Impedimetric Gram-Positive Bacteria Biosensor Using Vancomycin-Coated Silica Nanoparticles with a Gold Nanocluster-Deposited Electrode.

We introduce a swift, label-free electrochemical biosensor designed for the precise on-site detection of Gram-positive bacteria via electrochemical impedance spectroscopy. The biosensor was prepared by electroplating the electrode surface with gold nanoclusters (AuNCs) on the gold-interdigitated wave-shaped electrode with a printed circuit board (Au-PCB) electrode, which plays a role in cost-effective and promising lab-on-a-chip microsystems and integrated biosensing systems. This was followed by the application of silica nanoparticle-modified vancomycin (SiNPs-VAN) that binds to Gram-positive bacteria and facilitates their detection on the AuNC-coated surface. The biosensor demonstrated remarkable sensitivity and specificity. It could detect as few as 102 colony-forming units (CFU)/mL of Staphylococcus aureus, 101 CFU/mL of Bacillus cereus, and 102 CFU/mL of Micrococcus luteus within 20 min. Additionally, SiNPs-VAN is also known for its high stability, low cost, and ease of preparation. It is effective in identifying Gram-positive bacteria in water samples across a concentration range of 102-105 CFU/mL and shows selective identification of Gram-positive bacteria with minimal interference from Gram-negative bacteria like Escherichia coli. The ability of the biosensor to quantify Gram-positive bacteria aligns well with the results obtained from the quantitative real-time polymerase chain reaction (qRT-PCR). These findings highlight the potential of electrochemical biosensors for the detection of pathogens and other biological entities, marking a significant advancement in this field.

Differential growth enhancement followed by notable microbiota modulation in growing-finishing pigs by Bacillus subtilis strains ps4060, ps4100, and a 50:50 strain mixture.

A 50:50 blend of two Bacillus subtilis strains positively impacted the productivity of finishing pigs. Given this observed effect, we hypothesized that each strain has distinct effects on weight gain and their influence on gut microbiota. In a 16-week test, 160 pigs were divided into four groups: basal diet, B. subtilis ps4100, B. subtilis ps4060, and 50:50 mixture supplemented. Subsequently, we compared body weight and fecal microbiota. Among the supplements, ps4100, ps4060, and the 50:50 mix yielded respective average daily weight gains (ADG) of 3.6%, 4.6%, and 3.9% by the 6th week. The weight difference was maintained through the 16th week. At the 11th week, the difference in α-diversity among the fecal microbiota was marginal, and 17 of 229 genera showed differential abundance between the control and either of the treatment groups. A total of 12 of the 17 genera, including Lactobacillus, showed differential abundance between the ps4100 and ps4060-fed groups, and only Eubacterium consistently decreased in abundance in both the ps4100 and ps4060 groups. In comparison, microbial diversity was significantly different at the 16th week (p < 0.05), with 96 out of 229 genera exhibiting differential abundance. A total of 42 of the 96 genera exhibited similar patterns in both the ps4100 and ps4060 groups compared to the control group. Additionally, 236 of 687 microbial enzymes with differential abundance deduced from 16S rRNA reads showed similar differential abundance in both groups compared to the control group. We concluded that the overall microbial balance, rather than the dominance or significant decrease of a few specific genera, likely caused the enhanced ADG until the 11th week. Substantial changes in microbiota manifested at the 16th week did not cause dramatically increased ADG but were a consequence of weight gain and could positively affect animal physiology and health afterward.

Mechanisms of Plasma Ozone and UV-C Sterilization of SARS-CoV-2 Explored through Atomic Force Microscopy.

Ultraviolet-C (UV-C) radiation and ozone gas are potential mechanisms employed to inactivate the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), each exhibiting distinct molecular-level modalities of action. To elucidate these disparities and deepen our understanding, we delve into the intricacies of SARS-CoV-2 inactivation via UV-C and ozone gas treatments, exploring their distinct molecular-level impacts utilizing a suite of advanced techniques, including biological atomic force microscopy (Bio-AFM) and single virus force spectroscopy (SVFS). Whereas UV-C exhibited no perceivable alterations in virus size or surface topography, ozone gas treatment elucidated pronounced changes in both parameters, intensifying with prolonged exposure. Furthermore, a nuanced difference was observed in virus-host cell binding post-treatment: ozone gas distinctly reduced SARS-CoV-2 binding to host cells, while UV-C maintained the status quo. The results derived from these methodical explorations underscore the pivotal role of advanced Bio-AFM techniques and SVFS in enhancing our understanding of virus inactivation mechanisms, offering invaluable insights for future research and applications in viral contamination mitigation.

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.

Engineering Stem Cell Fate Controlling Biomaterials to Develop Muscle Connective Tissue Layered Myofibers.

Skeletal muscle connective tissue (MCT) surrounds myofiber bundles to provide structural support, produce force transduction from tendons, and regulate satellite cell differentiation during muscle regeneration. Engineered muscle tissue composed of myofibers layered within MCT has not yet been developed. Herein, a bioengineering strategy to create MCT-layered myofibers through the development of stem cell fate-controlling biomaterials that achieve both myogenesis and fibroblast differentiation in a locally controlled manner at the single construct is introduced. The reciprocal role of transforming growth factor-beta 1 (TGF-ß1) and its inhibitor as well as 3D matrix stiffness to achieve co-differentiation of MCT fibroblasts and myofibers from a human-induced pluripotent stem cell (hiPSC)-derived paraxial mesoderm is studied. To avoid myogenic inhibition, TGF-ß1 is conjugated on the gelatin-based hydrogel to control the fibroblasts' populations locally; the TGF-ß1 degrades after 2 weeks, resulting in increased MCT-specific extracellular matrix (ECM) production. The locations of myofibers and fibroblasts are precisely controlled by using photolithography and co-axial wet spinning techniques, which results in the formation of MCT-layered functional myofibers in 3D constructs. This advanced engineering strategy is envisioned as a possible method for obtaining biomimetic human muscle grafts for various biomedical applications.

Plant-Derived Anti-Human Epidermal Growth Factor Receptor 2 Antibody Suppresses Trastuzumab-Resistant Breast Cancer with Enhanced Nanoscale Binding.

Traditional monoclonal antibodies such as Trastuzumab encounter limitations when treating Human Epidermal Growth Factor Receptor 2 (HER2)-positive breast cancer, particularly in cases that develop resistance. This study introduces plant-derived anti-HER2 variable fragments of camelid heavy chain domain (VHH) fragment crystallizable region (Fc) KEDL(K) antibody as a potent alternative for overcoming these limitations. A variety of biophysical techniques, in vitro assays, and in vivo experiments uncover the antibody's nanoscale binding dynamics with transmembrane HER2 on living cells. Single-molecule force spectroscopy reveals the rapid formation of two robust bonds, exhibiting approximately 50 pN force resistance and bond lifetimes in the second range. The antibody demonstrates a specific affinity for HER2-positive breast cancer cells, including those that are Trastuzumab-resistant. Moreover, in immune-deficient mice, the plant-derived anti-HER2 VHH-FcK antibody exhibits superior antitumor activity, especially against tumors that are resistant to Trastuzumab. These findings underscore the plant-derived antibody's potential as an impactful immunotherapeutic strategy for treating Trastuzumab-resistant HER2-positive breast cancer.

Exploration of drug resistance mechanisms in triple negative breast cancer cells using a microfluidic device and patient tissues.

Chemoresistance is a major cause of treatment failure in many cancers. However, the life cycle of cancer cells as they respond to and survive environmental and therapeutic stress is understudied. In this study, we utilized a microfluidic device to induce the development of doxorubicin-resistant (DOXR) cells from triple negative breast cancer (TNBC) cells within 11 days by generating gradients of DOX and medium. In vivo chemoresistant xenograft models, an unbiased genome-wide transcriptome analysis, and a patient data/tissue analysis all showed that chemoresistance arose from failed epigenetic control of the nuclear protein-1 (NUPR1)/histone deacetylase 11 (HDAC11) axis, and high NUPR1 expression correlated with poor clinical outcomes. These results suggest that the chip can rapidly induce resistant cells that increase tumor heterogeneity and chemoresistance, highlighting the need for further studies on the epigenetic control of the NUPR1/HDAC11 axis in TNBC.

PIV-MyoMonitor: an accessible particle image velocimetry-based software tool for advanced contractility assessment of cardiac organoids.

Induced pluripotent stem cell (iPSC)-derived cardiac organoids offer a versatile platform for personalized cardiac toxicity assessment, drug screening, disease modeling, and regenerative therapies. While previous image-based contractility analysis techniques allowed the assessment of contractility of two-dimensional cardiac models, they face limitations, including encountering high noise levels when applied to three-dimensional organoid models and requiring expensive equipment. Additionally, they offer fewer functional parameters compared to commercial software. To address these challenges, we developed an open-source, particle image velocimetry-based software (PIV-MyoMonitor) and demonstrated its capacity for accurate contractility analysis in both two- and three-dimensional cardiac models using standard lab equipment. Comparisons with four other open-source software programs highlighted the capability of PIV-MyoMonitor for more comprehensive quantitative analysis, providing 22 functional parameters and enhanced video outputs. We showcased its applicability in drug screening by characterizing the response of cardiac organoids to a known isotropic drug, isoprenaline. In sum, PIV-MyoMonitor enables reliable contractility assessment across various cardiac models without costly equipment or software. We believe this software will benefit a broader scientific community.

Antibody functionalized capacitance sensor for label-free and real-time detection of bacteria and antibiotic susceptibility.

The effective management of infectious diseases and the growing concern of antibiotic resistance necessitates accurate and targeted therapies, highlighting the importance of antibiotic susceptibility testing. This study aimed to develop a real-time impedimetric biosensor for identifying and monitoring bacterial growth and antibiotic susceptibility. The biosensor employed a gold 8-channel disk-shaped microelectrode array with specific antibodies as bio-recognition elements. This setup was allowed for the analysis of bacterial samples, including Staphylococcus aureus, Bacillus cereus, and Micrococcus luteus. These microorganisms were successfully cultured and detected within 1 h of incubation even with a minimal bacterial concentration of 10 CFU/ml. Overall, the developed biosensor array exhibits promising capabilities for monitoring S. aureus, B. cereus and M. luteus, showcasing an excellent linear response ranging from 10 to 104 CFU/ml with a detection limit of 0.95, 1.22 and 1.04 CFU/mL respectively. Moreover, real-time monitoring of antibiotic susceptibility was facilitated by changes in capacitance, which dropped when bacteria were exposed to antibiotic doses higher than their minimum inhibitory concentration (MIC), indicating suppressed bacterial growth. The capacitance measurements enabled determination of half-maximal cytotoxic concentrations (CC50) values for each bacteria-antibiotic pair. As a proof-of-concept application, the developed sensor array was employed as a sensing platform for the real time detection of bacteria in milk samples, which ensured the reliability of the sensor for in-field detection of foodborne pathogens and rapid antimicrobial susceptibility tests (ASTs).

Automated sepsis detection with vancomycin- and allantoin-polydopamine magnetic nanoparticles.

Rapid and accurate identification of the bacteria responsible for sepsis is paramount for effective patient care. Molecular diagnostic methods, such as polymerase chain reaction (PCR), encounter challenges in sepsis due to inhibitory compounds in the blood, necessitating their removal for precise analysis. In this study we present an innovative approach that utilizes vancomycin (Van) and allantoin (Al)-conjugated polydopamine (PDA)-coated magnetic nanoparticles (MNPs) for the rapid and automated enrichment of bacteria and their DNA extraction from blood without inducing clumping and aggregation of blood. Al/Van-PDA-MNPs, facilitated by IMS, eliminate the need for preliminary sample treatments, providing a swift and efficient method for bacterial concentration and DNA extraction within an hour. Employing Al/Van-PDA-MNPs within an automated framework has markedly improved our ability to pre-concentrate various Gram-negative and Gram-positive bacteria directly from blood samples. This advancement has effectively reduced the detection threshold to 102 colony-forming unit/mL by both PCR and quantitative PCR. The method's expedited processing time, combined with its precision, positions it as a feasible diagnostic tool for diverse healthcare settings, ranging from small clinics to large hospitals. Furthermore, the innovative application of nanoparticles for DNA extraction holds promising potential for advancing sepsis diagnostics, enabling earlier interventions and improving patient outcomes.

Three-Dimensional Hanging Drop Spheroid Plates for Easy Chimeric Antigen Receptor (CAR) T Cytotoxicity Assay.

Chimeric antigen receptor (CAR) T cell therapy shows a highly effective therapeutic effect on B-cell malignancies. The tumor microenvironment (TME) of solid tumors in vivo poses a great challenge to CAR T cell therapy due to its complexity. Recently, tumor spheroids have attracted much attention because of their ability to recapitulate TME. However, the use of tumor spheroids for the CAR T cytotoxicity assay involves the difficult task of separating unbound T cells and dead tumor cells from the spheroids. Therefore, we developed a three-dimensional hanging spheroid plate (3DHSP) that facilitates spheroid formation and separation of unbound and dead cells from spheroids during cytotoxicity assays. In this work, detailed steps have been described for fabrication and operation of the 3DHSP. This new 3DHSP device is a 96-well plate in which each well consists of a hanging dripper and a spheroid separation plate. A tumor spheroid forms in a droplet hanging in the dripper and is mixed with CAR T cells. The mixture in the droplet is deposited into the spheroid separation plate by pipetting, and unbound and dead CAR T and tumor cells are detached from the spheroid and moved to the waste well in the plate by tilting the 3DHSP at 20°. The size of the spheroid can be used as a readout for CAR T cell cytotoxicity assay, suggesting that the 3DHSP does not require cumbersome fluorescent staining.

A 3D hanging spheroid-filter plate for high-throughput drug testing and CAR T cell cytotoxicity assay.

Tumour spheroids are widely used in immune cell cytotoxicity assays and anticancer drug testing, providing a physiologically relevant model replicating the tumour microenvironment. However, co-culture of immune and tumour cells complicates quantification of immune cell killing efficiency. We present a novel 3D hanging spheroid-filter plate that efficiently facilitates spheroid formation and separates unbound/dead cells during cytotoxicity assays. Optical imaging directly measures the cytotoxic effects of anti-cancer drugs on tumour spheroids, eliminating the need for live/dead fluorescent staining. This approach enables cost-effective evaluation of T-cell cytotoxicity with specific chimeric antigen receptors (CARs), enhancing immune cell-based assays and drug testing in three-dimensional tumour models.

Nano-biomaterials and advanced fabrication techniques for engineering skeletal muscle tissue constructs in regenerative medicine.

Engineered three-dimensional (3D) tissue constructs have emerged as a promising solution for regenerating damaged muscle tissue resulting from traumatic or surgical events. 3D architecture and function of the muscle tissue constructs can be customized by selecting types of biomaterials and cells that can be engineered with desired shapes and sizes through various nano- and micro-fabrication techniques. Despite significant progress in this field, further research is needed to improve, in terms of biomaterials properties and fabrication techniques, the resemblance of function and complex architecture of engineered constructs to native muscle tissues, potentially enhancing muscle tissue regeneration and restoring muscle function. In this review, we discuss the latest trends in using nano-biomaterials and advanced nano-/micro-fabrication techniques for creating 3D muscle tissue constructs and their regeneration ability. Current challenges and potential solutions are highlighted, and we discuss the implications and opportunities of a future perspective in the field, including the possibility for creating personalized and biomanufacturable platforms.

Phenotypic assay for cytotoxicity assessment of Balamuthia mandrillaris against human neurospheroids.

Introduction: The phenotypic screening of drugs against Balamuthia mandrillaris, a neuropathogenic amoeba, involves two simultaneous phases: an initial step to test amoebicidal activity followed by an assay for cytotoxicity to host cells. The emergence of three-dimensional (3D) cell cultures has provided a more physiologically relevant model than traditional 2D cell culture for studying the pathogenicity of B. mandrillaris. However, the measurement of ATP, a critical indicator of cell viability, is complicated by the overgrowth of B. mandrillaris in coculture with host cells during drug screening, making it challenging to differentiate between amoebicidal activity and drug toxicity to human cells. Methods: To address this limitation, we introduce a novel assay that utilizes three-dimensional hanging spheroid plates (3DHSPs) to evaluate both activities simultaneously on a single platform. Results and discussion: Our study showed that the incubation of neurospheroids with clinically isolated B. mandrillaris trophozoites resulted in a loss of neurospheroid integrity, while the ATP levels in the neurospheroids decreased over time, indicating decreased host cell viability. Conversely, ATP levels in isolated trophozoites increased, indicating active parasite metabolism. Our findings suggest that the 3DHSP-based assay can serve as an endpoint for the phenotypic screening of drugs against B. mandrillaris, providing a more efficient and accurate approach for evaluating both parasite cytotoxicity and viability.

Subcellular Force Imbalance in Actin Bundles Induces Nuclear Repositioning and Durotaxis.

Durotaxis is a phenomenon in which cells migrate toward substrates of increasing stiffness. However, how cells assimilate substrate stiffness as a directional cue remains poorly understood. In this study, we experimentally show that mouse embryonic fibroblasts can discriminate between different substrate stiffnesses and develop higher traction forces at regions of the cell adhering to the stiffer pillars. In this way, the cells generate a force imbalance between adhesion sites. It is this traction force imbalance that drives durotaxis by providing directionality for cell migration. Significantly, we found that traction forces are transmitted via LINC complexes to the cell nucleus, which serves to maintain the global force imbalance. In this way, LINC complexes play an essential role in anterograde nuclear movement and durotaxis. This conclusion is supported by the fact that LINC complex-deficient cells are incapable of durotaxis and instead migrate randomly on substrates featuring a stiffness gradient.

3D printed fluidic swab for COVID-19 testing with improved diagnostic yield and user comfort.

The current standard method of diagnosing coronavirus disease 2019 (COVID-19) involves uncomfortable and invasive nasopharyngeal (NP) sampling using cotton swabs (CS), which can be unsuitable for self-testing. Although mid-turbinate sampling is an alternative, it has a lower diagnostic yield than NP sampling. Nasal wash (NW) has a similar diagnostic yield to NP sampling, but is cumbersome to perform. In this study, we introduce a 3D printed fluidic swab (3DPFS) that enables easy NW sampling for COVID-19 testing with improved diagnostic yield. The 3DPFS comprises a swab head, microchannel, and socket that can be connected to a syringe containing 250 µL of NW solution. The 3DPFS efficiently collects nasal fluid from the surface of the nasal cavity, resulting in higher sensitivity than CS for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This was confirmed by both reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and lateral flow assays (LFA) in virus-spiked nasal samples and clinical samples. Additionally, users reported greater comfort when using the 3DPFS compared to CS. These findings suggest that the 3DPFS can improve the performance of COVID-19 testing by facilitating efficient and less painful nasal sample collection.

Mobile Efficient Diagnostics of Infectious Diseases via On-Chip RT-qPCR: MEDIC-PCR.

The COVID-19 outbreak has caused public and global health crises. However, the lack of on-site fast, reliable, sensitive, and low-cost reverse transcription polymerase chain reaction (RT-PCR) testing limits early detection, timely isolation, and epidemic prevention and control. Here, the authors report a rapid mobile efficient diagnostics of infectious diseases via on-chip -RT-quantitative PCR (RT-qPCR): MEDIC-PCR. First, the authors use a roll-to-roll printing process to accomplish low-cost carbon-black-based disposable PCR chips that enable rapid LED-induced photothermal PCR cycles. The MEDIC-PCR can perform RT (3 min), and PCR (9 min) steps. Further, the cohort of 89 COVID-19 and 103 non-COVID-19 patients testing is completed by the MEDIC-PCR to show excellent diagnostic accuracy of 97%, sensitivity of 94%, and specificity of 98%. This MEDIC-PCR can contribute to the preventive global health in the face of a future pandemic.

Multi-Inlet Spheroid Generator for High-Throughput Combinatorial Drug Screening Based on the Tumor Microenvironment.

Tumor spheroids are powerful tools for drug screening and understanding tumor physiology. Among spheroid formation methods, the hanging drop method is considered most suitable for high-throughput screening (HTS) of anticancer drugs because it does not require surface treatment. However, it still needs to increase the liquid-holding capacity because hanging drops often fall due to the increased pressure caused by the addition of drugs, cells, etc. Here, we report a multi-inlet spheroid generator (MSG) enabling the stable addition of liquid-containing drugs or cells into a spheroid through its side inlet. The MSG was able to load additional solutions through the side inlet without increasing the force applied to the hanging drop. The volume of the additional liquid was easily controlled by varying the diameter of the side inlet. Furthermore, the sequences of the solution injections were manipulated using multiple side inlets. The feasibility of the MSG in clinical application was demonstrated by testing the efficacy of drugs in patient-derived cancer (PDC) cells and controlling the stromal cell ratio in the tumor microenvironment (TME) containing spheroids. Our results suggest that the MSG is a versatile platform for HTS of anticancer drugs and recapitulating the TME.

Mechanobiological Analysis of Nanoparticle Toxicity.

Nanoparticles (NPs) are commonly used in healthcare and nanotherapy, but their toxicity at high concentrations is well-known. Recent research has shown that NPs can also cause toxicity at low concentrations, disrupting various cellular functions and leading to altered mechanobiological behavior. While researchers have used different methods to investigate the effects of NPs on cells, including gene expression and cell adhesion assays, the use of mechanobiological tools in this context has been underutilized. This review emphasizes the importance of further exploring the mechanobiological effects of NPs, which could reveal valuable insights into the mechanisms behind NP toxicity. To investigate these effects, different methods, including the use of polydimethylsiloxane (PDMS) pillars to study cell motility, traction force production, and rigidity sensing contractions, have been employed. Understanding how NPs affect cell cytoskeletal functions through mechanobiology could have significant implications, such as developing innovative drug delivery systems and tissue engineering techniques, and could improve the safety of NPs for biomedical applications. In summary, this review highlights the significance of incorporating mechanobiology into the study of NP toxicity and demonstrates the potential of this interdisciplinary field to advance our knowledge and practical use of NPs.