High-throughput microbead assay system with a portable, cost-effective Wi-Fi imaging module, and disposable multi-layered microfluidic cartridges for virus and microparticle detection, and tracking.

In recent years biomedical scientific community has been working towards the development of high-throughput devices that allow a reliable, rapid and parallel detection of several strains of virus or microparticles simultaneously. One of the complexities of this problem lies on the rapid prototyping of new devices and wireless rapid detection of small particles and virus alike. By reducing the complexity of microfluidics microfabrication and using economic materials along with makerspace tools (Kundu et al. 2018) it is possible to provide an affordable solution to both the problems of high-throughput devices and detection technologies. We present the development of a wireless, standalone device and disposable microfluidics chips that rapidly generate parallel readouts for selected, possible virus variants from a nasal or saliva sample, based on motorized and non-motorized microbeads detection, and imaging processing of the motion tracks of these beads in micrometers. Microbeads and SARS-CoV-2 COVID-19 Delta variant were tested as proof-of-concept for testing the microfluidic cartridges and wireless imaging module. The Microbead Assay (MA) system kit consists of a Wi-Fi readout module, a microfluidic chip, and a sample collection/processing sub-system. Here, we focus on the fabrication and characterization of the microfluidic chip to multiplex various micrometer-sized beads for economic, disposable, and simultaneous detection of up to six different viruses, microparticles or variants in a single test, and data collection using a commercially available, Wi-Fi-capable, and camera integrated device (Fig. 1).

Lung-on-a-Chip Models of the Lung Parenchyma.

Since the publication of the first lung-on-a-chip in 2010, research has made tremendous progress in mimicking the cellular environment of healthy and diseased alveoli. As the first lung-on-a-chip products have recently reached the market, innovative solutions to even better mimic the alveolar barrier are paving the way for the next generation lung-on-chips. The original polymeric membranes made of PDMS are being replaced by hydrogel membranes made of proteins from the lung extracellular matrix, whose chemical and physical properties exceed those of the original membranes. Other aspects of the alveolar environment are replicated, such as the size of the alveoli, their three-dimensional structure, and their arrangement. By tuning the properties of this environment, the phenotype of alveolar cells can be tuned, and the functions of the air-blood barrier can be reproduced, allowing complex biological processes to be mimicked. Lung-on-a-chip technologies also provide the possibility of obtaining biological information that was not possible with conventional in vitro systems. Pulmonary edema leaking through a damaged alveolar barrier and barrier stiffening due to excessive accumulation of extracellular matrix proteins can now be reproduced. Provided that the challenges of this young technology are overcome, there is no doubt that many application areas will benefit greatly.

Microfluidic devices for the detection of disease-specific proteins and other macromolecules, disease modelling and drug development: A review.

Microfluidics is a revolutionary technology that has promising applications in the biomedical field.Integrating microfluidic technology with the traditional assays unravels the innumerable possibilities for translational biomedical research. Microfluidics has the potential to build up a novel platform for diagnosis and therapy through precise manipulation of fluids and enhanced throughput functions. The developments in microfluidics-based devices for diagnostics have evolved in the last decade and have been established for their rapid, effective, accurate and economic advantages. The efficiency and sensitivity of such devices to detect disease-specific macromolecules like proteins and nucleic acids have made crucial impacts in disease diagnosis. The disease modelling using microfluidic systems provides a more prominent replication of the in vivo microenvironment and can be a better alternative for the existing disease models. These models can replicate critical microphysiology like the dynamic microenvironment, cellular interactions, and biophysical and biochemical cues. Microfluidics also provides a promising system for high throughput drug screening and delivery applications. However, microfluidics-based diagnostics still encounter related challenges in the reliability, real-time monitoring and reproducibility that circumvents this technology from being impacted in the healthcare industry. This review highlights the recent microfluidics developments for modelling and diagnosing common diseases, including cancer, neurological, cardiovascular, respiratory and autoimmune disorders, and its applications in drug development.

Vessel-on-a-Chip: A Powerful Tool for Investigating Endothelial COVID-19 Fingerprints.

Coronavirus disease (COVID-19) causes various vascular and blood-related reactions, including exacerbated responses. The role of endothelial cells in this acute response is remarkable and may remain important beyond the acute phase. As we move into a post-COVID-19 era (where most people have been or will be infected by the SARS-CoV-2 virus), it is crucial to define the vascular consequences of COVID-19, including the long-term effects on the cardiovascular system. Research is needed to determine whether chronic endothelial dysfunction following COVID-19 could lead to an increased risk of cardiovascular and thrombotic events. Endothelial dysfunction could also serve as a diagnostic and therapeutic target for post-COVID-19. This review covers these topics and examines the potential of emerging vessel-on-a-chip technology to address these needs. Vessel-on-a-chip would allow for the study of COVID-19 pathophysiology in endothelial cells, including the analysis of SARS-CoV-2 interactions with endothelial function, leukocyte recruitment, and platelet activation. "Personalization" could be implemented in the models through induced pluripotent stem cells, patient-specific characteristics, or genetic modified cells. Adaptation for massive testing under standardized protocols is now possible, so the chips could be incorporated for the personalized follow-up of the disease or its sequalae (long COVID) and for the research of new drugs against COVID-19.

Mini-thermal platform integrated with microfluidic device with on-site detection for real-time DNA amplification.

The recent cases of COVID-19 have brought the prospect of and requirement for point-of-care diagnostic devices into the limelight. Despite all the advances in point-of-care devices, there is still a huge requirement for a rapid, accurate, easy-to-use, low-cost, field-deployable and miniaturized PCR assay device to amplify and detect genetic material. This work aims to develop an Internet-of-Things automated, integrated, miniaturized and cost-effective microfluidic continuous flow-based PCR device capable of on-site detection. As a proof of application, the 594-bp GAPDH gene was successfully amplified and detected on a single system. The presented mini thermal platform with an integrated microfluidic device has the potential to be used for the detection of several infectious diseases.

Modeling Heart Diseases on a Chip: Advantages and Future Opportunities.

Heart disease is a significant burden on global health care systems and is a leading cause of death each year. To improve our understanding of heart disease, high quality disease models are needed. These will facilitate the discovery and development of new treatments for heart disease. Traditionally, researchers have relied on 2D monolayer systems or animal models of heart disease to elucidate pathophysiology and drug responses. Heart-on-a-chip (HOC) technology is an emerging field where cardiomyocytes among other cell types in the heart can be used to generate functional, beating cardiac microtissues that recapitulate many features of the human heart. HOC models are showing great promise as disease modeling platforms and are poised to serve as important tools in the drug development pipeline. By leveraging advances in human pluripotent stem cell-derived cardiomyocyte biology and microfabrication technology, diseased HOCs are highly tuneable and can be generated via different approaches such as: using cells with defined genetic backgrounds (patient-derived cells), adding small molecules, modifying the cells' environment, altering cell ratio/composition of microtissues, among others. HOCs have been used to faithfully model aspects of arrhythmia, fibrosis, infection, cardiomyopathies, and ischemia, to name a few. In this review, we highlight recent advances in disease modeling using HOC systems, describing instances where these models outperformed other models in terms of reproducing disease phenotypes and/or led to drug development.

Microfluidics for COVID-19: From Current Work to Future Perspective.

Spread of coronavirus disease 2019 (COVID-19) has significantly impacted the public health and economic sectors. It is urgently necessary to develop rapid, convenient, and cost-effective point-of-care testing (POCT) technologies for the early diagnosis and control of the plague's transmission. Developing POCT methods and related devices is critical for achieving point-of-care diagnosis. With the advantages of miniaturization, high throughput, small sample requirements, and low actual consumption, microfluidics is an essential technology for the development of POCT devices. In this review, according to the different driving forces of the fluid, we introduce the common POCT devices based on microfluidic technology on the market, including paper-based microfluidic, centrifugal microfluidic, optical fluid, and digital microfluidic platforms. Furthermore, various microfluidic-based assays for diagnosing COVID-19 are summarized, including immunoassays, such as ELISA, and molecular assays, such as PCR. Finally, the challenges of and future perspectives on microfluidic device design and development are presented. The ultimate goals of this paper are to provide new insights and directions for the development of microfluidic diagnostics while expecting to contribute to the control of COVID-19.

Biosensors for healthcare: current and future perspectives.

Biosensors are utilized in several different fields, including medicine, food, and the environment; in this review, we examine recent developments in biosensors for healthcare. These involve three distinct types of biosensor: biosensors for in vitro diagnosis with blood, saliva, or urine samples; continuous monitoring biosensors (CMBs); and wearable biosensors. Biosensors for in vitro diagnosis have seen a significant expansion recently, with newly reported clustered regularly interspaced short palindromic repeats (CRISPR)/Cas methodologies and improvements to many established integrated biosensor devices, including lateral flow assays (LFAs) and microfluidic/electrochemical paper-based analytical devices (µPADs/ePADs). We conclude with a discussion of two novel groups of biosensors that have drawn great attention recently, continuous monitoring and wearable biosensors, as well as with perspectives on the commercialization and future of biosensors.

Microscale combinatorial stimulation of human myeloid cells reveals inflammatory priming by viral ligands.

Cells sense a wide variety of signals and respond by adopting complex transcriptional states. Most single-cell profiling is carried out today at cellular baseline, blind to cells' potential spectrum of functional responses. Exploring the space of cellular responses experimentally requires access to a large combinatorial perturbation space. Single-cell genomics coupled with multiplexing techniques provide a useful tool for characterizing cell states across several experimental conditions. However, current multiplexing strategies require programmatic handling of many samples in macroscale arrayed formats, precluding their application in large-scale combinatorial analysis. Here, we introduce StimDrop, a method that combines antibody-based cell barcoding with parallel droplet processing to automatically formulate cell population × stimulus combinations in a microfluidic device. We applied StimDrop to profile the effects of 512 sequential stimulation conditions on human dendritic cells. Our results demonstrate that priming with viral ligands potentiates hyperinflammatory responses to a second stimulus, and show transcriptional signatures consistent with this phenomenon in myeloid cells of patients with severe COVID-19.

Recent approaches to mRNA vaccine delivery by lipid-based vectors prepared by continuous-flow microfluidic devices.

Advancements in nanotechnology have resulted in the introduction of several nonviral delivery vectors for the nontoxic, efficient delivery of encapsulated mRNA-based vaccines. Lipid- and polymer-based nanoparticles (NP) have proven to be the most potent delivery systems, providing increased delivery efficiency and protection of mRNA molecules from degradation. Here, the authors provide an overview of the recent studies carried out using lipid NPs and their functionalized forms, polymeric and lipid-polymer hybrid nanocarriers utilized mainly for the encapsulation of mRNAs for gene and immune therapeutic applications. A microfluidic system as a prevalent methodology for the preparation of NPs with continuous flow enables NP size tuning, rapid mixing and production reproducibility. Continuous-flow microfluidic devices for lipid and polymeric encapsulated RNA NP production are specifically reviewed.

Hyperviscosity syndromes; hemorheology for physicians and the use of microfluidic devices.

PURPOSE OF REVIEW: Hyperviscosity syndromes can lead to significant morbidity and mortality. Existing methods to measure microcirculatory rheology are not readily available and limited in relevance and accuracy at this level. In this review, we review selected hyperviscosity syndromes and the advancement of their knowledge using microfluidic platforms. RECENT FINDINGS: Viscosity changes drastically at the microvascular level as the physical properties of the cells themselves become the major determinants of resistance to blood flow. Current, outdated viscosity measurements only quantify whole blood or serum. Changes in blood composition, cell number, or the physical properties themselves lead to increased blood viscosity. Given the significant morbidity and mortality from hyperviscosity syndromes, new biophysical tools are needed and being developed to study microvascular biophysical and hemodynamic conditions at this microvascular level to help predict those at risk and guide therapeutic treatment. SUMMARY: The use of 'lab-on-a-chip' technology continues to rise to relevance with point of care, personalized testing and medicine as customizable microfluidic platforms enable independent control of many in vivo factors and are a powerful tool to study microcirculatory hemorheology.

3D-printed capillaric ELISA-on-a-chip with aliquoting.

Sandwich immunoassays such as the enzyme-linked immunosorbent assay (ELISA) have been miniaturized and performed in a lab-on-a-chip format, but the execution of the multiple assay steps typically requires a computer or complex peripherals. Recently, an ELISA for detecting antibodies was encoded structurally in a chip thanks to the microfluidic chain reaction (Yafia et al. Nature, 2022, 605, 464-469), but the need for precise pipetting and intolerance to commonly used surfactant concentrations limit the potential for broader adoption. Here, we introduce the ELISA-on-a-chip with aliquoting functionality that simplifies chip loading and pipetting, accommodates higher surfactant concentrations, includes barrier channels that delay the contact between solutions and prevent undesired mixing, and that executed a quantitative, high-sensitivity assay for the SARS-CoV-2 nucleocapsid protein in 4×-diluted saliva. Upon loading the chip using disposable pipettes, capillary flow draws each reagent and the sample into a separate volumetric measuring reservoir for detection antibody (70 µL), enzyme conjugate (50 µL), substrate (80 µL), and sample (210 µL), and splits washing buffer into 4 different reservoirs of 40, 40, 60, and 20 µL. The excess volume is autonomously drained via a structurally encoded capillaric aliquoting circuit, creating aliquots with an accuracy of >93%. Next, the user click-connects the assay module, comprising a nitrocellulose membrane with immobilized capture antibodies and a capillary pump, to the chip which triggers the step-by-step, timed flow of all aliquoted solutions to complete the assay in 1.5 h. A colored precipitate forming a line on a nitrocellulose strip serves as an assay readout, and upon digitization, yielded a binding curve with a limit of detection of 54 and 91 pg mL-1 for buffer and diluted saliva respectively, vastly outperforming rapid tests. The ELISA chip is 3D-printed, modular, adaptable to other targets and assays, and could be used to automate ELISA in the lab; or as a diagnostic test at the point of care with the convenience and form factor of rapid tests while preserving the protocol and performance of central laboratory ELISA.

Automated, flow-based chemiluminescence microarray immunoassay for the rapid multiplex detection of IgG antibodies to SARS-CoV-2 in human serum and plasma (CoVRapid CL-MIA).

In the face of the COVID-19 pandemic, the need for rapid serological tests that allow multiplexing emerged, as antibody seropositivity can instruct about individual immunity after an infection with SARS-CoV-2 or after vaccination. As many commercial antibody tests are either time-consuming or tend to produce false negative or false positive results when only one antigen is considered, we developed an automated, flow-based chemiluminescence microarray immunoassay (CL-MIA) that allows for the detection of IgG antibodies to SARS-CoV-2 receptor-binding domain (RBD), spike protein (S1 fragment), and nucleocapsid protein (N) in human serum and plasma in less than 8 min. The CoVRapid CL-MIA was tested with a set of 65 SARS-CoV-2 serology positive or negative samples, resulting in 100% diagnostic specificity and 100% diagnostic sensitivity, thus even outcompeting commercial tests run on the same sample set. Additionally, the prospect of future quantitative assessments (i.e., quantifying the level of antibodies) was demonstrated. Due to the fully automated process, the test can easily be operated in hospitals, medical practices, or vaccination centers, offering a valuable tool for COVID-19 serosurveillance. Graphical abstract.

A film-lever actuated switch technology for multifunctional, on-demand, and robust manipulation of liquids.

A lab-on-a-chip system with Point-of-Care testing capability offers rapid and accurate diagnostic potential and is useful in resource-limited settings where biomedical equipment and skilled professionals are not readily available. However, a Point-of-Care testing system that simultaneously possesses all required features of multifunctional dispensing, on-demand release, robust operations, and capability for long-term reagent storage is still a major challenge. Here, we describe a film-lever actuated switch technology that can manipulate liquids in any direction, provide accurate and proportional release response to the applied pneumatic pressure, as well as sustain robustness during abrupt movements and vibrations. Based on the technology, we also describe development of a polymerase chain reaction system that integrates reagent introduction, mixing and reaction functions all in one process, which accomplishes "sample-in-answer-out" performance for all clinical nasal samples from 18 patients with Influenza and 18 individual controls, in good concordance of fluorescence intensity with standard polymerase chain reaction (Pearson coefficients > 0.9). The proposed platform promises robust automation of biomedical analysis, and thus can accelerate the commercialization of a range of Point-of-Care testing devices.

A Miniaturized Silicon Lab-on-Chip for Integrated PCR and Hybridization Microarray for High Multiplexing Nucleic Acids Analysis.

A silicon lab-on-chip, for the detection of nucleic acids through the integrated PCR and hybridization microarray, was developed. The silicon lab-on-chip manufactured through bio-MEMS technology is composed of two PCR microreactors (each volume 11.2 µL) and a microarray-hybridization microchamber (volume 30 µL), fluidically connected by buried bypass. It contains heaters and temperature sensors for the management and control of the temperature cycles during the PCR amplification and hybridization processes. A post-silicon process based on (i) plasmo-O2 cleaning/activation, (ii) vapor phase epoxy silanization, (iii) microarray fabrication and (iv) a protein-based passivation step was developed and fully characterized. The ssDNA microarray (4 rows × 10 columns) composed of 400 spots (spot size-70 ± 12 µm; spot-to-spot distance-130 ± 13 µm) was manufactured by piezo-dispense technology. A DNA microarray probe density in the range of 1310 to 2070 probe µm-2 was observed, together with a limit of detection of about 19 target µm-2. The performances of the silicon lab-on-chip were validated by the detection of the beta-globin gene directly from human blood. Remarkable sensitivity, multiplexing analysis and specificity were demonstrated for the detection of beta-globin and mycobacterium tuberculosis sequences.

A lab-on-a-chip for the concurrent electrochemical detection of SARS-CoV-2 RNA and anti-SARS-CoV-2 antibodies in saliva and plasma.

Rapid, accurate and frequent detection of the RNA of SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) and of serological host antibodies to the virus would facilitate the determination of the immune status of individuals who have Coronavirus disease 2019 (COVID-19), were previously infected by the virus, or were vaccinated against the disease. Here we describe the development and application of a 3D-printed lab-on-a-chip that concurrently detects, via multiplexed electrochemical outputs and within 2 h, SARS-CoV-2 RNA in saliva as well as anti-SARS-CoV-2 immunoglobulins in saliva spiked with blood plasma. The device automatedly extracts, concentrates and amplifies SARS-CoV-2 RNA from unprocessed saliva, and integrates the Cas12a-based enzymatic detection of SARS-CoV-2 RNA via isothermal nucleic acid amplification with a sandwich-based enzyme-linked immunosorbent assay on electrodes functionalized with the Spike S1, nucleocapsid and receptor-binding-domain antigens of SARS-CoV-2. Inexpensive microfluidic electrochemical sensors for performing multiplexed diagnostics at the point of care may facilitate the widespread monitoring of COVID-19 infection and immunity.

All-in-One Optofluidic Chip for Molecular Biosensing Assays.

Integrated biosensor platforms have become subjects of high interest for consolidated assay preparation and analysis to reduce sample-to-answer response times. By compactly combining as many biosensor processes and functions as possible into a single lab-on-chip device, all-in-one point-of-care devices can aid in the accessibility and speed of deployment due to their compact size and portability. Biomarker assay preparation and sensing are functionalities that are often carried out on separate devices, thus increasing opportunity of contamination, loss of sample volume, and other forms of error. Here, we demonstrate a complete lab-on-chip system combining sample preparation, on-chip optofluidic dye laser, and optical detection. We first show the integration of an on-chip distributed feedback dye laser for alignment-free optical excitation of particles moving through a fluidic channel. This capability is demonstrated by using Rhodamine 6G as the gain medium to excite single fluorescent microspheres at 575 nm. Next, we present an optofluidic PDMS platform combining a microvalve network (automaton) for sample preparation of nanoliter volumes, on-chip distributed feedback dye laser for target excitation, and optical detection. We conduct concurrent capture and fluorescence tagging of Zika virus nucleic acid on magnetic beads in 30 min. Target-carrying beads are then optically excited using the on-chip laser as they flow through an analysis channel, followed by highly specific fluorescence detection. This demonstration of a complete all-in-one biosensor is a tangible step in the development of a rapid, point-of-care device that can assist in limiting the severity of future outbreaks.

Point-of-Care Diagnostics for Farm Animal Diseases: From Biosensors to Integrated Lab-on-Chip Devices.

Zoonoses and animal diseases threaten human health and livestock biosecurity and productivity. Currently, laboratory confirmation of animal disease outbreaks requires centralized laboratories and trained personnel; it is expensive and time-consuming, and it often does not coincide with the onset or progress of diseases. Point-of-care (POC) diagnostics are rapid, simple, and cost-effective devices and tests, that can be directly applied on field for the detection of animal pathogens. The development of POC diagnostics for use in human medicine has displayed remarkable progress. Nevertheless, animal POC testing has not yet unfolded its full potential. POC devices and tests for animal diseases face many challenges, such as insufficient validation, simplicity, and portability. Emerging technologies and advanced materials are expected to overcome some of these challenges and could popularize animal POC testing. This review aims to: (i) present the main concepts and formats of POC devices and tests, such as lateral flow assays and lab-on-chip devices; (ii) summarize the mode of operation and recent advances in biosensor and POC devices for the detection of farm animal diseases; (iii) present some of the regulatory aspects of POC commercialization in the EU, USA, and Japan; and (iv) summarize the challenges and future perspectives of animal POC testing.