Development and validation study of compact biophotonic platform for detection of serum biomarkers.

The application of advances in personalized medicine requires the support of in vitro diagnostic techniques aimed at the accurate, fast, sensitive, and precise determination of selected biomarkers. Herein, a novel optical centrifugal microfluidic device is developed for clinical analysis and point-of-care diagnostics. Based on compact disc technology, the integrated biophotonic system enables multiple immunoassays in miniaturized mode. The disposable microfluidic discs are made in cyclic olefin copolymer (COP), containing arrays of immobilized probes. In the developed approach, up to six patient samples can each be tested simultaneously. A portable instrument (<2 kg) controls the assay and the high-sensitive reproducible optical detection in transmission mode. Also, the instrument incorporates specific functionalities for personalized telemedicine. The device (analytical method, disc platform, reader, and software) has been validated to diagnose IgE-mediated drug allergies, such as amoxicillin and penicillin G. The total and specific IgE to ß-lactam antibiotics were determined in human serum from patients (25 µL). The excellent analytical performances (detection limit 0.24 ng/mL, standard deviation 7-20 %) demonstrated that the developed system could have the potential for a broader impact beyond the allergy field, as it applies to other IVD tests.

A human pluripotent stem cell-based somitogenesis model using microfluidics.

Emerging human pluripotent stem cell (hPSC)-based embryo models are useful for studying human embryogenesis. Particularly, there are hPSC-based somitogenesis models using free-floating culture that recapitulate somite formation. Somitogenesis in vivo involves intricately orchestrated biochemical and biomechanical events. However, none of the current somitogenesis models controls biochemical gradients or biomechanical signals in the culture, limiting their applicability to untangle complex biochemical-biomechanical interactions that drive somitogenesis. Herein, we develop a human somitogenesis model by confining hPSC-derived presomitic mesoderm (PSM) tissues in microfabricated trenches. Exogenous microfluidic morphogen gradients imposed on the PSM tissues cause axial patterning and trigger spontaneous rostral-to-caudal somite formation. A mechanical theory is developed to explain the size dependency between somites and the PSM. The microfluidic somitogenesis model is further exploited to reveal regulatory roles of cellular and tissue biomechanics in somite formation. This study presents a useful microengineered, hPSC-based model for understanding the biochemical and biomechanical events that guide somite formation.

Arabidopsis Root Microbiome Microfluidic (ARMM) Device for Imaging Bacterial Colonization and Morphogenesis of Arabidopsis Roots.

Imaging the spatiotemporal dynamics of host-microbiota interactions is of particular interest for augmenting our understanding of these complex systems. This is especially true of plant-microbe interactions happening around, on, and inside plant roots where relatively little is understood about the dynamics of these systems. Over the past decade, a number of microfluidic devices have been developed to grow plants hydroponically in gnotobiotic conditions and image morphogenesis of the root and/or dynamics with fluorescently labeled bacteria from the plant root microbiome. Here we describe the construction and use of our Arabidopsis Root Microbiome Microfluidic (ARMM) device for imaging fluorescent protein expressing bacteria and their colonization of Arabidopsis roots. In contrast to other plant root imaging devices, we designed this device to have a larger chamber for observing Arabidopsis root elongation and plant-microbe interactions with older seedlings (between 1.5 and 4 weeks after germination) and a 200 µm chamber depth to specifically maintain thin Arabidopsis roots within the focal distance of the confocal microscope. Our device incorporates a new approach to growing Arabidopsis seedlings in screw-top tube caps for simplified germination and transfer to the device. We present representative images from the ARMM device including high resolution cross section images of bacterial colonization at the root surface.

Human Activity Recording Based on Skin-Strain-Actuated Microfluidic Pumping in Asymmetrically Designed Micro-Channels.

The capability to record data in passive, image-based wearable sensors can simplify data readouts and eliminate the requirement for the integration of electronic components on the skin. Here, we developed a skin-strain-actuated microfluidic pump (SAMP) that utilizes asymmetric aspect ratio channels for the recording of human activity in the fluidic domain. An analytical model describing the SAMP's operation mechanism as a wearable microfluidic device was established. Fabrication of the SAMP was achieved using soft lithography from polydimethylsiloxane (PDMS). Benchtop experimental results and theoretical predictions were shown to be in good agreement. The SAMP was mounted on human skin and experiments conducted on volunteer subjects demonstrated the SAMP's capability to record human activity for hundreds of cycles in the fluidic domain through the observation of a stable liquid meniscus. Proof-of-concept experiments further revealed that the SAMP could quantify a single wrist activity repetition or distinguish between three different shoulder activities.

Scalable microfluidic method for tunable liposomal production by a design of experiment approach.

Liposomes constitute a widespread drug delivery platform, gaining more and more attention from the pharmaceutical industry and process development scientists. Their large-scale production as medicinal products for human use is all but trivial, especially when parenteral administration is required. In this study an off-the-shelf microfluidic system and a methodological approach are presented for the optimization, validation and scale-up of highly monodisperse liposomes manufacturing. Starting from a Doxil®-like formulation (HSPC, MPEG-DSPE and cholesterol), a rational approach (Design of Experiments, DoE) was applied for the screening of the process parameters affecting the quality attributes of the product (mainly size and polydispersity). Additional DoEs were conducted to determine the effect of critical process parameters (cholesterol concentration, total flow rate TFR and flow rate ratio FRR), thus assessing the formulation and process robustness. A scale-up was then successfully accomplished. The procedure was applied to a Marqibo®-like formulation as well (sphingomyelin and cholesterol) to show the generality of the proposed formulation, process development and scale-up approach. The application of the system and method herein presented enables the large-scale manufacturing of liposomes, in compliance with the internationally recognized regulatory standards for pharmaceutical development (Quality by Design).

Challenges and Opportunities in Developing Tracheal Substitutes for the Recovery of Long-Segment Defects.

Tracheal resection and reconstruction procedures are necessary when stenosis, tracheomalacia, tumors, vascular lesions, or tracheal injury cause a tracheal blockage. Replacement with a tracheal substitute is often recommended when the trauma exceeds 50% of the total length of the trachea in adults and 30% in children. Recently, tissue engineering and other advanced techniques have shown promise in fabricating biocompatible tracheal substitutes with physical, morphological, biomechanical, and biological characteristics similar to native trachea. Different polymers and biometals are explored. Even with limited success with tissue-engineered grafts in clinical settings, complete healing of tracheal defects remains a substantial challenge due to low mechanical strength and durability of the graft materials, inadequate re-epithelialization and vascularization, and restenosis. This review has covered a range of reconstructive and regenerative techniques, design criteria, the use of bioprostheses and synthetic grafts for the recovery of tracheal defects, as well as the traditional and cutting-edge methods of their fabrication, surface modification for increased immuno- or biocompatibility, and associated challenges.

Diffusion-based culture and real-time impedance monitoring of tumor spheroids in hydrogel microwells of a suspended membrane under microfluidic conditions.

Tumor spheroids are widely studied for in vitro modeling of tumor growth and responses to anticancer drugs. However, current methods are mostly limited to static and perfusion-based cultures, which can be improved by more accurately mimicking pathological conditions. Here, we developed a diffusion-based dynamic culture system for tumor spheroids studies using a thin membrane of hydrogel microwells and a microfluidic device. This allows for effective exchange of nutrients and metabolites between the tumors and the culture medium flowing underneath, resulting in uniform tumor spheroids. To monitor the growth and drug response of the spheroids in real-time, we performed spectroscopic analyses of the system's impedance, demonstrating a close correlation between the tumor size and the resistance and capacitance of the system. Our results also indicate an enhanced drug effect on the tumor spheroids in the presence of a low AC electric field, suggesting a weakening mechanism of the spheroids induced by external perturbation.

Biocompatible Janus microparticle synthesis in a microfluidic device.

Janus particles are popular in recent years due to their anisotropic physical and chemical properties. Even though there are several established synthesis methods for Janus particles, microfluidics-based methods are convenient and reliable due to low reagent consumption, monodispersity of the resultant particles and efficient control over reaction conditions. In this work a simple droplet-based microfluidic technique is utilized to synthesize magnetically anisotropic TiO2-Fe2O3 Janus microparticles. Two droplets containing reagents for Janus particle were merged by using an asymmetric device such that the resulting droplet contained the constituents within its two hemispheres distinct from each other. The synthesized Janus particles were observed under the optical microscope and the scanning electron microscope. Moreover, a detailed in vitro characterization of these particles was completed, and it was shown that these particles have a potential use for biomedical applications.

Tongue-on-a-Chip: Parallel Recording of Sweet and Bitter Receptor Responses to Sequential Injections of Pure and Mixed Sweeteners.

A microfluidic tongue-on-a-chip platform has been evaluated relative to the known sensory properties of various sweeteners. Analogous metrics of typical sensory features reported by human panels such as sweet taste thresholds, onset, and lingering, as well as bitter off-flavor and blocking interactions were deduced from the taste receptor activation curves and then compared. To this end, a flow cell containing a receptor cell array bearing the sweet and six bitter taste receptors was transiently exposed to pure and mixed sweetener samples. The sample concentration gradient across time was separately characterized by the injection of fluorescein dye. Subsequently, cellular calcium responses to different doses of advantame, aspartame, saccharine, and sucrose were overlaid with the concentration gradient. Parameters describing the response kinetics compared to the gradient were quantified. Advantame at 15 µM recorded a significantly faster sweetness onset of 5 ± 2 s and a longer lingering time of 39 s relative to sucrose at 100 mM with an onset of 13 ± 2 s and a lingering time of 6 s. Saccharine was shown to activate the bitter receptors TAS2R8, TAS2R31, and TAS2R43, confirming its known off-flavor, whereas addition of cyclamate reduced or blocked this saccharine bitter response. The potential of using this tongue-on-a-chip to bridge the gap with in vitro assays and taste panels is discussed.

Enrichment of different taxa of the enigmatic candidate phyla radiation bacteria using a novel picolitre droplet technique.

The candidate phyla radiation (CPR) represents a distinct monophyletic clade and constitutes a major portion of the tree of life. Extensive efforts have focused on deciphering the functional diversity of its members, primarily using sequencing-based techniques. However, cultivation success remains scarce, presenting a significant challenge, particularly in CPR-dominated groundwater microbiomes characterized by low biomass. Here, we employ an advanced high-throughput droplet microfluidics technique to enrich CPR taxa from groundwater. Utilizing a low-volume filtration approach, we successfully harvested a microbiome resembling the original groundwater microbial community. We assessed CPR enrichment in droplet and aqueous bulk cultivation for 30 days using a novel CPR-specific primer to rapidly track the CPR fraction through the cultivation attempts. The combination of soil extract and microbial-derived necromass provided the most supportive conditions for CPR enrichment. Employing these supplemented conditions, droplet cultivation proved superior to bulk cultivation, resulting in up to a 13-fold CPR enrichment compared to a 1- to 2-fold increase in bulk cultivation. Amplicon sequencing revealed 10 significantly enriched CPR orders. The highest enrichment in CPRs was observed for some unknown members of the Parcubacteria order, Cand. Jorgensenbacteria, and unclassified UBA9983. Furthermore, we identified co-enriched putative host taxa, which may guide more targeted CPR isolation approaches in subsequent investigations.

In the flow of molecular miniaturized fungal diagnosis.

The diagnosis of fungal infections presents several challenges and limitations, stemming from the similarities in symptomatology, diversity of underlying pathogenic species, complexity of fungal biology, and scarcity of rapid, affordable, and point-of-care approaches. In this review, we assess technological advances enabling the conversion of cutting-edge laboratory molecular diagnostic methods to cost-effective microfluidic devices. The most promising strategies toward the design of DNA sequence-based fungal diagnostic systems, capable of capturing and deciphering the highly informative DNA of the pathogen and adapted for resource-limited settings, are discussed, bridging fungal biology, molecular genetics, microfluidics, and biosensors.

Boosting Microfluidic Enzymatic Cascade Reactions with pH-Responsive Polymersomes by Spatio-Chemical Activity Control.

Microfluidic flow reactors permit the implementation of sensitive biocatalysts in polymeric environments (e.g., hydrogel dots), mimicking nature through the use of diverse microstructures within defined confinements. However, establishing complex hybrid structures to mimic biological processes and functions under continuous flow with optimal utilization of all components involved in the reaction process represents a significant scientific challenge. To achieve spatial, chemical, and temporal control for any microfluidic application, compartmentalization is required, as well as the unification of different sensitive compartments in the reaction chamber for the microfluidic flow design. This study presents a self-regulating microfluidic system fabricated by a sequential photostructuring process with an intermediate chemical process step to realize pH-sensitive hybrid structures for the fabrication of a microfluidic double chamber reactor for controlled enzymatic cascade reaction (ECR). The key point is the adaptation and retention of the function of pH-responsive horseradish peroxidase-loaded polymersomes in a microfluidic chip under continuous flow. ECR is successfully triggered and controlled by an interplay between glucose oxidase-converted glucose, the membrane state of pH-responsive polymersomes, and other parameters (e.g., flow rate and fluid composition). This study establishes a promising noninvasive regulatory platform for extended spatio-chemical control of current and future ECR and other cascade reaction systems.

Using Microfluidics to Align Matrix Architecture and Generate Chemokine Gradients Promotes Directional Branching in a Model of Epithelial Morphogenesis.

The mechanical cue of fiber alignment plays a key role in the development of various tissues in the body. The ability to study the effect of these stimuli in vitro has been limited previously. Here, we present a microfluidic device capable of intrinsically generating aligned fibers using the microchannel geometry. The device also features tunable interstitial fluid flow and the ability to form a morphogen gradient. These aspects allow for the modeling of complex tissues and to differentiate cell response to different stimuli. To demonstrate the abilities of our device, we incorporated luminal epithelial cysts into our device and induced growth factor stimulation. We found the mechanical cue of fiber alignment to play a dominant role in cell elongation and the ability to form protrusions was dependent on cadherin-3. Together, this work serves as a springboard for future potential with these devices to answer questions in developmental biology and complex diseases such as cancers.

Parallelized Immunomagnetic Isolation of Basophils Directly from Whole Blood.

Basophils are the rarest circulating white blood cells (WBCs), but they play important roles in allergic disorders and other diseases. To enhance diagnostic capabilities, it would be desirable to isolate and analyze basophils efficiently from small blood samples. In 100 µL of whole blood, there are typically ~103 basophils, outnumbered by ~105 WBCs and ~108 red blood cells (RBCs). Basophils' low abundance has therefore presented a significant challenge in their isolation from whole blood. Conventional in-bulk basophil isolation methods require lengthy processing steps and cannot work with small volumes of blood. Here we report a parallelized integrated basophil isolation device (pi-BID) for the negative immunomagnetic selection of basophils directly from 4 samples of 100 µL of whole blood, in parallel, within 14 minutes including sample preparation time. The pi-BID interfaces directly with standard sample tubes, and uses a single pressure source to drive the flow in parallel microfluidic channels. Compared with conventional in-bulk basophil isolation, the pi-BID is >3× faster, and has higher purity (~93%) and similar recovery (~67%). Compared with other microfluidic devices for the immunomagnetic isolation of WBC sub-types, our pi-BID achieves 10× higher enrichment of target cells from whole blood, with no prior removal of RBCs necessary.

Architecture design and advanced manufacturing of heart-on-a-chip: scaffolds, stimulation and sensors.

Heart-on-a-chip (HoC) has emerged as a highly efficient, cost-effective device for the development of engineered cardiac tissue, facilitating high-throughput testing in drug development and clinical treatment. HoC is primarily used to create a biomimetic microphysiological environment conducive to fostering the maturation of cardiac tissue and to gather information regarding the real-time condition of cardiac tissue. The development of architectural design and advanced manufacturing for these "3S" components, scaffolds, stimulation, and sensors is essential for improving the maturity of cardiac tissue cultivated on-chip, as well as the precision and accuracy of tissue states. In this review, the typical structures and manufacturing technologies of the "3S" components are summarized. The design and manufacturing suggestions for each component are proposed. Furthermore, key challenges and future perspectives of HoC platforms with integrated "3S" components are discussed. Architecture design concepts of scaffolds, stimulation and sensors in chips.

Full-cycle study on developing a novel structured micromixer and evaluating the nanoparticle products as mRNA delivery carriers.

Achieving precise control of nanoparticle size while maintaining consistency and high uniformity is of paramount importance for improving the efficacy of nanoparticle-based therapies and minimizing potential side effects. Although microfluidic technologies are widely used for reliable nanoparticle synthesis, they face challenges in meeting critical homogeneity requirements, mainly due to imperfect mixing efficiency. Furthermore, channel clogging during continuous operation presents a significant obstacle in terms of quality control, as it progressively impedes the mixing behavior necessary for consistent nanoparticle production for therapeutic delivery and complicates the scaling-up process. This study entailed the development of a 3D-printed novel micromixer embedded with hemispherical baffle microstructures, a dual vortex mixer (DVM), which integrates Dean vortices to generate two symmetrical counter-rotating intensified secondary flows. The DVM with a relatively large mixer volume showed rapid mixing characteristics even at a flow rate of several mL min-1 and produced highly uniform lipids, liposomes, and polymer nanoparticles in a size range (50-130 nm) and polydispersity index (PDI) values below 0.15. For the evaluation of products, SARS-CoV-2 Spike mRNA-loaded lipid nanoparticles were examined to verify protein expression in vitro and in vivo using firefly luciferase (FLuc) mRNA. This showed that the performance of the system is comparable to that of a commercial toroidal mixer. Moreover, the vigorous in-situ dispersion of nanoparticles by harnessing the power of vortex physically minimizes the occurrence of aggregation, ensuring consistent production performance without internal clogging of a half-day operation and facilitating quality control of the nanoparticles at desired scales.

Progress and Outlook on Electrochemical Sensing of Lung Cancer Biomarkers.

Electrochemical biosensors have emerged as powerful tools for the ultrasensitive detection of lung cancer biomarkers like carcinoembryonic antigen (CEA), neuron-specific enolase (NSE), and alpha fetoprotein (AFP). This review comprehensively discusses the progress and potential of nanocomposite-based electrochemical biosensors for early lung cancer diagnosis and prognosis. By integrating nanomaterials like graphene, metal nanoparticles, and conducting polymers, these sensors have achieved clinically relevant detection limits in the fg/mL to pg/mL range. We highlight the key role of nanomaterial functionalization in enhancing sensitivity, specificity, and antifouling properties. This review also examines challenges related to reproducibility and clinical translation, emphasizing the need for standardization of fabrication protocols and robust validation studies. With the rapid growth in understanding lung cancer biomarkers and innovations in sensor design, nanocomposite electrochemical biosensors hold immense potential for point-of-care lung cancer screening and personalized therapy guidance. Realizing this goal will require strategic collaboration among material scientists, engineers, and clinicians to address technical and practical hurdles. Overall, this work provides valuable insight for developing next-generation smart diagnostic devices to combat the high mortality of lung cancer.

Detecting mir-155-3p through a Molecular Beacon Bead-Based Assay.

Lung cancer (LC) is recognized as one of the most prevalent and lethal cancers worldwide, underscoring an urgent need for innovative diagnostic and therapeutic approaches. MicroRNAs (miRNAs) have emerged as promising biomarkers for several diseases and their progression, such as LC. However, traditional methods for detecting and quantifying miRNAs, such as PCR, are time-consuming and expensive. Herein, we used a molecular beacon (MB) bead-based assay immobilized in a microfluidic device to detect miR-155-3p, which is frequently overexpressed in LC. The assay relies on the fluorescence enhancement of the MB upon binding to the target miRNA via Watson and Crick complementarity, resulting in a conformational change from a stem-loop to a linear structure, thereby bringing apart the fluorophores at each end. This assay was performed on a microfluidic platform enabling rapid and straightforward target detection. We successfully detected miR-155-3p in a saline solution, obtaining a limit of detection (LOD) of 42 nM. Furthermore, we evaluated the method's performance in more complex biological samples, including A549 cells' total RNA and peripheral blood mononuclear cells (PBMCs) spiked with the target miRNA. We achieved satisfactory recovery rates, especially in A549 cells' total RNA.

Fluid inertia controls mineral precipitation and clogging in pore to network-scale flows.

Mineral precipitation caused by fluid mixing presents complex control and predictability challenges in a variety of natural and engineering processes, including carbon mineralization, geothermal energy, and microfluidics. Precipitation dynamics, particularly under the influence of fluid flow, remain poorly understood. Combining microfluidic experiments and three-dimensional reactive transport simulations, we demonstrate that fluid inertia controls mineral precipitation and clogging at flow intersections, even in laminar flows. We observe distinct precipitation regimes as a function of Reynolds number (Re). At low Reynolds numbers (Re < 10), precipitates form a thin, dense layer along the mixing interface, which shuts precipitation off, while at high Reynolds numbers (Re > 50), strong three-dimensional flows significantly enhance precipitation over the entire intersection, resulting in rapid clogging. When injection rates from two inlets are uneven, flow symmetry-breaking leads to unexpected flow bifurcation phenomena, which result in enhanced concurrent precipitation in both downstream channels. Finally, we extend our findings to rough channel networks and demonstrate that the identified inertial effects on precipitation at the intersection scale are also present and even more dramatic at the network scale. This study sheds light on the fundamental mechanisms underlying mixing-induced mineral precipitation and provides a framework for designing and optimizing processes involving mineral precipitation.