Highly integrated automatic injection terahertz microfluidic biosensor based on metasurface and LT-GaAs photoconductive antenna.

In this paper, a highly integrated terahertz (THz) biosensor is proposed and implemented, which pioneered the preparation of low-temperature gallium arsenide (LT-GaAs) thin film photoconductive antenna (PCA) on the sensor for direct generation and detection of THz waves, simplifying complex terahertz time-domain spectroscopy (THz-TDS) systems. A latch type metasurface is deposited in the detection region to produce a resonance absorption peak at 0.6 THz that is independent of polarisation. Microfluidics is utilised and automatic injection is incorporated to mitigate the experimental effects of hydrogen bond absorption of THz waves in aqueous-based environment. Additionally, cell damage is minimised by regulating the cell flow rate. The biosensor was utilised to detect the concentration of three distinct sizes of bacteria with successful results. The assay was executed as a proof of concept to detect two distinct types of breast cancer cells. Based on the experimental findings, it has been observed that the amplitude and blueshift of the resonance absorption peaks have the ability to identify and differentiate various cancer cell types. The findings of this study introduce a novel approach for developing microfluidic THz metasurface biosensors that possess exceptional levels of integration, sensitivity, and rapid label-free detection capabilities.

Enhanced Sweat Biosensing with Thread-Embedded Microfluidic Devices.

BACKGROUND This study explored the integration of conductive threads into a microfluidic compact disc (CD), developed using the xurographic method, for a potential sweat biosensing platform. MATERIAL AND METHODS The microfluidic CD platform, fabricated using the xurographic method with PVC films, included venting channels and conductive threads linked to copper electrodes. With distinct microfluidic sets for load and metering, flow control, and measurement, the CD's operation involved spinning for sequential liquid movement. Impedance analysis using HIOKI IM3590 was conducted for saline and artificial sweat solutions on 4 identical CDs, ensuring reliable conductivity and measurements over a 1 kHz to 200 kHz frequency range. RESULTS Significant differences in |Z| values were observed between saline and artificial sweat treatments. 27.5 µL of saline differed significantly from 27.5 µL of artificial sweat, 72.5 µL of saline from 72.5 µL of artificial sweat, and 192.5 µL of saline from 192.5 µL of sweat. Significant disparities in |Z| values were observed between dry fibers and Groups 2, 3, and 4 (varying saline amounts). No significant differences emerged between dry fibers and Groups 6, 7, and 8 (distinct artificial sweat amounts). These findings underscore variations in fiber characteristics between equivalent exposures, emphasizing the nuanced response of the microfluidic CD platform to different liquid compositions. CONCLUSIONS This study shows the potential of integrating conductive threads in a microfluidic CD platform for sweat sensing. Challenges in volume control and thread coating degradation must be addressed for transformative biosensing devices in personalized healthcare.

Enhanced microfluidic multi-target separation by positive and negative magnetophoresis.

We introduce magnetophoresis-based microfluidics for sorting biological targets using positive Magnetophoresis (pM) for magnetically labeled particles and negative Magnetophoresis (nM) for label-free particles. A single, externally magnetized ferromagnetic wire induces repulsive forces and is positioned across the focused sample flow near the main channel's closed end. We analyze magnetic attributes and separation performance under two transverse dual-mode magnetic configurations, examining magnetic fields, hydrodynamics, and forces on microparticles of varying sizes and properties. In pM, the dual-magnet arrangement (DMA) for sorting three distinct particles shows higher magnetic gradient generation and throughput than the single-magnet arrangement (SMA). In nM, the numerical results for SMA sorting of red blood cells (RBCs), white blood cells (WBCs), and prostate cancer cells (PC3-9) demonstrate superior magnetic properties and throughput compared to DMA. Magnetized wire linear movement is a key design parameter, allowing device customization. An automated device for handling more targets can be created by manipulating magnetophoretic repulsion forces. The transverse wire and magnet arrangement accommodate increased channel depth without sacrificing efficiency, yielding higher throughput than other devices. Experimental validation using soft lithography and 3D printing confirms successful sorting and separation, aligning well with numerical results. This demonstrates the successful sorting and separating of injected particles within a hydrodynamically focused sample in all systems. Both numerical and experimental findings indicate a separation accuracy of 100% across various Reynolds numbers. The primary channel dimensions measure 100 µm in height and 200 µm in width. N52 permanent magnets were employed in both numerical simulations and experiments. For numerical simulations, a remanent flux density of 1.48 T was utilized. In the experimental setup, magnets measuring 0.5 × 0.5 × 0.125 inches and 0.5 × 0.5 × 1 inch were employed. The experimental data confirm the device's capability to achieve 100% separation accuracy at a Reynolds number of 3. However, this study did not explore the potential impact of increased flow rates on separation accuracy.

Recent Trends and Innovations in Bead-Based Biosensors for Cancer Detection.

Demand is strong for sensitive, reliable, and cost-effective diagnostic tools for cancer detection. Accordingly, bead-based biosensors have emerged in recent years as promising diagnostic platforms based on wide-ranging cancer biomarkers owing to the versatility, high sensitivity, and flexibility to perform the multiplexing of beads. This comprehensive review highlights recent trends and innovations in the development of bead-based biosensors for cancer-biomarker detection. We introduce various types of bead-based biosensors such as optical, electrochemical, and magnetic biosensors, along with their respective advantages and limitations. Moreover, the review summarizes the latest advancements, including fabrication techniques, signal-amplification strategies, and integration with microfluidics and nanotechnology. Additionally, the challenges and future perspectives in the field of bead-based biosensors for cancer-biomarker detection are discussed. Understanding these innovations in bead-based biosensors can greatly contribute to improvements in cancer diagnostics, thereby facilitating early detection and personalized treatments.

Microfluidics identify moso bamboo and henon bamboo by leaf protoplast subpopulations with single-cell analysis.

Current techniques identifying herbal medicine species require marker labeling or lack systematical accuracy (expert authentication). There is an emerging interest in developing an accurate and label-free tool for herbal medicine authentication. Here, a high-resolution microfluidic-based method is developed for identifying herbal species by protoplast subpopulations. Moso bamboo and henon bamboo are used as a model to be differentiated based on protoplast. Their biophysical properties factors are characterized to be 7.09 (± 0.39) × 108 V/m2 and 6.54 (± 0.26) × 108 V/m2, respectively. Their biophysical distributions could be distinguished by the Cramér-von Mises criterion with a 94.60% confidence level. The subpopulations of each were compared with conventional flow cytometry indicating the existence of subpopulations and the differences between the two species. The subsets divided by a biophysical factor of 8.05(± 0.51) × 108 V/m2 suggest good consistency with flow cytometry. The work demonstrated the possibility of microfluidics manipulation on protoplast for medication safety use taking advantage of dielectrophoresis. The device is promising in developing a reliable and accurate way of identifying herbal species with difficulties in authentication.

Self-Powered Microfluidics for Point-of-Care Solutions: From Sampling to Detection of Proteins and Nucleic Acids.

Self-powered microfluidics presents a revolutionary approach to address the challenges of healthcare in decentralized and point-of-care settings where limited access to resources and infrastructure prevails or rapid clinical decision-making is critical. These microfluidic systems exploit physical and chemical phenomena, such as capillary forces and surface tension, to manipulate tiny volumes of fluids without the need for external power sources, making them cost-effective and highly portable. Recent technological advancements have demonstrated the ability to preprogram complex multistep liquid operations within the microfluidic circuit of these standalone systems, which enabled the integration of sensitive detection and readout principles. This chapter first addresses how the accessibility to in vitro diagnostics can be improved by shifting toward decentralized approaches like remote microsampling and point-of-care testing. Next, the crucial role of self-powered microfluidic technologies to enable this patient-centric healthcare transition is emphasized using various state-of-the-art examples, with a primary focus on applications related to biofluid collection and the detection of either proteins or nucleic acids. This chapter concludes with a summary of the main findings and our vision of the future perspectives in the field of self-powered microfluidic technologies and their use for in vitro diagnostics applications.

Uniform sized cancer spheroids production using hydrogel-based droplet microfluidics: a review.

Three-dimensional (3D) cell culture models have been extensively utilized in various mechanistic studies as well as for drug development studies as superior in vitro platforms than conventional two-dimensional (2D) cell culture models. This is especially the case in cancer biology, where 3D cancer models, such as spheroids or organoids, have been utilized extensively to understand the mechanisms of cancer development. Recently, many sophisticated 3D models such as organ-on-a-chip models are emerging as advanced in vitro models that can more accurately mimic the in vivo tissue functions. Despite such advancements, spheroids are still considered as a powerful 3D cancer model due to the relatively simple structure and compatibility with existing laboratory instruments, and also can provide orders of magnitude higher throughput than complex in vitro models, an extremely important aspects for drug development. However, creating well-defined spheroids remain challenging, both in terms of throughputs in generation as well as reproducibility in size and shape that can make it challenging for drug testing applications. In the past decades, droplet microfluidics utilizing hydrogels have been highlighted due to their potentials. Importantly, core-shell structured gel droplets can avoid spheroid-to-spheroid adhesion that can cause large variations in assays while also enabling long-term cultivation of spheroids with higher uniformity by protecting the core organoid area from external environment while the outer porous gel layer still allows nutrient exchange. Hence, core-shell gel droplet-based spheroid formation can improve the predictivity and reproducibility of drug screening assays. This review paper will focus on droplet microfluidics-based technologies for cancer spheroid production using various gel materials and structures. In addition, we will discuss emerging technologies that have the potential to advance the production of spheroids, prospects of such technologies, and remaining challenges.

Microfluidic Cartridge for Bead-Based Affinity Assays.

Within the vast field of medical biotechnology, the biopharmaceutical industry is particularly fast-growing and highly competitive, so reducing time and costs associated to process optimization becomes instrumental to ensure speed to market and, consequently, profitability. The manufacturing of biopharmaceutical products, namely, monoclonal antibodies (mAbs), relies mostly on mammalian cell culture processes, which are highly dynamic and, consequently, difficult to optimize. In this context, there is currently an unmet need of analytical methods that can be integrated at-line in a bioreactor, for systematic monitoring and quantification of key metabolites and proteins. Microfluidic-based assays have been extensively and successfully applied in the field of molecular diagnostics; however, this technology remains largely unexplored for Process Analytical Technology (PAT), despite holding great potential for the at-line measurement of different analytes in bioreactor processes, combining low reagent/molecule consumption with assay sensitivity and rapid turnaround times.Here, the fabrication and handling of a microfluidic cartridge for protein quantification using bead-based affinity assays is described. The device allows geometrical multiplexed immunodetection of specific protein analytes directly from bioreactor samples within 2.5 h and minimal hands-on time. As a proof-of-concept, quantification of Chinese hamster ovary (CHO) host cell proteins (HCP) as key impurities, IgG as product of interest, and lactate dehydrogenase (LDH) as cell viability marker was demonstrated with limits of detection (LoD) in the low ng/mL range. Negligible matrix interference and no cross-reactivity between the different immunoassays on chip were found. The results highlight the potential of the miniaturized analytical method for PAT at reduced cost and complexity in comparison with sophisticated instruments that are currently the state-of-the-art in this context.

A Guide to the Quantitation of Protein Secretion Dynamics at the Single-Cell Level.

Protein secretion is a key cellular functionality, particularly in immunology, where cells can display large heterogeneity in this crucial activity in addition to binary secretion behavior. However, few methods enable quantitative secretion rate measurements at the single-cell level, and these methods are mostly based on microfluidics systems. Here, we describe such a microfluidic single-cell method for precisely measuring protein secretion rates in detail, building on the published droplet-based microfluidic platform DropMap. We give an updated, detailed guide toward quantifying protein secretion rates, discussing its setup and limitations. We illustrate the protocol on two key immunological analytes, immunoglobulin G, and interferon-γ.

Droplet Microfluidics with Capillary Electrophoresis for Glycan Biomarker Analysis.

Several glycoproteins are validated biomarkers of various diseases such as cancer, cardiovascular diseases, chronic alcohol abuse, or congenital disorders of glycosylation (CDG). In particular, CDG represent a group of more than 150 inherited diseases with varied symptoms affecting multiple organs. The distribution of glycans from target glycoprotein(s) can be used to extract information to help the diagnosis and possibly differentiate subtypes of CDG. Indeed, depending on the glycans and the proteins to which they are attached, glycans can play a very broad range of roles in both physical and biological properties of glycoproteins. For glycans in general, capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) has become a staple. Analysis of glycans with CE-LIF requires several sample preparation steps, including release of glycans from the target glycoprotein, fluorescent labeling of glycans, and purification of labeled glycans. Here, we describe the protocol for glycan sample treatment in a microfluidic droplet system prior to CE-LIF of labeled glycans. The microfluidic droplet approach offers full automation, sample, and reagent volume reduction and elimination of contamination from external environment.

Evaluating Nanoparticles Penetration by a New Microfluidic Hydrogel-Based Approach.

Reliable predictions for the route and accumulation of nanotherapeutics in vivo are limited by the huge gap between the 2D in vitro assays used for drug screening and the 3D physiological in vivo environment. While developing a standard 3D in vitro model for screening nanotherapeutics remains challenging, multi-cellular tumor spheroids (MCTS) are a promising in vitro model for such screening. Here, we present a straightforward and flexible 3D-model microsystem made out of agarose-based micro-wells, which enables the formation of hundreds of reproducible spheroids in a single pipetting. Immunostaining and fluorescent imaging, including live high-resolution optical microscopy, can be done in situ without manipulating spheroids.

Magnetic Microtweezers for High-Throughput Bioseparation in Sub-Nanoliter Droplets.

Multiomics studies at single-cell level require small volume manipulation, high throughput analysis, and multiplexed detection, characteristics that droplet microfluidics can tackle. However, the initial step of molecule bioseparation remains challenging. Here, we describe a unique magnetic device to trap and extract magnetic particles in sub-nanoliter droplets, for compartmentalisation of detection steps. Relying on electrodeposition of NiFe structures and microfluidic manipulation, the extraction of 1 µm diameter magnetic particles was achieved at high throughput (20 droplets per second) with an efficiency close to 100% in 450 pL droplets. The first demonstration of its adaptability to single-cell analysis is demonstrated with the extraction of mRNA. Using a purified nucleic acid solution, this unique magnetic configuration was able to reach a RNA extraction rate of 72%. This is the first demonstration of a physical separation in droplets at high throughput at single-cell scale.

Selective Targeting of Tumor Cells in a Microfluidic Tumor Model with Multiple Cell Types.

Organ-on-a-chip technology allows researchers to precisely monitor drug efficacy in 3D tissue culture systems that are physiologically more relevant to humans compared to 2D cultures and that allow better control over experimental conditions as compared to animal models. Specifically, the high control over microenvironmental conditions combined with the broad range of direct measurements that can be performed in these systems makes organ-on-a-chip devices a versatile tool to investigate tumor targeting and drug delivery. Here, we describe a detailed protocol for studying the cell-selective targeting of protein drugs to tumor cells on an organ-on-a-chip system using a co-culture consisting of BT-474 cancer cells and C5120 human fibroblasts as an example.

Microfluidic 3D Cytotoxic Assay.

Microfluidic-based cytotoxic assays provide high physiological relevance with the potential to replace conventional animal experiments and two-dimensional (2D) assays. Here, a 3D method utilizing a microfluidic platform for analysis of lymphocyte cytotoxicity is introduced in detail, including platform design, cell culture method, real-time cytotoxic assay setup, and image-based analysis. A 2D experimental method is used for comparison, which effectively demonstrates the advantages of 3D microfluidic platforms in closely recapitulating immune responses within the tumor microenvironment. Moreover, a wide range of experimental possibilities and applications using microfluidic 3D cytotoxic assays is introduced in this chapter, along with their capabilities, limitations, and future outlook.

Molecular fingerprinting of biological nanoparticles with a label-free optofluidic platform.

Label-free detection of multiple analytes in a high-throughput fashion has been one of the long-sought goals in biosensing applications. Yet, for all-optical approaches, interfacing state-of-the-art label-free techniques with microfluidics tools that can process small volumes of sample with high throughput, and with surface chemistry that grants analyte specificity, poses a critical challenge to date. Here, we introduce an optofluidic platform that brings together state-of-the-art digital holography with PDMS microfluidics by using supported lipid bilayers as a surface chemistry building block to integrate both technologies. Specifically, this platform fingerprints heterogeneous biological nanoparticle populations via a multiplexed label-free immunoaffinity assay with single particle sensitivity. First, we characterise the robustness and performance of the platform, and then apply it to profile four distinct ovarian cell-derived extracellular vesicle populations over a panel of surface protein biomarkers, thus developing a unique biomarker fingerprint for each cell line. We foresee that our approach will find many applications where routine and multiplexed characterisation of biological nanoparticles are required.

Microfluidic-Based dsRNA Delivery Nanoplatform for Efficient Spodoptera exigua Control.

Nanotechnology-based RNA interference (RNAi) offers a promising approach to pest control. However, current methods for producing RNAi nanopesticides are mainly implemented in a batch-to-batch manner, lacking consistent quality control. Herein, we present a microfluidic-based nanoplatform for RNA nanopesticide preparation using lipid nanoparticles (LNPs) as nanocarriers, taking advantage of the enhanced mass transfer and continuous processing capabilities of microfluidic technology. The dsRNA@LNPs were rapidly formed within seconds, which showed uniform size distribution, improved leaf wettability, and excellent dispersion properties. The delivery efficiency of dsRNA@LNPs was evaluated by targeting the chitin synthetase B (CHSB) gene ofSpodoptera exigua. The dsRNA@LNPs can effectively resist nuclease-rich midgut fluid degradation. Importantly, dsCHSB@LNPs exhibited increased mortality rates, significant reduction of larvae growth, and enhanced gene suppression efficiency. Therefore, a continuous nanoplatform for RNAi nanopesticide preparation is demonstrated by utilizing microfluidic technology, representing a new route to produce RNAi nanopesticides with enhanced quality control and might accelerate their practical applications.

Clinical evaluation of a novel digital microfluidic based point-of-care test platform for detection of SARS-Cov-2 and influenza A/B.

Respiratory pathogens, such as SARS-CoV-2 and influenza A/B, can cause severe illnesses in susceptible individuals. This research evaluated a novel digital microfluidic point-of-care testing platform designed to detect 23 pathogens, comparing its performance to conventional laboratory-based nucleic acid tests. The platform integrates nucleic acid extraction and amplification processes for rapid detection with only 2 min of hands-on time. Performance assays demonstrated that the platform has high sensitivity (87 %-100 %) and specificity (99 %-100 %) for the detection of the evaluated 3 viruses. Additionally, the platform can be adapted for the detection of other respiratory pathogens, aiding in the early diagnosis of respiratory diseases, identifying the source of an outbreak or epidemic, and curbing the spread of the disease.

Construction of dentin-on-a-chip based on microfluidic technology and tissue engineering.

AIM: Three-dimensional (3D) cell culture systems perform better in resembling tissue or organism structures compared with traditional 2D models. Organs-on-chips (OoCs) are becoming more efficient 3D models. This study aimed to create a novel simplified dentin-on-a-chip using microfluidic chip technology and tissue engineering for screening dental materials. METHODOLOGY: A microfluidic device with three channels was designed for creating 3D dental tissue constructs using stem cells from the apical papilla (SCAP) and gelatin methacrylate (GelMA). The study investigated the effect of varying cell densities and GelMA concentrations on the layer features formed within the microfluidic chip. Cell viability and distribution were evaluated through live/dead staining and nuclei/F-actin staining. The osteo/odontogenic potential was assessed through ALP staining and Alizarin red staining. The impact of GelMA concentrations (5 %, 10 %) on the osteo/odontogenic differentiation trajectory of SCAP was also studied. RESULTS: The 3D tissue constructs maintained high viability and favorable spreading within the microfluidic chip for 3-7 days. A cell seeding density of 2 × 104 cells/µL was found to be the most optimal choice, ensuring favorable cell proliferation and even distribution. GelMA concentrations of 5 % and 10 % proved to be most effective for promoting cell growth and uniform distribution. Within the 5 % GelMA group, SCAP demonstrated higher osteo/odontogenic differentiation than that in the 10 % GelMA group. CONCLUSION: In 3D culture, GelMA concentration was found to regulate the osteo/odontogenic differentiation of SCAP. The study recommends a seeding density of 2 × 104 cells/µL of SCAP within 5 % GelMA for constructing simplified dentin-on-a-chip. CLINICAL SIGNIFICANCE: This study built up the 3D culture protocol, and induced odontogenic differentiation of SCAP, thus forming the simplified dentin-on-a-chip and paving the way to be used as a well-defined biological model for regenerative endodontics. It may serve as a potential testing platform for cell differentiation.

Application of a microfluidic electronic tongue based on impedance spectroscopy for coconut water analysis.

The food industry has grown with the demands for new products and their authentication, which has not been accompanied by the area of analysis and quality control, thus requiring novel process analytical technologies for food processes. An electronic tongue (e-tongue) is a multisensor system that can characterize complex liquids in a fast and simple way. Here, we tested the efficacy of an impedimetric microfluidic e-tongue setup - comprised by four interdigitated electrodes (IDE) on a printed circuit board (PCB), with four pairs of digits each, being one bare sensor and three coated with different ultrathin nanostructured films with different electrical properties - in the analysis of fresh and industrialized coconut water. Principal Component Analysis (PCA) was applied to observe sample differences, and Partial Least Squares Regression (PLSR) was used to predict sample physicochemical parameters. Linear Discriminant Analysis (LDA) and Partial Least Square - Discriminant Analysis (PLS-DA) were compared to classify samples based on data from the e-tongue device. Results indicate the potential application of the microfluidic e-tongue in the identification of coconut water composition and determination of physicochemical attributes, allowing for classification of samples according to soluble solid content (SSC) and total titratable acidity (TTA) with over 90% accuracy. It was also demonstrated that the microfluidic setup has potential application in the food industry for quality assessment of complex liquid samples.

ATPSpin: A Single Microfluidic Platform that Produces Diversified ATPS-Alginate Microfibers.

Microfluidic spinning is emerging as a useful technique in the fabrication of alginate fibers, enabling applications in drug screening, disease modeling, and disease diagnostics. In this paper, by capitalizing on the benefits of aqueous two-phase systems (ATPS) to produce diverse alginate fiber forms, we introduce an ATPS-Spinning platform (ATPSpin). This ATPS-enabled method efficiently circumvents the rapid clogging challenges inherent to traditional fiber production techniques by regulating the interaction between alginate and cross-linking agents like Ba2+ ions. By varying system parameters under the guidance of a regime map, our system produces several fiber forms─solid, hollow, and droplet-filled─consistently and reproducibly from a single device. We demonstrate that the resulting alginate fibers possess distinct features, including biocompatibility. We also encapsulate HEK293 cells in the microfibers as a proof-of-concept that this versatile microfluidic fiber generation platform may have utility in tissue engineering and regenerative medicine applications.