Reagent storage and delivery on integrated microfluidic chips for point-of-care diagnostics.

Microfluidic-based point-of-care diagnostics offer several unique advantages over existing bioanalytical solutions, such as automation, miniaturisation, and integration of sensors to rapidly detect on-site specific biomarkers. It is important to highlight that a microfluidic POC system needs to perform a number of steps, including sample preparation, nucleic acid extraction, amplification, and detection. Each of these stages involves mixing and elution to go from sample to result. To address these complex sample preparation procedures, a vast number of different approaches have been developed to solve the problem of reagent storage and delivery. However, to date, no universal method has been proposed that can be applied as a working solution for all cases. Herein, both current self-contained (stored within the chip) and off-chip (stored in a separate device and brought together at the point of use) are reviewed, and their merits and limitations are discussed. This review focuses on reagent storage devices that could be integrated with microfluidic devices, discussing further issues or merits of these storage solutions in two different sections: direct on-chip storage and external storage with their application devices. Furthermore, the different microvalves and micropumps are considered to provide guidelines for designing appropriate integrated microfluidic point-of-care devices.

A PEDOT: PSS/GO fiber microelectrode fabricated by microfluidic spinning for dopamine detection in human serum and PC12 cells.

Rapid and accurate in situ determination of dopamine is of great significance in the study of neurological diseases. In this work, poly (3,4-ethylenedioxythiophene): poly (styrenesulfonic acid) (PEDOT: PSS)/graphene oxide (GO) fibers were fabricated by an effective method based on microfluidic wet spinning technology. The composite microfibers with stratified and dense arrangement were continuously prepared by injecting PEDOT: PSS and GO dispersion solutions into a microfluidic chip. PEDOT: PSS/GO fiber microelectrodes with high electrochemical activity and enhanced electrochemical oxidation activity of dopamine were constructed by controlling the structure composition of the microfibers with varying flow rate. The fabricated fiber microelectrode had a low detection limit (4.56 nM) and wide detection range (0.01-8.0 µM) for dopamine detection with excellent stability, repeatability, and reproducibility. In addition, the PEDOT: PSS/GO fiber microelectrode prepared was successfully used for the detection of dopamine in human serum and PC12 cells. The strategy for the fabrication of multi-component fiber microelectrodes is a new and effective approach for monitoring the intercellular neurotransmitter dopamine and has high potential as an implantable neural microelectrode.

Poloxamer-188 as a wetting agent for microfluidic resistive pulse sensing measurements of extracellular vesicles.

INTRODUCTION: Microfluidic resistive pulse sensing (MRPS) can determine the concentration and size distribution of extracellular vesicles (EVs) by measuring the electrical resistance of single EVs passing through a pore. To ensure that the sample flows through the pore, the sample needs to contain a wetting agent, such as bovine serum albumin (BSA). BSA leaves EVs intact but occasionally results in unstable MRPS measurements. Here, we aim to find a new wetting agent by evaluating Poloxamer-188 and Tween-20. METHODS: An EV test sample was prepared using an outdated erythrocyte blood bank concentrate. The EV test sample was diluted in Dulbecco's phosphate-buffered saline (DPBS) or DPBS containing 0.10% BSA (w/v), 0.050% Poloxamer-188 (v/v) or 1.00% Tween-20 (v/v). The effect of the wetting agents on the concentration and size distribution of EVs was determined by flow cytometry. To evaluate the precision of sample volume determination with MRPS, the interquartile range (IQR) of the particles transit time through the pore was examined. To validate that DPBS containing Poloxamer-188 yields reliable MRPS measurements, the repeatability of MRPS in measuring blood plasma samples was examined. RESULTS: Flow cytometry results show that the size distribution of EVs in Tween 20, in contrast to Poloxamer-188, differs from the control measurements (DPBS and DPBS containing BSA). MRPS results show that Poloxamer-188 improves the precision of sample volume determination compared to BSA and Tween-20, because the IQR of the transit time of EVs in the test sample is 11 µs, which is lower than 56 µs for BSA and 16 µs for Tween-20. Furthermore, the IQR of the transit time of particles in blood samples with Poloxamer-188 are 14, 16, and 14 µs, which confirms the reliability of MRPS measurements. CONCLUSION: The solution of 0.050% Poloxamer-188 in DPBS does not lyse EVs and results in repeatable and unimpeded MRPS measurements.

Synthesis and active manipulation of magnetic liquid beads.

We report the fabrication and characterisation of magnetic liquid beads with a solid magnetic shell and liquid core using microfluidic techniques. The liquid beads consist of a fluorinated oil core and a polymer shell with magnetite particles. The beads are generated in a flow-focusing polydimethylsiloxane (PDMS) device and cured by photo polymerisation. We investigated the response of the liquid beads to an external magnetic field by characterising their motion towards a permanent magnet. Magnetic sorting of liquid beads in a channel was achieved with 90% efficiency. The results show that the liquid beads can be controlled magnetically and have potential applications in digital microfluidics including nucleic acid amplification, drug delivery, cell culture, sensing, and tissue engineering. The present paper also discusses the magnetophoretic behaviour of the liquid bead by varying its mass and magnetite concentration in the shell. We also demonstrated the two-dimensional self-assembly of magnetic liquid beads for potential use in digital polymerase chain reaction and digital loop mediated isothermal amplification.

Micropump integrated white blood cell separation platform for detection of chronic granulomatous disease.

White blood cells (WBCs) are robust defenders during antigenic challenges and prime immune cell functioning indicators. High-purity WBC separation is vital for various clinical assays and disease diagnosis. Red blood cells (RBCs) are a major hindrance in WBC separation, constituting 1000 times the WBC population. The study showcases a low-cost micropump integrated microfluidic platform to provide highly purified WBCs for point-of-care testing. An integrated user-friendly microfluidic platform was designed to separate WBCs from finger-prick blood (⁓5 µL), employing an inertial focusing technique. We achieved an efficient WBC separation with 86% WBC purity and 99.99% RBC removal rate in less than 1 min. In addition, the microdevice allows lab-on-chip colorimetric evaluation of chronic granulomatous disease (CGD), a rare genetic disorder affecting globally. The assay duration, straight from separation to disease detection, requires only 20 min. Hence, the proposed microfluidic platform can further be implemented to streamline various clinical procedures involving WBCs in healthcare industries.

Integrating microfluidics and synthetic biology: advancements and diverse applications across organisms.

Synthetic biology is the design and modification of biological systems for specific functions, integrating several disciplines like engineering, genetics, and computer science. The field of synthetic biology is to understand biological processes within host organisms through the manipulation and regulation of their genetic pathways and the addition of biocontrol circuits to enhance their production capabilities. This pursuit serves to address global challenges spanning diverse domains that are difficult to tackle through conventional routes of production. Despite its impact, achieving precise, dynamic, and high-throughput manipulation of biological processes is still challenging. Microfluidics offers a solution to those challenges, enabling controlled fluid handling at the microscale, offering lower reagent consumption, faster analysis of biochemical reactions, automation, and high throughput screening. In this review, we diverge from conventional focus on automating the synthetic biology design-build-test-learn cycle, and instead, focus on microfluidic platforms and their role in advancing synthetic biology through its integration with host organisms - bacterial cells, yeast, fungi, animal cells - and cell-free systems. The review illustrates how microfluidic devices have been instrumental in understanding biological systems by showcasing microfluidics as an essential tool to create synthetic genetic circuits, pathways, and organisms within controlled environments. In conclusion, we show how microfluidics expedite synthetic biology applications across diverse domains including but not limited to personalized medicine, bioenergy, and agriculture.

Microfluidic Immunosensor Platform for Sensitive Detection of Human Epidermal Growth Factor Receptor-2 Based on Enhanced Cathode Electrochemiluminescence of Bimetallic Nanoclusters.

In this work, a microfluidic immunosensor chip was developed by incorporating microfluidic technology with electrochemiluminescence (ECL) for sensitive detection of human epidermal growth factor receptor-2 (HER2). The immunosensor chip can achieve robust reproducibility in mass production by integrating multiple detection units in a series. Notably, nanoscale materials can be better adapted to microfluidic systems, greatly enhancing the accuracy of the immunosensor chip. Ag@Au NCs closed by glutathione (GSH) were introduced in the ECL microfluidic immunosensor system with excellent and stable ECL performance. The synthesized CeO2-Au was applied as a coreaction promoter in the ECL signal amplification system, which made the result of HER2 detection more reliable. In addition, the designed microfluidic immunosensor chip integrated the biosensing system into a microchip, realizing rapid and accurate detection of HER2 by its high throughput and low usage. The developed short peptide ligand NARKFKG (NRK) achieved an effective connection between the antibody and nanocarrier for improving the detection efficiency of the sensor. The immunosensor chip had better storage stability and sensitivity than traditional detection methods, with a wide detection range from 10 fg·mL-1 to 100 ng·mL-1 and a low detection limit (LOD) of 3.29 fg·mL-1. In general, a microfluidic immunosensor platform was successfully constructed, providing a new idea for breast cancer (BC) clinical detection.

Antigen density and applied force control enrichment of nanobody-expressing yeast cells in microfluidics.

In vitro display technologies such as yeast display have been instrumental in developing the selection of new antibodies, antibody fragments or nanobodies that bind to a specific target, with affinity towards the target being the main factor that influences selection outcome. However, the roles of mechanical forces are being increasingly recognized as a crucial factor in the regulation and activation of effector cell function. It would thus be of interest to isolate binders behaving optimally under the influence of mechanical forces. We developed a microfluidic assay allowing the selection of yeast displaying nanobodies through antigen-specific immobilization on a surface under controlled hydrodynamic flow. This approach enabled enrichment of model yeast mixtures using tunable antigen density and applied force. This new force-based selection method opens the possibility of selecting binders by relying on both their affinity and force resistance, with implications for the design of more efficient immunotherapeutics.

On the compatibility of single-cell microcarriers (nanovials) with microfluidic impedance cytometry.

We investigate for the first time the compatibility of nanovials with microfluidic impedance cytometry (MIC). Nanovials are suspendable crescent-shaped single-cell microcarriers that enable specific cell adhesion, the creation of compartments for undisturbed cell growth and secretion, as well as protection against wall shear stress. MIC is a label-free single-cell technique that characterizes flowing cells based on their electrical fingerprints and it is especially targeted to cells that are naturally in suspension. Combining nanovial technology with MIC is intriguing as it would represent a robust framework for the electrical analysis of single adherent cells at high throughput. Here, as a proof-of-concept, we report the MIC analysis of mesenchymal stromal cells loaded in nanovials. The electrical analysis is supported by numerical simulations and validated by means of optical analysis. We demonstrate that the electrical diameter can discriminate among free cells, empty nanovials, cell-loaded nanovials, and clusters, thus grounding the foundation for the use of nanovials in MIC. Furthermore, we investigate the potentiality of MIC to assess the electrical phenotype of cells loaded in nanovials and we draw directions for future studies.

OMEF biochip for evaluating red blood cell deformability using dielectrophoresis as a diagnostic tool for type 2 diabetes mellitus.

Type 2 diabetes mellitus (T2DM) is a prevalent and debilitating disease with numerous health risks, including cardiovascular diseases, kidney dysfunction, and nerve damage. One important aspect of T2DM is its association with the abnormal morphology of red blood cells (RBCs), which leads to increased blood viscosity and impaired blood flow. Therefore, evaluating the mechanical properties of RBCs is crucial for understanding the role of T2DM in cellular deformability. This provides valuable insights into disease progression and potential diagnostic applications. In this study, we developed an open micro-electro-fluidic (OMEF) biochip technology based on dielectrophoresis (DEP) to assess the deformability of RBCs in T2DM. The biochip facilitates high-throughput single-cell RBC stretching experiments, enabling quantitative measurements of the cell size, strain, stretch factor, and post-stretching relaxation time. Our results confirm the significant impact of T2DM on the deformability of RBCs. Compared to their healthy counterparts, diabetic RBCs exhibit ∼27% increased size and ∼29% reduced stretch factor, suggesting potential biomarkers for monitoring T2DM. The observed dynamic behaviors emphasize the contrast between the mechanical characteristics, where healthy RBCs demonstrate notable elasticity and diabetic RBCs exhibit plastic behavior. These differences highlight the significance of mechanical characteristics in understanding the implications for RBCs in T2DM. With its ∼90% sensitivity and rapid readout (ultimately within a few minutes), the OMEF biochip holds potential as an effective point-of-care diagnostic tool for evaluating the deformability of RBCs in individuals with T2DM and tracking disease progression.

Microfluidic device for the high-throughput and selective encapsulation of single target cells.

Droplet-based microfluidic technologies for encapsulating single cells have rapidly evolved into powerful tools for single-cell analysis. In conventional passive single-cell encapsulation techniques, because cells arrive randomly at the droplet generation section, to encapsulate only a single cell with high precision, the average number of cells per droplet has to be decreased by reducing the average frequency at which cells arrive relative to the droplet generation rate. Therefore, the encapsulation efficiency for a given droplet generation rate is very low. Additionally, cell sorting operations are required prior to the encapsulation of target cells for specific cell type analysis. To address these challenges, we developed a cell encapsulation technology with a cell sorting function using a microfluidic chip. The microfluidic chip is equipped with an optical detection section to detect the optical information of cells and a sorting section to encapsulate cells into droplets by controlling a piezo element, enabling active encapsulation of only the single target cells. For a particle population including both targeted and non-targeted particles arriving at an average frequency of up to 6000 particles per s, with an average number of particles per droplet of 0.45, our device maintained a high purity above 97.9% for the single-target-particle droplets and achieved an outstanding throughput, encapsulating up to 2900 single target particles per s. The proposed encapsulation technology surpasses the encapsulation efficiency of conventional techniques, provides high efficiency and flexibility for single-cell research, and shows excellent potential for various applications in single-cell analysis.

Millifluidic magnetophoresis-based chip for age-specific fractionation: evaluating the impact of age on metabolomics and gene expression in yeast.

A novel millifluidic process introduces age-based fractionation of S. pastorianus var. carlsbergensis yeast culture through magnetophoresis. Saccharomyces yeast is a model organism for aging research used in various industries. Traditional age-based cell separation methods were labor-intensive, but techniques like magnetic labeling have eased the process by being non-invasive and scalable. Our approach introduces an age-specific fractionation using a 3D-printed millfluidic chip in a two-step process, ensuring efficient cell deflection in the magnetic field and counteracting magnetic induced convection. Among various channel designs, the pinch-shaped channel proved most effective for age differentiation based on magnetically labeled bud scar numbers. Metabolomic analyses revealed changes in certain amino acids and increased NAD+ levels, suggesting metabolic shifts in aging cells. Gene expression studies further underlined these age-related metabolic changes. This innovative platform offers a high-throughput, non-invasive method for age-specific yeast cell fractionation, with potential applications in industries ranging from food and beverages to pharmaceuticals.

Toward microfluidic continuous-flow and intelligent downstream processing of biopharmaceuticals.

Biopharmaceuticals have emerged as powerful therapeutic agents, revolutionizing the treatment landscape for various diseases, including cancer, infectious diseases, autoimmune and genetic disorders. These biotherapeutics pave the way for precision medicine with their unique and targeted capabilities. The production of high-quality biologics entails intricate manufacturing processes, including cell culture, fermentation, purification, and formulation, necessitating specialized facilities and expertise. These complex processes are subject to rigorous regulatory oversight to evaluate the safety, efficacy, and quality of biotherapeutics prior to clinical approval. Consequently, these drugs undergo extensive purification unit operations to achieve high purity by effectively removing impurities and contaminants. The field of personalized precision medicine necessitates the development of novel and highly efficient technologies. Microfluidic technology addresses unmet needs by enabling precise and compact separation, allowing rapid, integrated and continuous purification modules. Moreover, the integration of intelligent biomanufacturing systems with miniaturized devices presents an opportunity to significantly enhance the robustness of complex downstream processing of biopharmaceuticals, with the benefits of automation and advanced control. This allows seamless data exchange, real-time monitoring, and synchronization of purification steps, leading to improved process efficiency, data management, and decision-making. Integrating autonomous systems into biopharmaceutical purification ensures adherence to regulatory standards, such as good manufacturing practice (GMP), positioning the industry to effectively address emerging market demands for personalized precision nano-medicines. This perspective review will emphasize on the significance, challenges, and prospects associated with the adoption of continuous, integrated, and intelligent methodologies in small-scale downstream processing for various types of biologics. By utilizing microfluidic technology and intelligent systems, purification processes can be enhanced for increased efficiency, cost-effectiveness, and regulatory compliance, shaping the future of biopharmaceutical production and enabling the development of personalized and targeted therapies.

Isolation of Extracellular Vesicles from Cell Culture and Blood Through Nano-Targeted DLD Microfluidic Device.

Extracellular vesicles (EVs) are secreted by cells and found in biological fluids such as blood, with concentration correlated with oncogenic signals, making them attractive biomarkers for liquid biopsy. The current gold-standard method for EVs isolation requires an ultracentrifugation (UC) step among others. The cost and complexity of this technique are forbiddingly high for many researchers, as well as for routine use in biological laboratories and hospitals. This chapter reports on a simple microfluidic method for EVs isolation, based on a microfluidic size sorting technique named Deterministic Lateral Displacement (DLD). With the design of micrometric DLD array, we demonstrated the potential of our DLD devices for the isolation of nano-biological objects such as EVs, with main population size distribution consistent with UC technique.

Microchip for Immunomagnetic Sorting of Circulating Tumor Cells (CTCs).

Circulating tumor cells (CTCs) isolated directly from whole blood opens new perspectives for cancer monitoring and the development of personalized treatments. However, due to their rarity among the multitude of blood cells, it remains a challenge to recover them alive with high level of purity, i.e., with few remaining white blood cells, and in a time frame compatible with the clinical context. Microfluidic chips have emerged as promising tools to address these challenges. We propose a two-step workflow including a pre-enrichment step, performed by a size-based pre-enrichment system, and a purification step, performed by an immunomagnetic chip. Here, we describe the protocol for the fabrication of the immunomagnetic microchip, the preparation of the sample, and the procedure for injection into the microchip allowing the sorting of the CTCs.

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.

On-Chip Magnetic Extraction of Circulating Cell-Free DNA from Biological Samples.

In recent years, the analysis of circulating cell-free DNA (cfDNA) containing tumor-derived DNA has emerged as a noninvasive means for cancer monitoring and personalized medicine. However, the isolation of cfDNA from peripheral blood has remained a challenge due to the low abundance and high fragmentation of these molecules. Here, we present a dynamic Magnetic ExTRactiOn (METRO) protocol using microfluidic fluidized bed technology to isolate circulating cfDNA from raw biological materials such as undiluted serum. This protocol maximizes the surface area for DNA binding within the chip in order to capture short DNA fragments. It uses only a few µL of sample and reagents. The protocol can be automated, and it is fully compatible with sensitive DNA amplification methods such as droplet-based digital PCR (ddPCR).

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.

Enhancing the Efficiency of Conventional Surface Immunoassays Within Standard Labware Using Microscale Flows.

In this chapter, we present the design and fabrication of a device and implementation of a protocol to realize increased efficiency of immunoassays within microtiter plates. The device, WellProbe, is a 3D-structured probe that can be used to deliver precise flows at the bottom of standard well plates to establish concentric areas of shear stress intensities using hydrodynamically confined flows. The protocols involve both operation and data analysis.

A Thermoplastic Microsystem to Perform Antibiotic Susceptibility Testing by Monitoring Oxygen Consumption.

Antibiotic susceptibility testing (AST) is a routine procedure in diagnostic laboratories to determine pathogen resistance profiles toward antibiotics. The need for fast and accurate resistance results is rapidly increasing with a global rise in pathogen antibiotic resistance over the past years. Microfluidic technologies can enable AST with lower volumes, lower cell numbers, and a reduction in the sample-to-result time compared to state-of-the-art systems. We present a protocol to perform AST on a miniaturized nanoliter chamber array platform. The chambers are filled with antibiotic compounds and oxygen-sensing nanoprobes that serve as a viability indicator. The growth of bacterial cells in the presence of different concentrations of antibiotics is monitored; living cells consume oxygen, which can be observed as an increase of a luminesce signal within the growth chambers. Here, we demonstrate the technique using a quality control Escherichia coli strain, ATCC 35218. The AST requires 20 µL of a diluted bacterial suspension (OD600 = 0.02) and provides resistance profiles about 2-3 h after the inoculation. The microfluidic method can be adapted to other aerobic pathogens and is of particular interest for slow-growing strains.