terahertz (THz) biosensor is proposed and implemented, which pioneered the preparation of low-temperature gallium arsenide (LT-GaAs) thin film photoconductive antenna (PCA) on the sensor for direct generation and detection of THz waves, simplifying complex terahertz time-domain spectroscopy (THz-TDS) systems. A latch type metasurface is deposited in the detection region to produce a resonance absorption peak at 0.6 THz that is independent of polarisation. Microfluidics is utilised and automatic injection is incorporated to mitigate the experimental effects of hydrogen bond absorption of THz waves in aqueous-based environment. Additionally, cell damage is minimised by regulating the cell flow rate. The biosensor was utilised to detect the concentration of three distinct sizes of bacteria with successful results. The assay was executed as a proof of concept to detect two distinct types of breast cancer cells. Based on the experimental findings, it has been observed that the amplitude and blueshift of the resonance absorption peaks have the ability to identify and differentiate various cancer cell types. The findings of this study introduce a novel approach for developing microfluidic THz metasurface biosensors that possess exceptional levels of integration, sensitivity, and rapid label-free detection capabilities.
Topics: Gallium; Arsenicals; Biosensing Techniques; Terahertz Spectroscopy; Humans; Equipment Design; Microfluidics
PubMed: 38858883
DOI: 10.1364/OE.518638 -
Microbial Cell Factories Jun 2024In vitro expression involves the utilization of the cellular transcription and translation machinery in an acellular context to produce one or more proteins of interest...
BACKGROUND
In vitro expression involves the utilization of the cellular transcription and translation machinery in an acellular context to produce one or more proteins of interest and has found widespread application in synthetic biology and in pharmaceutical biomanufacturing. Most in vitro expression systems available are active at moderate temperatures, but to screen large libraries of natural or artificial genetic diversity for highly thermostable enzymes or enzyme variants, it is instrumental to enable protein synthesis at high temperatures.
OBJECTIVES
Develop an in vitro expression system operating at high temperatures compatible with enzymatic assays and with technologies that enable ultrahigh-throughput protein expression in reduced volumes, such as microfluidic water-in-oil (w/o) droplets.
RESULTS
We produced cell-free extracts from Thermus thermophilus for in vitro translation including thermostable enzymatic cascades for energy regeneration and a moderately thermostable RNA polymerase for transcription, which ultimately limited the temperature of protein synthesis. The yield was comparable or superior to other thermostable in vitro expression systems, while the preparation procedure is much simpler and can be suited to different Thermus thermophilus strains. Furthermore, these extracts have enabled in vitro expression in microfluidic droplets at high temperatures for the first time.
CONCLUSIONS
Cell-free extracts from Thermus thermophilus represent a simpler alternative to heavily optimized or pure component thermostable in vitro expression systems. Moreover, due to their compatibility with droplet microfluidics and enzyme assays at high temperatures, the reported system represents a convenient gateway for enzyme screening at higher temperatures with ultrahigh-throughput.
Topics: Thermus thermophilus; Transcription, Genetic; Protein Biosynthesis; Microfluidics; Cell-Free System; DNA-Directed RNA Polymerases; Temperature; Hot Temperature; Bacterial Proteins
PubMed: 38858677
DOI: 10.1186/s12934-024-02440-y -
Scientific Reports Jun 2024We introduce magnetophoresis-based microfluidics for sorting biological targets using positive Magnetophoresis (pM) for magnetically labeled particles and negative...
We introduce magnetophoresis-based microfluidics for sorting biological targets using positive Magnetophoresis (pM) for magnetically labeled particles and negative Magnetophoresis (nM) for label-free particles. A single, externally magnetized ferromagnetic wire induces repulsive forces and is positioned across the focused sample flow near the main channel's closed end. We analyze magnetic attributes and separation performance under two transverse dual-mode magnetic configurations, examining magnetic fields, hydrodynamics, and forces on microparticles of varying sizes and properties. In pM, the dual-magnet arrangement (DMA) for sorting three distinct particles shows higher magnetic gradient generation and throughput than the single-magnet arrangement (SMA). In nM, the numerical results for SMA sorting of red blood cells (RBCs), white blood cells (WBCs), and prostate cancer cells (PC3-9) demonstrate superior magnetic properties and throughput compared to DMA. Magnetized wire linear movement is a key design parameter, allowing device customization. An automated device for handling more targets can be created by manipulating magnetophoretic repulsion forces. The transverse wire and magnet arrangement accommodate increased channel depth without sacrificing efficiency, yielding higher throughput than other devices. Experimental validation using soft lithography and 3D printing confirms successful sorting and separation, aligning well with numerical results. This demonstrates the successful sorting and separating of injected particles within a hydrodynamically focused sample in all systems. Both numerical and experimental findings indicate a separation accuracy of 100% across various Reynolds numbers. The primary channel dimensions measure 100 µm in height and 200 µm in width. N52 permanent magnets were employed in both numerical simulations and experiments. For numerical simulations, a remanent flux density of 1.48 T was utilized. In the experimental setup, magnets measuring 0.5 × 0.5 × 0.125 inches and 0.5 × 0.5 × 1 inch were employed. The experimental data confirm the device's capability to achieve 100% separation accuracy at a Reynolds number of 3. However, this study did not explore the potential impact of increased flow rates on separation accuracy.
Topics: Humans; Microfluidic Analytical Techniques; Cell Separation; Erythrocytes; Microfluidics; Leukocytes; Hydrodynamics; Cell Line, Tumor
PubMed: 38858424
DOI: 10.1038/s41598-024-64330-y -
Current Opinion in Structural Biology Jun 2024Proteins execute numerous cell functions in concert with one another in protein-protein interactions (PPI). While essential in each cell, such interactions are not... (Review)
Review
Proteins execute numerous cell functions in concert with one another in protein-protein interactions (PPI). While essential in each cell, such interactions are not identical from cell to cell. Instead, PPI heterogeneity contributes to cellular phenotypic heterogeneity in health and diseases such as cancer. Understanding cellular phenotypic heterogeneity thus requires measurements of properties of PPIs such as abundance, stoichiometry, and kinetics at the single-cell level. Here, we review recent, exciting progress in single-cell PPI measurements. Novel technology in this area is enabled by microscale and microfluidic approaches that control analyte concentration in timescales needed to outpace PPI disassembly kinetics. We describe microscale innovations, needed technical capabilities, and methods poised to be adapted for single-cell analysis in the near future.
PubMed: 38848654
DOI: 10.1016/j.sbi.2024.102860 -
ACS Sensors Jun 2024Health and security concerns have made it essential to develop integrated, continuous collection and sensing platforms that are compact and capable of real-time...
Health and security concerns have made it essential to develop integrated, continuous collection and sensing platforms that are compact and capable of real-time detection. In this study, we numerically investigate the flow physics associated with the single-step collection and enrichment of aerosolized polystyrene microparticles into a flowing liquid using a stratified air-water flow in a U-shaped microchannel. We validate our simulation results by comparing them to experimental data from the literature. Additionally, we fabricate an identical microfluidic device using PDMS-based soft lithography and test it to corroborate the previously published experimental data. Diversion and entrapment efficiencies are used as evaluation metrics, both of which increase with increasing particle diameter and superficial air inlet velocity. Overall, our ANSYS Fluent two-dimensional (2D) and three-dimensional (3D) multiphase flow simulations exhibit a good agreement with our experimental data and data in the literature (average deviation of ∼11%) in terms of diversion efficiency. Simulations also found the entrapment efficiency to be lower than the diversion efficiency, indicating discrepancies in the literature in terms of captured particles. The effect of the Dean force on the flow physics was also investigated using 3D simulations. We found that the effect of the Dean flow was more dominant relative to the centrifugal force on the smaller particles (e.g., 0.65 μm) compared to the larger particles (e.g., 2.1 μm). Increasing the superficial air inlet velocity also increases the effect of the centrifugal forces relative to the Dean forces. Overall, this experimentally validated multiphase model decouples and investigates the multiple and simultaneous forces on aerosolized particles flowing through a curved microchannel, which is crucial for designing more efficient capture devices. Once integrated with a microfluidic-based biosensor, this stratified flow-based microfluidic biothreat capture platform should deliver continuous sensor-ready enriched biosamples for real-time sensing.
Topics: Aerosols; Polystyrenes; Particle Size; Microfluidic Analytical Techniques; Lab-On-A-Chip Devices; Microfluidics
PubMed: 38848499
DOI: 10.1021/acssensors.4c00042 -
Physiological Measurement Jun 2024Vascular ageing is the deterioration of arterial structure and function which occurs naturally with age, and which can be accelerated with disease. Measurements of...
Vascular ageing is the deterioration of arterial structure and function which occurs naturally with age, and which can be accelerated with disease. Measurements of vascular ageing are emerging as markers of cardiovascular risk, with potential applications in disease diagnosis and prognosis, and for guiding treatments. However, vascular ageing is not yet routinely assessed in clinical practice. A key step towards this is the development of technologies to assess vascular ageing. In this Roadmap, experts discuss several aspects of this process, including: measurement technologies; the development pipeline; clinical applications; and future research directions. The Roadmap summarises the state of the art, outlines the major challenges to overcome, and identifies potential future research directions to address these challenges.
PubMed: 38838703
DOI: 10.1088/1361-6579/ad548e -
RSC Advances May 2024Diana Berman, Agnieszka Jastrzebska, Massimiliano Papi, and Andreas Rosenkranz introduce the themed issue on 2D materials and their applications.
Diana Berman, Agnieszka Jastrzebska, Massimiliano Papi, and Andreas Rosenkranz introduce the themed issue on 2D materials and their applications.
PubMed: 38831772
DOI: 10.1039/d4ra90059f -
The European Physical Journal. E, Soft... Jun 2024In this study, we demonstrate the fabrication of polymersomes, protein-blended polymersomes, and polymeric microcapsules using droplet microfluidics. Polymersomes with...
In this study, we demonstrate the fabrication of polymersomes, protein-blended polymersomes, and polymeric microcapsules using droplet microfluidics. Polymersomes with uniform, single bilayers and controlled diameters are assembled from water-in-oil-in-water double-emulsion droplets. This technique relies on adjusting the interfacial energies of the droplet to completely separate the polymer-stabilized inner core from the oil shell. Protein-blended polymersomes are prepared by dissolving protein in the inner and outer phases of polymer-stabilized droplets. Cell-sized polymeric microcapsules are assembled by size reduction in the inner core through osmosis followed by evaporation of the middle phase. All methods are developed and validated using the same glass-capillary microfluidic apparatus. This integrative approach not only demonstrates the versatility of our setup, but also holds significant promise for standardizing and customizing the production of polymer-based artificial cells.
Topics: Artificial Cells; Polymers; Emulsions; Capsules; Microfluidics; Water; Microfluidic Analytical Techniques; Proteins
PubMed: 38829453
DOI: 10.1140/epje/s10189-024-00428-5 -
STAR Protocols Jun 2024Microfluidic single-cell cultivation (MSCC) is a powerful tool for investigating the cellular behavior of various cell types at the single-cell level. Here, we present a...
Microfluidic single-cell cultivation (MSCC) is a powerful tool for investigating the cellular behavior of various cell types at the single-cell level. Here, we present a protocol specifically developed for the reliable and reproducible MSCC of industrially relevant Chinese hamster ovary (CHO) suspension cell lines. We summarize critical experimental steps from the initial seed train up to the final MSCC experiment, with a special focus on pre-culture management and medium preparation, device inoculation, and the establishment of a constant medium perfusion.
Topics: Animals; CHO Cells; Cricetulus; Cell Culture Techniques; Single-Cell Analysis; Cricetinae; Microfluidics; Microfluidic Analytical Techniques
PubMed: 38824641
DOI: 10.1016/j.xpro.2024.103106 -
Biomaterials Oct 2024The pre-clinical animal models often fail to predict intrinsic and idiosyncratic drug induced liver injury (DILI), thus contributing to drug failures in clinical trials,...
The pre-clinical animal models often fail to predict intrinsic and idiosyncratic drug induced liver injury (DILI), thus contributing to drug failures in clinical trials, black box warnings and withdrawal of marketed drugs. This suggests a critical need for human-relevant in vitro models to predict diverse DILI phenotypes. In this study, a porcine liver extracellular matrix (ECM) based biomaterial ink with high printing fidelity, biocompatibility and tunable rheological and mechanical properties is formulated for supporting both parenchymal and non-parenchymal cells. Further, we applied 3D printing and microfluidic technology to bioengineer a human physiomimetic liver acinus model (HPLAM), recapitulating the radial hepatic cord-like structure with functional sinusoidal microvasculature network, biochemical and biophysical properties of native liver acinus. Intriguingly, the human derived hepatic cells incorporated HPLAM cultured under physiologically relevant microenvironment, acts as metabolic biofactories manifesting enhanced hepatic functionality, secretome levels and biomarkers expression over several weeks. We also report that the matured HPLAM reproduces dose- and time-dependent hepatotoxic response of human clinical relevance to drugs typically recognized for inducing diverse DILI phenotypes as compared to conventional static culture. Overall, the developed HPLAM emulates in vivo like functions and may provide a useful platform for DILI risk assessment to better determine safety and human risk.
Topics: Humans; Chemical and Drug Induced Liver Injury; Liver; Animals; Swine; Printing, Three-Dimensional; Microfluidics; Models, Biological; Drug Evaluation, Preclinical; Extracellular Matrix; Hepatocytes; Biomimetics
PubMed: 38823194
DOI: 10.1016/j.biomaterials.2024.122627