Single-cell magnetotaxis in mucus-mimicking polymeric solutions.

Magnetotactic bacteria (MTB) are promising candidates for use as biomicrorobots in biomedical applications due to their motility, self-propulsion, and the ability to direct their navigation with an applied magnetic field. When in the body, the MTB may encounter non-Newtonian fluids such as blood plasma or mucus. However, their motility and the effectiveness of directed navigation in non-Newtonian fluids has yet to be studied on a single-cell level. In this work, we investigate motility of Magnetospirillum magneticum AMB-1 in three concentrations of polyacrylamide (PAM) solution, a mucus-mimicking fluid. The swimming speeds increase from 44.0 ± 13.6 µm/s in 0 mg/mL of PAM to 52.73 ± 15.6 µm/s in 1 mg/mL then decreases to 24.51 ± 11.7 µm/s in 2 mg/mL and 21.23 ± 10.5 µm/s in 3 mg/mL. This trend of a speed increase in low polymer concentrations followed by a decrease in speed as the concentration increases past a threshold concentration is consistent with other studies of motile, flagellated bacteria. Past this threshold concentration of PAM, there is a higher percentage of cells with an overall trajectory angle deviating from the angle of the magnetic field lines. There is also less linearity in the trajectories and an increase in reversals of swimming direction. Altogether, we show that MTB can be directed in polymer concentrations mimicking biological mucus, demonstrating the influence of the medium viscosity on the linearity of their trajectories which alters the effective path that could be predefined in Newtonian fluids when transport is achieved by magnetotaxis.

Concepts and new developments in droplet-based single cell multi-omics.

Single cell sequencing technologies have become a fixture in the molecular profiling of cells due to their ease, flexibility, and commercial availability. In particular, partitioning individual cells inside oil droplets via microfluidic reactions enables transcriptomic or multi-omic measurements for thousands of cells in parallel. Complementing the multitude of biological discoveries from genomics analyses, the past decade has brought new capabilities from assay baselines to enable a deeper understanding of the complex data from single cell multi-omics. Here, we highlight four innovations that have improved the reliability and understanding of droplet microfluidic assays. We emphasize new developments that further orient principles of technology development and guidelines for the design, benchmarking, and implementation of new droplet-based methodologies.

Upscaling Osteoclast Generation by Enhancing Macrophage Aggregation Using Hollow Microgels.

Osteoclasts, the bone resorbing cells of hematopoietic origin formed by macrophage fusion, are essential in bone health and disease. However, in vitro research on osteoclasts remains challenging due to heterogeneous cultures that only contain a few multinucleated osteoclasts. Indeed, a strategy to generate homogeneous populations of multinucleated osteoclasts in a scalable manner has remained elusive. Here, the investigation focuses on whether microencapsulation of human macrophages in microfluidically generated hollow, sacrificial tyramine-conjugated dextran (Dex-TA) microgels could facilitate macrophage precursor aggregation and formation of multinucleated osteoclasts. Therefore, human mononuclear cells are isolated from buffy coats and differentiated toward macrophages. Macrophages are encapsulated in microgels using flow focus microfluidics and outside-in enzymatic oxidative phenolic crosslinking, and differentiated toward osteoclasts. Morphology, viability, and osteoclast fusion of microencapsulated cells are assessed. Furthermore, microgels are degraded to allow cell sorting of released cells based on osteoclastic marker expression. The successful encapsulation and osteoclast formation of human macrophages in Dex-TA microgels are reported for the first time using high-throughput droplet microfluidics. Intriguingly, osteoclast formation within these 3D microenvironments occurs at a significantly higher level compared to the conventional 2D culture system. Furthermore, the feasibility of establishing a pure osteoclast culture from cell transfer and release from degradable microgels is demonstrated.

High-Throughput Absorbance-Activated Droplet Sorting for Engineering Aldehyde Dehydrogenases.

Recent decades have seen a dramatic increase in the commercial use of biocatalysts, transitioning from energy-intensive traditional chemistries to more sustainable methods. Current enzyme engineering techniques, such as directed evolution, require the generation and testing of large mutant libraries to identify optimized variants. Unfortunately, conventional screening methods are unable to screen such large libraries in a robust and timely manner. Droplet-based microfluidic systems have emerged as a powerful high-throughput tool for library screening at kilohertz rates. Unfortunately, almost all reported systems are based on fluorescence detection, restricting their use to a limited number of enzyme types that naturally convert fluorogenic substrates or require the use of surrogate substrates. To expand the range of enzymes amenable to evolution using droplet-based microfluidic systems, we present an absorbance-activated droplet sorter that allows of droplet sorting at kilohertz rates without the need for optical monitoring of the microfluidic system. To demonstrate the utility of the sorter, we rapidly screen a 105-member aldehyde dehydrogenase library towards D-glyceraldehyde using a NADH mediated coupled assay that generates WST-1 formazan as the colorimetric product. We successfully identify a variant with a 51% improvement in catalytic efficiency and a significant increase in overall activity across a broad substrate spectrum.

Selective Segregation of Thermo-Responsive Microgels via Microfluidic Technology.

Separation of equally sized particles distinguished solely by material properties remains still a very challenging task. Here a simple separation of differently charged, thermo-responsive polymeric particles (for example microgels) but equal in size, via the combination of pressure-driven microfluidic flow and precise temperature control is proposed. The separation principle relies on forcing thermo-responsive microgels to undergo the volume phase transition during heating and therefore changing its size and correspondingly the change in drift along a pressure driven shear flow. Different thermo-responsive particle types such as different grades of ionizable groups inside the polymer matrix have different temperature regions of volume phase transition temperature (VPTT). This enables selective control of collapsed versus swollen microgels, and accordingly, this physical principle provides a simple method for fractioning a binary mixture with at least one thermo-responsive particle, which is achieved by elution times in the sense of particle chromatography. The concepts are visualized in experimental studies, with an intend to improve the purification strategy of the broad distribution of charged microgels into fractioning to more narrow distribution microgels distinguished solely by slight differences in net charge.

Activation of Mitochondria in Mesenchymal Stem Cells by Mitochondrial Delivery of Coenzyme Q10.

The efficacy of mesenchymal stem cell (MSC) transplantation has been reported for various diseases. We previously developed a drug delivery system targeting mitochondria (MITO-Porter) by using a microfluidic device to encapsulate Coenzyme Q10 (CoQ10) on a large scale. The current study aimed to confirm if treatment with CoQ10 encapsulated by MITO-Porter enhanced mitochondrial functions in MSCs, with the potential to improve MSC transplantation therapy. We used highly purified human bone marrow-derived MSCs, described as rapidly expanding clones (RECs), and attempted to control and increase the amount of CoQ10 encapsulated in the MITO-Porter using microfluidic device system. We treated these RECs with CoQ10 encapsulated MITO-Porter, and evaluated its cellular uptake, co-localization with mitochondria, changes in mitochondrial respiratory capacity, and cellular toxicity. There was no significant change in mitochondrial respiratory capacity following treatment with the previous CoQ10 encapsulated MITO-Porter; however, mitochondrial respiratory capacity in RECs was significantly increased by treatment with CoQ10-rich MITO-Porter. Utilization of a microfluidic device enabled the amount of CoQ10 encapsulated in MITO-Porter to be controlled, and treatment with CoQ10-rich MITO-Porter successfully activated mitochondrial functions in MSCs. The MITO-Porter system thus provides a promising tool to improve MSC cell transplantation therapy.

Open-source spring-driven syringe pump with 3D-printed components for microfluidic applications.

The operation of microfluidic devices requires precise and constant fluid flow. Microfluidic systems in low-resource settings require a portable, inexpensive, and electricity-free pumping approach due to the rising demand for microfluidics in point-of-care testing (POCT). Open-source alternatives, employing 3D printing and motors, offer affordability. However, using motors require electrical power, which often relies on external sources, hindering the on-site use of open-source pumps. This study introduces a spring-driven, 3D-printed syringe pump, eliminating the need for an external power source. The syringe pump is operated by the flat spiral spring's torque. By manually winding up the mainspring, the syringe pump can be operated without electricity. Various flow rates can be achieved by utilizing different syringe sizes and choosing the right gear combinations. All the parts of the syringe pump can be fabricated by 3D printing, requiring no additional components that require electricity. It operates by winding a mainspring and is user-friendly, allowing flow rate adjustments by assembling gears that modulate syringe plunger pushing velocity. The fabrication cost is $25-30 and can be assembled easily by following the instructions. We expect that the proposed syringe pump will enable the utilization of microfluidic technologies in resource-limited settings, promoting the adoption of microfluidics. Detailed information and results are available in the original research paper (https://doi.org/10.1016/j.snb.2024.135289).

Drop-by-Drop Addition of Reagents to a Double Emulsion.

Developments in droplet microfluidics have facilitated an era of high-throughput, sensitive single-cell, or single-molecule measurements capable of tackling the heterogeneity present in biological systems. Relying on single emulsion (SE) compartments, droplet assays achieve absolute quantification of nucleic acids, massively parallel single-cell profiling, and more. Double emulsions (DEs) have seen recent interest for their potential to build upon SE techniques. DEs are compatible with flow cytometry enabling high-throughput multi-parameter drop screening and eliminate content mixing due to coalescence during lengthy workflows. Despite these strengths, DEs lack important technical functions that exist in SEs such as methods for adding reagents to droplets on demand. Consequently, DEs cannot be used for multistep workflows which has limited their adoption in assay development. Here, strategies to enable reagent addition and other active manipulations on DEs are reported by converting DE inputs to SEs on chip. After conversion, drops are manipulated using existing SE techniques, including reagent addition, before reforming a DE at the outlet. Device designs and operation conditions achieving drop-by-drop reagent addition to DEs are identified and used as part of a multi-step aptamer screening assay performed entirely in DE drops. This work enables the further development of multistep DE droplet assays.

Harnessing the power of clustered regularly interspaced short palindromic repeats (CRISPR) based microfluidics for next-generation molecular diagnostics.

CRISPR-based (Clustered regularly interspaced short palindromic repeats-based) technologies have revolutionized molecular biology and diagnostics, offering unprecedented precision and versatility. However, challenges remain, such as high costs, demanding technical expertise, and limited quantification capabilities. To overcome these limitations, innovative microfluidic platforms are emerging as powerful tools for enhancing CRISPR diagnostics. This review explores the exciting intersection of CRISPR and microfluidics, highlighting their potential to revolutionize healthcare diagnostics. By integrating CRISPR's specificity with microfluidics' miniaturization and automation, researchers are developing more sensitive and portable diagnostic tools for a range of diseases. These microfluidic devices streamline sample processing, improve diagnostic performance, and enable point-of-care applications, allowing for rapid and accurate detection of pathogens, genetic disorders, and other health conditions. The review discusses various CRISPR/Cas systems, including Cas9, Cas12, and Cas13, and their integration with microfluidic platforms. It also examines the advantages and limitations of these systems, highlighting their potential for detecting DNA and RNA biomarkers. The review also explores the key challenges in developing and implementing CRISPR-driven microfluidic diagnostics, such as ensuring robustness, minimizing cross-contamination, and achieving robust quantification. Finally, it highlights potential future directions for this rapidly evolving field, emphasizing the transformative potential of these technologies for personalized medicine and global health.

Nanogels: Recent Advances in Synthesis and Biomedical Applications.

In the context of advanced nanomaterials research, nanogels (NGs) have recently gained broad attention for their versatility and promising biomedical applications. To date, a significant number of NGs have been developed to meet the growing demands in various fields of biomedical research. Summarizing preparation methods, physicochemical and biological properties, and recent applications of NGs may be useful to help explore new directions for their development. This article presents a comprehensive overview of the latest NG synthesis methodologies, highlighting advances in formulation with different types of hydrophilic or amphiphilic polymers. It also underlines recent biomedical applications of NGs in drug delivery and imaging, with a short section dedicated to biosafety considerations of these innovative nanomaterials. In conclusion, this article summarizes recent innovations in NG synthesis and their numerous applications, highlighting their considerable potential in the biomedical field.

Dynamic Live Cell Imaging of Budding Yeast Meiosis.

For over a century, major advances in understanding meiosis have come from the use of microscopy-based methods. Studies using the budding yeast, Saccharomyces cerevisiae, have made important contributions to our understanding of meiosis because of the facility with which budding yeast can be manipulated as a genetic model organism. In contrast, imaging-based approaches with budding yeast have been constrained by the small size of its chromosomes. The advent of advances in fluorescent chromosome tagging techniques has made it possible to use yeast more effectively for imaging-based approaches as well. This protocol describes live cell imaging methods that can be used to monitor chromosome movements throughout meiosis in living yeast cells.

Microfluidic device: A versatile biosensor platform to multiplex aptamer-based detection of malaria biomarkers.

Plasmodium falciparum malaria remains a dominant infectious disease that affects Africa than the rest of the world, considering its associated cases and death rates. It's a febrile illness that produces several reliable biomarkers, for example, P. falciparum lactate dehydrogenase (PfLDH), P. falciparum Plasmodium glutamate dehydrogenase (PfGDH), and P. falciparum histidine-rich proteins (HRP-II) in blood circulatory system that can easily be employed as targets in rapid diagnostic tests (RDTs). In recent times, several DNA aptamers have been developed via SELEX technology to detect some specific malaria biomarkers (PfLDH, PvLDH, HRP-II, PfGDH) in a biosensor mode with good binding affinity properties to overcome the trend of cross-reactivity, limited sensitivity and stability problems that have been observed with immunodiagnostics. In this review, we summarized existing diagnostic methods and relevant biomarkers to suggest promising approaches to develop sensitive and species-specific multiplexed diagnostic devices enabling effective detection of malaria in complex biological matrices and surveillance in the endemic region.

Computational-Aided Approach for the Optimization of Microfluidic-Based Nanoparticles Manufacturing Process.

In the last few years, the microfluidic production of nanoparticles (NPs) is becoming a promising alternative to conventional industrial approaches (e.g., nanoprecipitation, salting out, and emulsification-diffusion) thanks to the production efficiency, low variability, and high controllability of the production parameters. Nevertheless, the development of new formulations and the switching of the production process toward microfluidic platforms requires expensive and time-consuming number of experiments for the tuning of the formulation to obtain NPs with specific morphological and functional characteristics. In this work, we developed a computational fluid dynamic pipeline, validated through an ad hoc experimental strategy, to reproduce the mixing between the solvent and anti-solvent (i.e., acetonitrile and TRIS-HCl, respectively). Moreover, beyond the classical variables able to describe the mixing performances of the microfluidic chip, novel variables were described in order to assess the region of the NPs formation and the changing of the amplitude of the precipitation region according to different hydraulic conditions. The numerical approach proved to be able to capture a progressive reduction of the nanoprecipitation region due to an increment of the flow rate ratio; in parallel, through the experimental production, a progressive increment of the NPs size heterogeneity was observed with the same fluid dynamic conditions. Hence, the preliminary comparison between numerical and experimental evidence proved the effectiveness of the computational strategy to optimize the NPs manufacturing process.

Microfluidics in smart food safety.

The evolution of food safety practices is crucial in addressing the challenges posed by a growing global population and increasingly complex food supply chains. Traditional methods are often labor-intensive, time-consuming, and susceptible to human error. This chapter explores the transformative potential of integrating microfluidics into smart food safety protocols. Microfluidics, involving the manipulation of small fluid volumes within microscale channels, offers a sophisticated platform for developing miniaturized devices capable of complex tasks. Combined with sensors, actuators, big data analytics, artificial intelligence, and the Internet of Things, smart microfluidic systems enable real-time data acquisition, analysis, and decision-making. These systems enhance control, automation, and adaptability, making them ideal for detecting contaminants, pathogens, and chemical residues in food products. The chapter covers the fundamentals of microfluidics, its integration with smart technologies, and its applications in food safety, addressing the challenges and future directions in this field.

Stem cell recruitment polypeptide hydrogel microcarriers with exosome delivery for osteoarthritis treatment.

With the accelerated aging tendency, osteoarthritis (OA) has become an intractable global public health challenge. Stem cells and their derivative exosome (Exo) have shown great potential in OA treatment. Research in this area tends to develop functional microcarriers for stem cell and Exo delivery to improve the therapeutic effect. Herein, we develop a novel system of Exo-encapsulated stem cell-recruitment hydrogel microcarriers from liquid nitrogen-assisted microfluidic electrospray for OA treatment. Benefited from the advanced droplet generation capability of microfluidics and mild cryogelation procedure, the resultant particles show uniform size dispersion and excellent biocompatibility. Moreover, acryloylated stem cell recruitment peptides SKPPGTSS are directly crosslinked within the particles by ultraviolet irradiation, thus simplifying the peptide coupling process and preventing its premature release. The SKPPGTSS-modified particles can recruit endogenous stem cells to promote cartilage repair and the released Exo from the particles further enhances the cartilage repair performance through synergistic effects. These features suggest that the proposed hydrogel microcarrier delivery system is a promising candidate for OA treatment.

Implementation of Rapid Nucleic Acid Amplification Based on the Super Large Thermoelectric Cooler Rapid Temperature Rise and Fall Heating Module.

In the rapid development of molecular biology, nucleic acid amplification detection technology has received more and more attention. The traditional polymerase chain reaction (PCR) instrument has poor refrigeration performance during its transition from a high temperature to a low temperature in the temperature cycle, resulting in a longer PCR amplification cycle. Peltier element equipped with both heating and cooling functions was used, while the robust adaptive fuzzy proportional integral derivative (PID) algorithm was also utilized as the fundamental temperature control mechanism. The heating and cooling functions were switched through the state machine mode, and the PCR temperature control module was designed to achieve rapid temperature change. Cycle temperature test results showed that the fuzzy PID control algorithm was used to accurately control the temperature and achieve rapid temperature rise and fall (average rising speed = 11 °C/s, average falling speed = 8 °C/s) while preventing temperature overcharging, maintaining temperature stability, and achieving ultra-fast PCR amplification processes (45 temperature cycle time < 19 min). The quantitative results show that different amounts of fluorescence signals can be observed according to the different concentrations of added viral particles, and an analytical detection limit (LoD) as low as 10 copies per µL can be achieved with no false positive in the negative control. The results show that the TEC amplification of nucleic acid has a high detection rate, sensitivity, and stability. This study intended to solve the problem where the existing thermal cycle temperature control technology finds it difficult to meet various new development requirements, such as the rapid, efficient, and miniaturization of PCR.

Impedance Characteristics of Microfluidic Channels and Integrated Coplanar Parallel Electrodes as Design Parameters for Whole-Channel Analysis in Organ-on-Chip Micro-Systems.

Microfluidics have revolutionized cell culture by allowing for precise physical and chemical environmental control. Coupled with electrodes, microfluidic cell culture can be activated or have its changes sensed in real-time. We used our previously developed reliable and stable microfluidic device for cell growth and monitoring to design, fabricate, and characterize a whole-channel impedance-based sensor and used it to systematically assess the electrical and electrochemical influences of microfluidic channel boundaries coupled with varying electrode sizes, distances, coatings, and cell coverage. Our investigation includes both theoretical and experimental approaches to investigate how design parameters and insulating boundary conditions change impedance characteristics. We examined the system with various solutions using a frequency range of 0.5 Hz to 1 MHz and a modulation voltage of 50 mV. The results show that impedance is directly proportional to electrode distance and inversely proportional to electrode coating, area, and channel size. We also demonstrate that electrode spacing is a dominant factor contributing to impedance. In the end, we summarize all the relationships found and comment on the appropriateness of using this system to investigate barrier cells in blood vessel models and organ-on-a-chip devices. This fundamental study can help in the careful design of microfluidic culture constructs and models that require channel geometries and impedance-based biosensing.

A Miniature Modular Fluorescence Flow Cytometry System.

Fluorescence flow cytometry is a powerful instrument to distinguish cells or particles labelled with high-specificity fluorophores. However, traditional flow cytometry is complex, bulky, and inconvenient for users to adjust fluorescence channels. In this paper, we present a modular fluorescence flow cytometry (M-FCM) system in which fluorescence channels can be flexibly arranged. Modules for particle focusing and fluorescence detection were developed. After hydrodynamical focusing, the cells were measured in the detection modules, which were integrated with in situ illumination and fluorescence detection. The signal-to-noise ratio of the detection reached to 33.2 dB. The crosstalk among the fluorescence channels was eliminated. The M-FCM system was applied to evaluate cell viability in drug screening, agreeing well with the commercial cytometry. The modular cytometry presents several outstanding features: flexibility in setting fluorescence channels, cost efficiency, compact construction, ease of operation, and the potential to upgrade for multifunctional measurements. The modular cytometry provides a multifunctional platform for various biophysical measurements, e.g., electrical impedance and refractive-index detection. The proposed work paves an innovative avenue for the multivariate analysis of cellular characteristics.

Centrifugal Microfluidic Cell Culture Platform for Physiologically Relevant Virus Infection Studies: A Case Study with HSV-1 Infection of Periodontal Cells.

Static well plates remain the gold standard to study viral infections in vitro, but they cannot accurately mimic dynamic viral infections as they occur in the human body. Therefore, we established a dynamic cell culture platform, based on centrifugal microfluidics, to study viral infections in perfusion. To do so, we used human primary periodontal dental ligament (PDL) cells and herpes simplex virus-1 (HSV-1) as a case study. By microscopy, we confirmed that the PDL cells efficiently attached and grew in the chip. Successful dynamic viral infection of perfused PDL cells was monitored using fluorescent imaging and RT-qPCR-based experiments. Remarkably, viral infection in flow resulted in a gradient of HSV-1-infected cells gradually decreasing from the cell culture chamber entrance towards its end. The perfusion of acyclovir in the chip prevented HSV-1 spreading, demonstrating the usefulness of such a platform for monitoring the effects of antiviral drugs. In addition, the innate antiviral response of PDL cells, measured by interferon gene expression, increased significantly over time in conventional static conditions compared to the perfusion model. These results provide evidence suggesting that dynamic viral infections differ from conventional static infections, which highlights the need for more physiologically relevant in vitro models to study viral infections.

Advances in Microflow Cytometry-Based Molecular Detection Methods for Improved Future MDS Cancer Diagnosis.

Myelodysplastic syndromes (MDS) are a rare form of early-stage blood cancer that typically leads to leukemia and other deadly complications. The typical diagnosis for MDS involves a mixture of blood tests, a bone marrow biopsy, and genetic analysis. Flow cytometry has commonly been used to analyze these types of samples, yet there still seems to be room for advancement in several areas, such as the limit of detection, turnaround time, and cost. This paper explores recent advancements in microflow cytometry technology and how it may be used to supplement conventional methods of diagnosing blood cancers, such as MDS and leukemia, through flow cytometry. Microflow cytometry, a more recent adaptation of the well-researched and conventional flow cytometry techniques, integrated with microfluidics, demonstrates significant potential in addressing many of the shortcomings flow cytometry faces when diagnosing a blood-related disease such as MDS. The benefits that this platform brings, such as portability, processing speed, and operating cost, exemplify the importance of exploring microflow cytometry as a point-of-care (POC) diagnostic device for MDS and other forms of blood cancer.