Development of Nano-Micro Fused LSPR Chip for In Situ Single-Cell Secretion Analysis.

Single-cell analysis has become increasingly important in uncovering cell heterogeneity, which has great implications in medicine and biology for a deep understanding of cell characteristics. Owing to its significance, it is vital to create novel devices that can reveal special or unique cells. In this work, we developed a single-cell secretion detection chip consisting of microwells that can trap single cells. Each well is surrounded by Au nanopillars capable of localized surface plasmon resonance (LSPR) measurement. Using microfabrication and nanofabrication techniques, Au nanopillar and microwell structures were fabricated on a COP film. The Au nanopillar was modified with IL-6 antibodies for the direct detection of single-cell secreted IL-6 via LSPR absorbance peak shift. Specific IL-6 detection was successfully demonstrated using a null and IL-6 oversecreting Jurkat cell. A high single-cell trapping efficiency of over 80% was also achieved. Overall, the development of this single-cell secretion detection chip with a simple LSPR measurement setup represents a significant development in the field of cell biology and immunology, providing researchers with a powerful tool for studying individual cells and their secreted cytokines, and is useful for point-of-care testing (POCT) diagnostics.

A High-Throughput Single-Cell Assay on a Valve-Based Microfluidic Platform Applied to Protein Quantification, Immune Response Monitoring, and Drug Discovery.

The use of microfluidic technology in single-cell assay has shown potential in biomedical applications like protein quantification, immune response monitoring, and drug discovery. Because of the details of information that can be obtained at single-cell resolution, the single-cell assay has been applied to tackle challenging issues such as cancer treatment. Information like the levels of protein expression, cellular heterogeneity, and unique behaviors within subsets are very important in the biomedical field. For a single-cell assay system, a high-throughput platform that can do on-demand media exchange and real-time monitoring is advantageous in single-cell screening and profiling. In this work, a high-throughput valve-based device is presented, its use in single-cell assay, particularly in protein quantification and surface-marker analysis, and its potential application to immune response monitoring and drug discovery are laid down in detail.

Linking antigen specific T-cell dynamics in a microfluidic chip to single cell transcription patterns.

A new non-invasive screening profile has been realized that can aid in determining T-cell activation state at single-cell level. Production of activated T-cells with good specificity and stable proliferation is greatly beneficial for advancing adoptive immunotherapy as innate immunological cells are not effective in recognizing and eliminating cancer as expected. The screening method is realized by relating intracellular Ca2+ intensity and motility of T-cells interacting with APC (Antigen Presenting Cells) in a microfluidic chip. The system is tested using APC pulsed with OVA257-264 peptide and its modified affinities (N4, Q4, T4 and V4), and the T-cells from OT-1 mice. In addition, single cell RNA sequencing reveals the activation states of the cells and the clusters from the derived profiles can be indicative of the T-cell activation state. The presented system here can be versatile for a comprehensive application to proceed with T-cell-based immunotherapy and screen the antigen-specific T-cells with excellent efficiency and high proliferation.

A design and optimization of a high throughput valve based microfluidic device for single cell compartmentalization and analysis.

The need for high throughput single cell screening platforms has been increasing with advancements in genomics and proteomics to identify heterogeneity, unique cell subsets or super mutants from thousands of cells within a population. For real-time monitoring of enzyme kinetics and protein expression profiling, valve-based microfluidics or pneumatic valving that can compartmentalize single cells is advantageous by providing on-demand fluid exchange capability for several steps in assay protocol and on-chip culturing. However, this technique is throughput limited by the number of compartments in the array. Thus, one big challenge lies in increasing the number of microvalves to several thousand that can be actuated in the microfluidic device to confine enzymes and substrates in picoliter volumes. This work explores the design and optimizations done on a microfluidic platform to achieve high-throughput single cell compartmentalization as applied to single-cell enzymatic assay for protein expression quantification. Design modeling through COMSOL Multiphysics was utilized to determine the circular microvalve's optimized parameters, which can close thousands of microchambers in an array at lower sealing pressure. Multiphysical modeling results demonstrated the relationships of geometry, valve dimensions, and sealing pressure, which were applied in the fabrication of a microfluidic device comprising of up to 5000 hydrodynamic traps and corresponding microvalves. Comparing the effects of geometry, actuation media and fabrication technique, a sealing pressure as low as 0.04 MPa was achieved. Applying to single cell enzymatic assay, variations in granzyme B activity in Jurkat and human PBMC cells were observed. Improvement in the microfluidic chip's throughput is significant in single cell analysis applications, especially in drug discovery and treatment personalization.

Low levels of Trichinella spp. antibodies detected in domestic pigs at selected slaughterhouses with farm-based exposure assessment in Bulacan, Philippines.

Trichinella spp. is considered as one of the most widespread food-borne zoonotic parasites globally. The disease it causes impacts human public health, pig production, and food safety. Unfortunately in the Philippines, there is still insufficient research on the presence of Trichinella among livestock. This study aims to update its status and records in the country, by verifying the presence of Trichinella spp. IgG antibodies from the selected province, Bulacan, and link its potential presence to known animal husbandry and farm practices. This study was conducted in purposively selected slaughterhouses. Pigs were randomly selected for each slaughterhouse. Blood samples were collected and serum samples were harvested from each pig samples (n = 555). Sera were tested using ELISA for the detection of Trichinella spp. IgG antibodies. For serologically positive pigs, farm-based exposure assessment was conducted to evaluate potential routes of infection. For this study, a total of 555 blood sera, wherein three blood sera were detected to be serologically positive (low prevalence of 0.54 %, 95 % CI = 0.11-1.57). Potential infection routes point towards outdoor housing management, pigs with unknown origin, pig farms presence with rodents, and pigs fed with waste as important risks. In summary, the present paper confirms that Trichinella spp. antibodies were detected in very low prevalence in Bulacan, Philippines and demonstrated the potential utilization of antibody detection as an efficient and complementary early screening tool in Trichinella detection among pigs without immediately sacrificing livestock for the sake of testing. These results merit calls for a wider screening, testing, and isolation of Trichinella spp. in pigs from other Philippine provinces.

The future of microfluidics in immune checkpoint blockade.

Recent advances in microfluidic techniques have enabled researchers to study sensitivities to immune checkpoint therapy, to determine patients' response to particular antibody treatment. Utilization of this technology is helpful in antibody discovery and in the design of personalized medicine. A variety of microfluidic approaches can provide several functions in processes such as immunologic, genomic, and/or transcriptomic analysis with the aim of improving the efficacy and coverage of immunotherapy, particularly immune checkpoint blockade (ICB). To achieve this requires researchers to overcome the challenges in the current state of the technology. This review looks into the advancements in microfluidic technologies applied to researches on immune checkpoint blockade treatment and its potential shift from proof-of-principle stage to clinical application.

A Microfluidic Platform for Single Cell Fluorometric Granzyme B Profiling.

Granzyme B (GrB) is an essential cytotoxic effector in cancer immunotherapy as it can be a potential biomarker to predict the efficacy of immunotherapies including checkpoint inhibitors. Monitoring the Granzyme B activity in cells would help determine a patient's clinical response to treatment and lead to better treatment strategies by preventing administration of ineffective therapies and avoid adverse events resulting in a delay in subsequent treatment. Methods: A microfluidic device with hydrodynamic traps and pneumatic valving system was fabricated using photo and soft lithography. Single cell Granzyme B (GrB) activity was detected and measured fluorometrically using a commercial assay kit with a peptide substrate containing GrB recognition sequence (Ac-IEPD-AFC) and AFC (7-Amino-4-trifluoromethylcoumarin) label. Fluorescence was observed and measured using a confocal microscope with CSU-W1 scanner unit and CCD camera as well as an inverted microscope with photodetector. Model cells (NK-92, GrB-transduced Jurkat, and THP1 cells) and human PBMCs from healthy donor and lung cancer patients including an anti-PD-1 antibody treated patient were profiled of its GrB activity as proof of concept. Results: GrB expression from the model cells was found to be markedly different. NK-92 cells were found to have higher GrB activity than the GrB-transduced Jurkat cells. THP-1 was found to have relatively no significant activity. A marked increase in GrB expression was also observed in anti-PD-1 treated lung cancer patient sample in comparison to PBMC from a healthy donor. TCR+ Ig-G4+ PBMC cells were found to have high activity which signifies a clear response to PD-1 blockade. Conclusion: As proof of concept, we have shown the capability of a microfluidic platform to measure GrB production through a single cell enzymatic activity assay. Our platform might be a promising tool for evaluating the sensitivity of immunotherapies and identifying specific T cell subset responsible for the anti-tumor response.

The curious case of the endemic freshwater crocodile Crocodylus mindorensis as incidental host of marine fish acanthocephalan.

We performed the first host-parasite survey of the Philippine crocodile, Crocodylus mindorensis, a critically endangered species for which ecological information is lacking. We collected by gastric lavage samples of the stomach contents of crocodiles (n = 10) residing at the Palawan Wildlife Rescue and Conservation Center in Puerto Princesa, Palawan, Philippines. The only parasite detected was an acanthocephalan, which was identified as Neorhadinorhynchus nudus (n = 68), a parasite typically found in the marine fish species consumed by three crocodile individuals. Given the known hosts of N. nudus, its parasitism of C. mindorensis in captivity is likely established by consumption of marine fish. Our findings have implications for the conservation management of C. mindorensis, particularly in terms of preventing introduction of parasites that could lead to development of infectious disease or alter the fitness of captive animals.

Long-term changes in the diet of Gymnogobius isaza from Lake Biwa, Japan: effects of body size and environmental prey availability.

Body size and environmental prey availability are both key factors determining feeding habits of gape-limited fish predators. However, our understanding of their interactive or relative effects is still limited. In this study, we performed quantitative dietary analysis of different body sizes of goby (Gymnogobius isaza) specimens collected from Lake Biwa between 1962 and 2004. First, we report that the diet was composed mainly of zooplankton (cladocerans and copepods) before the 1980s, and thereafter, shifted to zoobenthos (gammarids). This foraging shift coincided with, and thus can be linked to, known historical events in the lake at that time: decrease in zooplankton abundance with the alleviation of eutrophication, increase in fish body size resulting from fish population collapse, and increase in gammarid abundance due to reduced fish predation pressure. Supporting this view, our data analyses revealed how the long-term changes in the diet composition would be co-mediated by changes in fish body size and environmental prey availability. Specifically, while zoobenthos abundance strongly affected the fish diet composition, larger (smaller) fish preferred zoobenthos (zooplankton). Furthermore, the body size effects were stronger than those of prey availability. These results provide the best long-term evidence that fish feeding habits vary over decades with its body size and prey community due to anthropogenic disturbances.