BioChip Journal最新文献

Among human CD8⁺ T cells, senescent cells are marked by the expression of CD57. The frequency of senescent CD57⁺CD8⁺ T cells is significantly correlated with aging and age-associated disorders, and it can be measured by multi-color flow cytometry. However, multi-color flow cytometry presents challenges in terms of accessibility and requires significant resource allocation. Therefore, developing a rapid and straightforward method for quantifying CD57⁺CD8⁺ T cells remains a key challenge. This study introduces a microfluidic device composed of a PDMS microfluidic channel with a pre-modified glass substrate for anti-CD8 antibody immobilization. This design allows blood samples to flow through, enabling the selective capture of CD8⁺ T cells while minimizing the required blood sample volume. This technology enables accurate and reliable quantification of CD57⁺ cells among captured CD8⁺ T cells through fluorescence image analysis. The ability of the device to easily quantify senescent CD57⁺CD8⁺ T cells is anticipated to contribute significantly to both immunological research and clinical applications.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleocapsid protein (NP) participates in viral genome packaging and abundantly produced when infected. In this work, SPR biosensor for the detection of SARS-CoV-2 in viral fluid using Fv-antibodies with the binding affinity to nucleocapsid protein (NP) of SARS-CoV-2. The F_V-antibodies with a specific binding activity to the SARS-CoV-2 NP were screened using the F_V-antibody library, which was expressed on the outer membrane of E. coli. F_V-antibodies comprised three complementarity-determining regions (CDRs) and four frame regions (FRs) of the heavy chain at the binding pocket of IgG. The F_V-antibody library was prepared by performing site-directed mutagenesis and by using the autodisplay technology; F_V-antibodies with specific binding activities to the nucleocapsid protein (NP) of SARS-CoV-2 were screened using NP-immobilized magnetic beads. First, E. coli isolates with the target F_V-antibody were screened, and the binding affinity (K_D) was estimated for the screened E. coli clones using FACS analysis. Then, the outer membrane (OM) of the screened E. coli clones with autodisplayed Fv-antibodies was obtained and layered on an SPR biosensor, and the binding curves of four different coronavirus (CoV) culture fluids, SARS-CoV-2, SARS-CoV, MERS-CoV, and CoV strain 229E, were compared. Finally, the F_V-antibodies of the screened E. coli clones were synthesized as peptides (11 amino acid residues), and the binding constants (K_D) to NP as well as the binding curves of the CoV strains in culture fluids were estimated. Using docking simulation, binding sites and interaction types between NP and each synthetic peptide were investigated.

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Abstract

Due to its similarity to in vivo conditions, tumor spheroids are actively used in research areas, such as drug screening and cell–cell interactions. A substantial quantity of spheroids is crucial for obtaining dependable results in high-throughput screening. Conventional fabrication methods of spheroid have limitations in low yield and morphological variation. Droplet-based microfluidic system capable of mass-producing uniformed spheroids can overcome these limitations. In this study, we investigated the optimal culture conditions, which allows to researchers provide guidelines for producing spheroids with the desired diameter and quantity. Mass-produced spheroids were employed to analyze compaction, which is crucial for evaluating the remission effects of drugs, as well as the formation of a necrotic core, which induces a bias in the analysis of drug response and viability. The time point at which compaction is completed and the diameter begins to increase was measured using spheroids with diameters of both > 400 μm and < 400 μm, and spheroids do not proliferate a linear growth trend. Spheroid with diameters ranging from 73.4 ± 11.42 μm to 371 ± 5.11 μm was used to predict the formation of the necrotic core based on live cell counting, and diameter of 300–330 μm was mathematically calculated as the diameter where a necrotic core forms. Additionally, the use of artificial intelligence (AI) for high-throughput analysis is crucial for obtaining time-saving and reproducible data. We produced BT474 and MCF-7 spheroids with diameters of 100, 200, and 300 μm and obtained morphological indicators from an AI-based program to compare the differences in heterogeneous breast tumor spheroids. Through this study, we optimized the diameter of spheroids and the initiation timing for drug screening and emphasized the importance of AI-based morphological analysis in high-throughput screening.

Functionalized carbon quantum dots with tunable optical properties widely used in sensor applications. In this study, carbon quantum dots were synthesized from Zelkova serrata leaves (ZCQDs) in an aqueous medium via a single-step hydrothermal reaction with essential reactive functional groups. As-synthesized ZCQDs (average size, 3–7 nm) were characterized and confirmed to contain hydroxyl/amine and carboxylic acid functional groups. Photoluminescence spectral analysis revealed that the fluorescence intensity of ZCQDs drastically decreased after the addition of different concentrations of normetanephrine (NM) in deionized water and geriatric plasma samples. Our developed strategy could detect NM with a limit of detection of 7.96 and 86.2 nM in deionized water and geriatric plasma samples, respectively. Interestingly, Fourier transform infrared spectroscopy revealed a new peak at 1660 cm⁻¹, confirming the formation of the keto (C=O) group in NM. In addition, significant peak shifts were observed in the C 1s, and O 1s deconvoluted X-ray photoelectron spectra. Bandgap calculations also revealed significant interactions between NM and ZCQDs. Antibacterial activities of ZCQDs were investigated in Escherichia coli and Staphylococcus aureus, and potent activities were observed in Staphylococcus aureus at a half-maximal inhibitory concentration of 32 μg/mL via the generation of intracellular reactive oxygen species. By enabling specific therapies and improving our understanding of intricate biological processes at the nanoscale, these materials have the potential to completely transform the biomedical field. Our findings suggest the involvement of a working mechanism in transferring electrons between the conductance band of ZCQDs and the acidic protons of N, to produce the oxidized form NM for photoluminescence quenching of ZCQDs.

Separation of micro- and nano-sized bioparticles is essential for efficient diagnostics, chemical and biological analyses, drug development, food and chemical processing, and environmental monitoring. However, most of the currently available bio-separation techniques are based on the membrane filtration approach, whose efficiency is restricted by membrane-related disadvantages, including pore size, surface charge density, and biocompatibility, which results in a reduction in the isolation resolution. To address these issues, till now, many microfluidic devices have been developed for particle/cell profiling due to their excellent sensitivity and specificity, less sample consumption, shortened processing time, and high throughput features. Of the various microfluidic systems, the spiral inertial microfluidic technique has recently attracted attention as an innovative strategy and advanced cutting-edge technology toward bioparticle separation. Depending on the needs of the microfluidic device, the spiral inertial chip can be customized to separate bioparticles owing to their sizes and different shapes. In this review, we discuss the kinematics of microchannel particle separation mechanisms, recent developments in the inertial microfluidic device realm, and their applications for the separation of several types of bioparticles, including blood cells, stem cells, sperm cells, pathogens, and algae. Finally, we highlight challenges and economical perspectives associated with guidelines for further development of spiral inertial microfluidic devices in the future.

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Bead-based fluorescence immunoassay is drawing attention as a next-generation technology in disease diagnosis owing to its high sensitivity and multiplexing capability. Fluorescence imaging of beads is typically used to determine their mean fluorescence intensity. However, the mean intensity can be evaluated differently depending on the analysis methods [such as the shape and size of the region of interest (ROI)]. To address these problems, this study proposes a highly reliable and reproducible image analysis method utilizing a fluorescence intensity-based effective pixel extraction technique. Various potential sources of defective signals (e.g., fluorescence aggregation, non-specific antigen–antibody reactions, and bead defects) can be prevented from contributing to the average value by selectively extracting pixels representing the specific reactions of antigens and antibodies in the ROI. In this study, we fabricated a microfluidic chip composed of multiple bead-based detection lines, performed fluorescence immunoassay, and then compared the mean fluorescence intensity calculated from the fluorescence images with that of a conventional analysis method. Using the conventional method, the evaluated average mean intensity value of beads varied significantly based on the size of the ROI with the coefficients of variation ranging from approximately 29–95%. In contrast, the effective pixel extraction method resulted in a coefficient of variation of approximately 3–7% under varying ROI size. Furthermore, the coefficients of variation for four detection lines containing various types of defective signals significantly decreased from approximately 7.1% to 2.6%. The proposed technique will help in minimizing the analysis deviation caused by different ROI selections or defective signals in fluorescent image-based immunoassays.

Abstract

We present a numerical simulation method for designing a buffer system in microchip electrophoresis (MCE) equipped with capacitively-coupled-contactless-conductivity detection (C4D). One of the key design considerations for MCE-C4D is background electrolyte (BGE). This is because a C4D typically exhibits low sensitivity, and optimizing BGE conditions (e.g., base and acid species, pH, and ionic strength) can improve its sensitivity. However, BGE has been traditionally designed through experience or trial and error, which is time- and reagent-consuming. In this study, we employ Simul 5, an open-source electrophoresis simulation software, for rational BGE design. Four BGEs including trimethylamine (TEA)/acetic acid (AcOH, pH 10.6), MES/His (pH 6.1), MES/TRIS (pH 8.1), and TRIS/HCl (pH 7.4), previously used in electrophoresis-C4D of amino acids and protein, were selected for evaluation of our numerical method. Glutamic acid (Glu) was selected as a model analyte for initial simulation verification. Our numerical simulation revealed that the best achievable detection sensitivity was 1.046 × 10^–5 S/(m µM) in the TRIS/HCl buffer because anionic Glu species with a low mobility (27 × 10^–9 m²/Vs) replaced Cl⁻ co-ion of a high mobility (79.1 × 10^–9 m²/Vs) in the analyte zone, leading to a significant negative conductivity peak. TEA/AcOH, MES/His, and MES/TRIS buffers exhibited progressively lower sensitivity. After the initial evaluation, trypsin inhibitor (TI), a more complex proteinous analyte was tested in the MES/His and MES/TRIS BGEs. The best detection sensitivity was 1.032 × 10^–4 S/(m µM) in the MES/TRIS buffer because counter-ionic species TRIS⁺ of a high mobility (29.5 × 10^–9 m²/Vs) was replaced by the ionic TI, characterized by a large charge (− 11.5) and a low mobility (8.08 × 10^–9 m²/Vs), resulting in a strong negative peak. Based on a comprehensive analysis of the impacts of compositional changes in each ionic species of the analyte zone on conductivity-peak height, we propose a BGE design guideline for enhanced sensitivity. Subsequent MCE-C4D confirmation experiments demonstrated excellent qualitative agreement with the simulation results for the Glu and TI analytes. We anticipate that our numerical analysis method will find wide application in designing BGEs for portable MCE-C4D systems by enhancing sensitivity.

Macrophages are immune cells that play important roles in the human body’s initial immune responses against pathogens and tumor cells. We investigated the use of electrical impedance monitoring to assess the differentiation of THP-1 monocyte into macrophages, which is necessary for immunotherapy research conducted. The change in resistance at 1 kHz and capacitance at 100 kHz measured were proportionally increased according to not only the increase in the density of resting macrophages differentiated by Phorbol-12-myristate-13-acetate treatment but also the initial number of THP-1 cells seeded on the electrode. Additionally, real-time impedance data from THP-1 cells after 48 h of cultivation demonstrated greater recognition of the resting macrophage phenotypes (adhesion cells) covered microelectrode surface with a significant increase of impedance signal in comparison with monocytes phenotypes (suspended cells). Furthermore, during the polarization phase of macrophages, the alternatively activated macrophage phenotype was larger and flatter than that of classically activated macrophage and resting macrophage phenotypes, indicating a correlation with a higher resistance and lower capacitance impedances at 1 kHz and 100 kHz of alternatively activated macrophages (4750 Ω and – 3.5 nF) than that of classically activated macrophages (2000 Ω and – 1.5 nF) and resting macrophages (3500 Ω and – 2.0 nF), respectively. The study’s findings demonstrated that the impedance measurement system is high sensitivity and confidence in monitoring macrophages differentiation and polarization. The electrical impedance, which has significance for each macrophage phenotype, is compatible with macrophages characteristic features observed using flow cytometry and a microscope.

Soft actuators have played an indispensable part in the field of biosensors and soft robotics as such systems offer solutions that cannot be addressed with rigid actuators due to the lack of both flexibility and sensitivity. However, soft actuators have certain limitations when it comes to their durability and longevity. In recent years, quite a few versatile fabrication techniques and innovative solutions have been developed that have played an essential role in the development of soft robotics. An exemplary innovation involves the integration of nanomaterials into polymers that act as a host in the fabrication of inorganic/organic actuators. These actuators have shown significant enhancement both in their physical and chemical properties. Consequently, it paves the way for the development of sophisticated soft actuator-based devices that can find broader applications in the field of biomedical sciences. However, biocompatibility has been a matter of concern for inorganic/organic soft actuators. Addressing this issue, studies on the development of biomaterial-based soft actuators that incorporate nanomaterials have been conducted for biohybrid robots. This review aims to provide a comprehensive understanding of diverse stimulus-trigger actuation alongside exploring the influence of nanomaterials in inorganic/organic actuators. Further, it gives valuable insights into the implication of biomaterials in soft actuators for the development of biohybrid robot.

Yellow fever virus (YFV) is an acute infectious virus with high morbidity and mortality risks during the toxic phase. Early diagnosis and suppression are essential because YFV has no precise treatment. With the aim of detecting YFV, we fabricated a highly sensitive electrochemical biosensor comprised with a truncated DNA aptamer/MXene heterolayer. The synthesized DNA aptamer was prepared by systematic evolution of ligands using the exponential enrichment (SELEX) technique, which can specifically detect the YFV NS1 protein. MXenes increase the electrical sensitivity and the possibility of attachment of aptamers by widening the surface area. The aptamer-cutting process which called a truncation process can reduce the production cost of biosensors. The biosensor performance was evaluated using electrochemical methods, cyclic voltammetry (CV), and electrochemical impedance spectroscopy (EIS). The limit of detection (LOD) was 2.757 pM for YFV diluted in phosphate-buffered saline (PBS) and 2.366 pM for YFV diluted in 10% human serum, proving that the biosensor specifically binds to YFV through selectivity evaluation. This biosensor can be a valuable tool for the early diagnosis of YFV, enabling timely intervention as well as facilitating the control and prevention of yellow fever outbreaks.