Humanized brain organoids-on-chip integrated with sensors for screening neuronal activity and neurotoxicity.

The complex structure and function of the human central nervous system that develops from the neural tube made in vitro modeling quite challenging until the discovery of brain organoids. Human-induced pluripotent stem cells-derived brain organoids offer recapitulation of the features of early human neurodevelopment in vitro, including the generation, proliferation, and differentiation into mature neurons and micro-macroglial cells, as well as the complex interactions among these diverse cell types of the developing brain. Recent advancements in brain organoids, microfluidic systems, real-time sensing technologies, and their cutting-edge integrated use provide excellent models and tools for emulation of fundamental neurodevelopmental processes, the pathology of neurological disorders, personalized transplantation therapy, and high-throughput neurotoxicity testing by bridging the gap between two-dimensional models and the complex three-dimensional environment in vivo. In this review, we summarize how bioengineering approaches are applied to mitigate the limitations of brain organoids for biomedical and clinical research. We further provide an extensive overview and future perspectives of the humanized brain organoids-on-chip platforms with integrated sensors toward brain organoid intelligence and biocomputing studies. Such approaches might pave the way for increasing approvable clinical applications by solving their current limitations.

Analytical Validation of a Spiral Microfluidic Chip with Hydrofoil-Shaped Pillars for the Enrichment of Circulating Tumor Cells.

The isolation of circulating tumor cells (CTCs) from peripheral blood with high efficiency remains a challenge hindering the utilization of CTC enrichment methods in clinical practice. Here, we propose a microfluidic channel design for the size-based hydrodynamic enrichment of CTCs from blood in an epitope-independent and high-throughput manner. The microfluidic channel comprises a spiral-shaped part followed by a widening part, incorporating successive streamlined pillars, that improves the enrichment efficiency. The design was tested against two benchmark designs, a spiral microfluidic channel and a spiral microfluidic channel followed by a widening channel without the hydrofoils, by processing 5 mL of healthy blood samples spiked with 100 MCF-7 cells. The results proved that the design with hydrofoil-shaped pillars perform significantly better in terms of recovery (recovery rate of 67.9% compared to 23.6% in spiral and 56.7% in spiral with widening section), at a cost of slightly lower white blood cell (WBC) depletion (depletion rate of 94.2% compared to 98.6% in spiral and 94.2% in spiral with widening section), at 1500 µL/min flow rate. For analytical validation, the design was further tested with A549, SKOV-3, and BT-474 cell lines, yielding recovery rates of 62.3 ± 8.4%, 71.0 ± 6.5%, and 82.9 ± 9.9%, respectively. The results are consistent with the size and deformability variation in the respective cell lines, where the increasing size and decreasing deformability affect the recovery rate in a positive manner. The analysis before and after the microfluidic chip process showed that the process does not affect cell viability.

Platform-agnostic electrochemical sensing app and companion potentiostat.

Electrochemical sensing is ubiquitous in a number of fields ranging from biosensing, to environmental monitoring through to food safety and battery or corrosion characterisation. Whereas conventional potentiostats are ideal to develop assays in laboratory settings, they are in general, not well-suited for field work due to their size and power requirements. To address this need, a number of portable battery-operated potentiostats have been proposed over the years. However, most open source solutions do not take full advantage of integrated circuit (IC) potentiostats, a rapidly evolving field. This is partly due to the constraining requirements inherent to the development of dedicated interfaces, such as apps, to address and control a set of common electrochemical sensing parameters. Here we propose the PocketEC, a universal app that has all the functionalities to interface with potentiostat ICs through a user defined property file. The versatility of PocketEC, developed with an assay developer mindset, was demonstrated by interfacing it, via Bluetooth, to the ADuCM355 evaluation board, the open-source DStat potentiostat and the Voyager board, a custom-built, small footprint potentiostat based around the LMP91000 chip. The Voyager board is presented here for the first time. Data obtained using a standard redox probe, Ferrocene Carboxylic Acid (FCA) and a silver ion assay using anodic stripping multi-step amperometry were in good agreement with analogous measurements using a bench top potentiostat. Combined with its Voyager board companion, the PocketEC app can be used directly for a number of wearable or portable electrochemical sensing applications. Importantly, the versatility of the app makes it a candidate of choice for the development of future portable potentiostats. Finally, the app is available to download on the Google Play store and the source codes and design files for the PocketEC app and the Voyager board are shared via Creative Commons license (CC BY-NC 3.0) to promote the development of novel portable or wearable applications based on electrochemical sensing.

Microfluidic-based blood immunoassays.

Microfluidics enables the integration of whole protocols performed in a laboratory, including sample loading, reaction, extraction, and measurement steps on a single system, which offers significant advantages thanks to small-scale operation combined with precise fluid control. These include providing efficient transportation mechanisms and immobilization, reduced sample and reagent volumes, fast analysis and response times, lower power requirements, lower cost and disposability, improved portability and sensitivity, and greater integration and automation capability. Immunoassay is a specific bioanalytical method based on the interaction of antigens and antibodies, which is utilized to detect bacteria, viruses, proteins, and small molecules in several areas such as biopharmaceutical analysis, environmental analysis, food safety, and clinical diagnostics. Because of the advantages of both techniques, the combination of immunoassays and microfluidic technology is considered one of the most potential biosensor systems for blood samples. This review presents the current progress and important developments in microfluidic-based blood immunoassays. After providing several basic information about blood analysis, immunoassays, and microfluidics, the review points out in-depth information about microfluidic platforms, detection techniques, and commercial microfluidic blood immunoassay platforms. In conclusion, some thoughts and future perspectives are provided.

Assessment of silicon, glass, FR4, PDMS and PMMA as a chip material for acoustic particle/cell manipulation in microfluidics.

In the present study, the capabilities of different chip materials for acoustic particle manipulation have been assessed with the same microfluidic device architecture, under the same actuator and flow conditions. Silicon, glass, epoxy with fiberglass filling (FR4), polydimethylsiloxane (PDMS) and polymethyl methacrylate (PMMA) are considered as chip materials. The acoustophoretic chips in this study were manufactured with four different fabrication methods: plasma etching, chemical etching, micromachining and molding. A novel chip material, FR4, has been employed as a microfluidic chip material in acoustophoretic particle manipulation for the first time in literature, which combines the ease of manufacturing of polymer materials with improved acoustic performance. The acoustic particle manipulation performance is evaluated through acoustophoretic focusing experiments with 2µm and 12µm polystyrene microspheres and cultured breast cancer cell line (MDA-MB-231). Unlike the common approach in the literature, the piezoelectric materials were actuated with partitioned cross-polarized electrodes which allowed effective actuation of different family of chip materials. Different from previous studies, this study evaluates the performance of each acoustophoretic device through the perspective of synchronization of electrical, vibrational and acoustical resonances, considers the thermal performance of the chip materials with their effects on cell viability as well as manufacturability and scalability of their fabrication methods. We believe our study is an essential work towards the commercialization of acoustophoretic devices since it brings a critical understanding of the effect of chip material on device performance as well as the cost of achieving that performance.

Escherichia coli Enumeration in a Capillary-Driven Microfluidic Chip with SERS.

Pathogen detection is still a challenging issue for public health, especially in food products. A selective preconcentration step is also necessary if the target pathogen concentration is very low or if the sample volume is limited in the analysis. Plate counting (24-48 h) methods should be replaced by novel biosensor systems as an alternative reliable pathogen detection technique. The usage of a capillary-driven microfluidic chip is an alternative method for pathogen detection, with the combination of surface-enhanced Raman scattering (SERS) measurements. Here, we constructed microchambers with capillary microchannels to provide nanoparticle-pathogen transportation from one chamber to the other. Escherichia coli (E. coli) was selected as a model pathogen and specific antibody-modified magnetic nanoparticles (MNPs) as a capture probe in a complex milk matrix. MNPs that captured E. coli were transferred in a capillary-driven microfluidic chip consisting of four chambers, and 4-aminothiophenol (4-ATP)-labelled gold nanorods (Au NRs) were used as the Raman probe in the capillary-driven microfluidic chip. The MNPs provided immunomagnetic (IMS) separation and preconcentration of analytes from the sample matrix and then, 4-ATP-labelled Au NRs provided an SERS response by forming sandwich immunoassay structures in the last chamber of the capillary-driven microfluidic chip. The developed SERS-based method could detect 101-107 cfu/mL of E. coli with the total analysis time of less than 60 min. Selectivity of the developed method was also tested by using Salmonella enteritidis (S. enteritidis) and Staphylococcus aureus (S. aureus) as analytes, and very weak signals were observed.

Development of a microfluidic platform to maintain viability of micro-dissected tumor slices in culture.

One of the issues limiting the development of personalized medicine is the absence of realistic models that reflect the nature and complexity of tumor tissues. We described a new tissue culture approach that combines a microfluidic chip with the microdissected breast cancer tumor. "Tumor-on-a-chip" devices are suitable for precision medicine since the viability of tissue samples is maintained during the culture period by continuously feeding fresh media and eliminating metabolic wastes from the tissue. However, the mass transport of oxygen, which arguably is the most critical nutrient, is rarely assessed. According to our results, transportation of oxygen provides satisfactory in vivo oxygenation within the system. A high level of dissolved oxygen, around 98%-100% for every 24 h, was measurable in the outlet medium. The microfluidic chip system developed within the scope of this study allows living and testing tumor tissues under laboratory conditions. In this study, tumors were generated in CD-1 mice using MDA-MB-231 and SKBR-3 cell lines. Microdissected tumor tissues were cultured both in the newly developed microfluidic chip system and in conventional 24-well culture plates. Two systems were compared for two different types of tumors. The confocal microscopy analyses, lactate dehydrogenase release, and glucose consumption values showed that the tissues in the microfluidic system remained more viable with respect to the conventional well plate culturing method, up to 96 h. The new culturing technique described here may be superior to conventional culturing techniques for developing new treatment strategies, such as testing chemotherapeutics on tumor samples from individual patients.

A capillary driven microfluidic chip for SERS based hCG detection.

In this study, a capillary driven microfluidic chip-based immunoassay was developed for the determination of Human Chorionic Gonadotropin (hCG) protein, which is prohibited by the World Anti-Doping Agency (WADA). Here, we used antibody modified magnetic metal organic framework nanoparticles (MMOFs) as a capture prob in urine sample. MMOF captured hCG was transferred in a capillary driven microfluidic chip consisting of four chambers, and the interaction of MMOF with gold nanorods labelled with 5,5'-Dithiobis-(2-nitrobenzoic acid) (DTNB) as a Raman label was carried out in the capillary driven microfluidic chip. The movement of MMOF through first chamber to the last chamber was achieved with a simple magnet. In the last chamber of capillary driven microfluidic chip, SERS signals of DTNB molecules from the sandwich complex were recorded using a Raman spectrophotometer. The selectivity of the developed method was demonstrated by applying the same procedure for the detection of Human Luteinizing Hormone (hLH), Human Chorionic Gonadotropin Hormone (hGH) and Immunoglobulin G (IgG) protein. The regression coefficient and limit of detection obtained from the standard addition method were found as 0,9985 and 0,61 IU/L, respectively. Furthermore, the conventional ELISA method confirmed that the results obtained by the presented method were acceptable with the similarity of 97.9% in terms of average recovery value, for the detection of hCG in urine samples. The analysis system developed for target proteins will be an alternative technique such as Western Blot used in routine analysis that is expensive and time consuming.

A microfluidic device enabling drug resistance analysis of leukemia cells via coupled dielectrophoretic detection and impedimetric counting.

We report the development of a lab-on-a-chip system, that facilitates coupled dielectrophoretic detection (DEP-D) and impedimetric counting (IM-C), for investigating drug resistance in K562 and CCRF-CEM leukemia cells without (immuno) labeling. Two IM-C units were placed upstream and downstream of the DEP-D unit for enumeration, respectively, before and after the cells were treated in DEP-D unit, where the difference in cell count gave the total number of trapped cells based on their DEP characteristics. Conductivity of the running buffer was matched the conductivity of cytoplasm of wild type K562 and CCRF-CEM cells. Results showed that DEP responses of drug resistant and wild type K562 cells were statistically discriminative (at p = 0.05 level) at 200 mS/m buffer conductivity and at 8.6 MHz working frequency of DEP-D unit. For CCRF-CEM cells, conductivity and frequency values were 160 mS/m and 6.2 MHz, respectively. Our approach enabled discrimination of resistant cells in a group by setting up a threshold provided by the conductivity of running buffer. Subsequent selection of drug resistant cells can be applied to investigate variations in gene expressions and occurrence of mutations related to drug resistance.

A Novel Microfluidic Method Utilizing a Hydrofoil Structure to Improve Circulating Tumor Cell Enrichment: Design and Analytical Validation.

Being one of the major pillars of liquid biopsy, isolation and characterization of circulating tumor cells (CTCs) during cancer management provides critical information on the evolution of cancer and has great potential to increase the success of therapies. In this article, we define a novel strategy to effectively enrich CTCs from whole blood based on size, utilizing a spiral microfluidic channel embedded with a hydrofoil structure at the downstream of the spiral channel. The hydrofoil increases the distance between the streams of CTCs and peripheral blood cells, which are already distributed about two focal axes by the spiral channel, thereby improving the resolution of the separation. Analytical validation of the system has been carried out using Michigan Cancer Foundation-7 (MCF7) breast cancer cell lines spiked into blood samples from healthy donors, and the performance of the system in terms of white blood cell (WBC) depletion, CTC recovery rate and cell viability has been shown in single or two-step process: by passing the sample once or twice through the microfluidic chip. Single step process yielded high recovery (77.1%), viable (84.7%) CTCs. When the collected cell suspension is re-processed by the same chip, recovery decreases to 65.5%, while the WBC depletion increases to 88.3%, improving the purity. Cell viability of >80% was preserved after two-step process. The novel microfluidic chip is a good candidate for CTC isolation applications requiring high recovery rate and viability, including functional downstream analyses for variety of cancer types.

Multiplex enumeration of Escherichia coli and Salmonella enteritidis in a passive capillary microfluidic chip.

Multiplex detection and quantification of bacteria in water by using portable devices are particularly essential in low and middle-income countries where access to clean drinking water is limited. Addressing this crucial problem, we report a highly sensitive immunoassay sensor system utilizing the fluorescence technique with magnetic nanoparticles (MNPs) to separate target bacteria and two different types of quantum dots (CdTe and Ni doped CdTe QDs) incorporated into a passive microfluidic chip to transport and to form sandwich complexes for the detection of two target bacteria, namely Escherichia coli (E. coli) and Salmonella enteritidis (S. enteritidis) in less than 60 min. The assay is carried out on a capillary driven microfluidic chip that can be operated by merely pipetting the samples and reagents, and fluorescence measurements are done by using a handheld fluorescence spectrophotometer, which renders the system portable. The linear range of the method was found to be 101 to 105 cfu mL-1 for both E. coli and S. enteritidis. The limit of detection (LOD) was calculated to be 5 and 3 cfu mL-1 for E. coli and S. enteritidis, respectively. The selectivity of the method was examined by testing Enterobacter dissolvens (E. dissolvens) and Staphylococcus aureus (S. aureus) samples, and no significant interference was observed. The method was also demonstrated to detect bacteria in tap water and lake water samples spiked with target bacteria.

Low-Cost Microfabrication Tool Box.

Microsystems are key enabling technologies, with applications found in almost every industrial field, including in vitro diagnostic, energy harvesting, automotive, telecommunication, drug screening, etc. Microsystems, such as microsensors and actuators, are typically made up of components below 1000 microns in size that can be manufactured at low unit cost through mass-production. Yet, their development for commercial or educational purposes has typically been limited to specialized laboratories in upper-income countries due to the initial investment costs associated with the microfabrication equipment and processes. However, recent technological advances have enabled the development of low-cost microfabrication tools. In this paper, we describe a range of low-cost approaches and equipment (below £1000), developed or adapted and implemented in our laboratories. We describe processes including photolithography, micromilling, 3D printing, xurography and screen-printing used for the microfabrication of structural and functional materials. The processes that can be used to shape a range of materials with sub-millimetre feature sizes are demonstrated here in the context of lab-on-chips, but they can be adapted for other applications. We anticipate that this paper, which will enable researchers to build a low-cost microfabrication toolbox in a wide range of settings, will spark a new interest in microsystems.

Fast fluorometric enumeration of E. coli using passive chip.

In this report, a passive microfluidic chip design was developed for fast and sensitive fluorometric determination of Escherichia coli (E. coli) based on sandwich immunoassay. Initially, magnetic nanoparticles (MNPs) and chitosan modified mercaptopropionic acid capped cadmium telluride (CdTe) quantum dots (QDs) were functionalized with E.coli specific antibody to form a sandwich immunoassay with the E. coli. The magnetic separation and preconcentration of the E.coli from the sample solution was performed in the vial. Conjugation of QDs to the magnetically captured E. coli and washing were performed using a passive type of microchip. The microfluidic chip consists of four microchambers connected to each other by microchannels which act as capillary valves. Signal measurement was performed at the last chamber by using a hand-held spectrofluorometer equipped with a fiber optic reflection probe. The selectivity of the method was tested with Enterobacter aerogenes (E. aerogenes) and Salmonella enteritidis (S. enteritidis), it was observed that these bacteria have no interference effect on E.coli determination. The calibration curve was found to be linear in the range of 101-105 cfu/mL with a correlation coefficient higher than 0.99. The limit of detection was calculated as 5 cfu/mL. The method was successfully applied to spiked tap and lake water samples. The results suggest that the developed method is applicable for on-site E. coli detection and offers several advantages such as large dynamic range, high sensitivity, high selectivity and short analysis time.

Phaseguides as tunable passive microvalves for liquid routing in complex microfluidic networks.

A microfluidic passive valving platform is introduced that has full control over the stability of each valve. The concept is based on phaseguides, which are small ridges at the bottom of a channel acting as pinning barriers. It is shown that the angle between the phaseguide and the channel sidewall is a measure of the stability of the phaseguide. The relationship between the phaseguide-wall angle and the stability is characterized numerically, analytically and experimentally. Liquid routing is enabled by using multiple phaseguide with different stability values. This is demonstrated by filling complex chamber matrices. As an ultimate demonstration of control, a 400-chamber network is used as a pixel array. It is the first time that differential stability is demonstrated in the realm of passive valving. It ultimately enables microfluidic devices for massive data generation in a low-cost disposable format.

Phaseguide assisted liquid lamination for magnetic particle-based assays.

We have developed a magnetic particle-based assay platform in which functionalised magnetic particles are transferred sequentially through laminated volumes of reagents and washing buffers. Lamination of aqueous liquids is achieved via the use of phaseguide technology; microstructures that control the advancing air-liquid interface of solutions as they enter a microfluidic chamber. This allows manual filling of the device, eliminating the need for external pumping systems, and preparation of the system requires only a few minutes. Here, we apply the platform to two on-chip strategies: (i) a one-step streptavidin-biotin binding assay, and (ii) a two-step C-reactive protein immunoassay. With these, we demonstrate how condensing multiple reaction and washing processes into a single step significantly reduces procedural times, with both assay procedures requiring less than 8 seconds.