Rapid automated extracellular vesicle isolation and miRNA preparation on a cost-effective digital microfluidic platform.

As a prerequisite for extracellular vesicle (EV) -based studies and diagnosis, effective isolation, enrichment and retrieval of EV biomarkers are crucial to subsequent analyses, such as miRNA-based liquid biopsy for non-small-cell lung cancer (NSCLC). However, most conventional approaches for EV isolation suffer from lengthy procedure, high cost, and intense labor. Herein, we introduce the digital microfluidic (DMF) technology to EV pretreatment protocols and demonstrate a rapid and fully automated sample preparation platform for clinical tumor liquid biopsy. Combining a reusable DMF chip technique with a low-cost EV isolation and miRNA preparation protocol, the platform completes automated sample processing in 20-30 min, supporting immediate RT-qPCR analyses on EV-derived miRNAs (EV-miRNAs). The utility and reliability of the platform was validated via clinical sample processing for EV-miRNA detection. With 23 tumor and 20 non-tumor clinical plasma samples, we concluded that EV-miR-486-5p and miR-21-5p are effective biomarkers for NSCLC with a small sample volumn (20-40 µL). The result was consistent to that of a commercial exosome miRNA extraction kit. These results demonstrate the effectiveness of DMF in EV pretreatment for miRNA detection, providing a facile solution to EV isolation for liquid biopsy.

Capacitive Bionic Magnetic Sensors Based on One-Step Biointerface Preparation.

Magnetic biomolecule-based bionic magnetic field sensors are anticipated to open up novel pathways for magnetic field detection. The detection range and accuracy of current bionic magnetic field sensors are limited, and little work is based on the capacitive response principle. We successfully developed a biochemical interface with an extralarge target-receptor size ratio, which can be manufactured in a single step for weak magnetic field detection across a wide frequency range, and we used electrochemical capacitance as a magnetic field change conduction strategy. The thickness-controllable nanoscale bovine serum albumin/graphene layer on an indium tin oxide working electrode combines with the one-step preparation method to immobilize the MagR/Cry4 complex. This capacitive bionic magnetic sensor can achieve the detection range of 0-120 mT. This biointerface design strategy obtains the further improvement of the performance of this bionic magnetic field sensor. Furthermore, the biointerface construction and optimization methodology in this proposal has potential applications in the design of other medical biosensors.

Monitoring of Sweat Ions and Physiological Parameters via a Reconfigurable Modular System.

In recent years, wearable sensors have revolutionized health monitoring by enabling continuous, real-time tracking of human health and performance. These noninvasive devices are usually designed to monitor human physical state and biochemical markers. However, enhancing their functionalities often demands intricate customization by designers and additional expenses for users. Here, we present a strategy using assembled modular circuits to customize health monitoring wearables. The modular circuits can be effortlessly reconfigured to meet various specific requirements, facilitating the incorporation of diverse functions at a lower cost. To validate this approach, modular circuits were employed to develop four distinct systems for in vitro evaluations. These systems enabled the detection of sweat biomarkers and physical signals under various scenarios, including sedentary state, exercise, and daily activities with or without incorporating iontophoresis to induce sweat. Four key sweat markers (K+, Ca2+, Na+, and pH) and three essential physical indicators (heart rate, blood oxygen levels, and skin temperature) are selected as the detection targets. Commercial methods were also used to evaluate the potential for effective health monitoring with our technique. This reconfigurable modular wearable (ReModuWear) system promises to provide more easy-to-use and comprehensive health assessments. Additionally, it may contribute to environmental sustainability by reusing modules.

Detecting EGFR gene amplification using a fluorescence in situ hybridization platform based on digital microfluidics.

Signal transduction mediated by epidermal growth factor receptor (EGFR) gene affects the proliferation, invasion, metastasis, and angiogenesis of tumor cells. In particular, non-small cell lung cancer (NSCLC) patients with increased in copy number of EGFR gene are often sensitive to tyrosine kinase inhibitors. Despite being the standard for detecting EGFR amplification in the clinic, fluorescence in situ hybridization (FISH) traditionally involves repetitive and complex benchtop procedures that are not only time consuming but also require well-trained personnel. To address these limitations, we develop a digital microfluidics-based FISH platform (DMF-FISH) that automatically implements FISH operations. This system mainly consists of a DMF chip for reagent operation, a heating array for temperature control and a signal processing system. With the capability of automatic droplet handling and efficient temperature control, DMF-FISH performs cell digestion, gradient elution, hybridization and DAPI staining without manual intervention. In addition to operational feasibility, DMF-FISH yields comparable performance with the benchtop FISH protocol but reducing the consumption of DNA probe by 87 % when tested with cell lines and clinical samples. These results highlight unique advantages of the fully automated DMF-FISH system and thus suggest its great potential for clinical diagnosis and personalized therapy of NSCLC.

A Cell State Monitoring System with Integrated In Situ Imaging and pH Detection.

Cell models are one of the most widely used basic models in biological research, and a variety of in vitro cell culture techniques and models have been developed recently to simulate the physiological microenvironment in vivo. However, regardless of the technique or model, cell culture is the most fundamental but crucial component. As a result, we have developed a cell culture monitoring system to assess the functional status of cells within a biochip. This article focuses on a mini-microscope made from a readily available camera for in situ continuous observation of cell growth within a biochip and a pH sensor based on optoelectronic sensing for measuring pH. With the aid of this monitoring system, scientists can keep an eye on cell growth in real time and learn how the pH of the culture medium affects it. This study offers a new approach for tracking cells on biochips and serves as a valuable resource for enhancing cell culture conditions.

Non-invasive detection of tumor markers in salivary extracellular vesicles based on digital PCR chips.

The World Health Organization (WHO) has stated that countless cancer patients could be saved if early detection and treatment were available. However, current clinical evaluation of tumors still relies primarily on imaging examinations and tissue biopsies. These methods not only require sophisticated equipment, but also have high false positive rates or invasive problems. Here, we describe a digital polymerase chain reaction (dPCR) chip for the detection of biomarkers in salivary extracellular vesicles (SEVs), which can be used to identify markers for the early diagnosis of tumors. Based on microfluidic technology fine microstructure and microfluidics operations, this dPCR chip can accurate quantitative SEVs in a variety of tumor markers, and shows extremely strong sensitivity (10 copies). In the detection of clinical samples, the chip can effectively distinguish lung cancer cases from normal controls (P < 0.001; two-tailed t-test), and in the detection of extremely low concentration samples, it shows considerably higher precise quantitative ability than quantitative real-time polymerase chain reaction (qPCR). Overall, this study may shed new light on non-invasive early screening of tumor markers by detecting extracellular vesicle-associated markers in saliva.

Cell spheroids culture array with modifiable chemical gradients.

Cancer cell spheroids have been shown to mimic in vivo tumour microenvironment and are therefore suitable for in vitro drug screening. Microfluidic technology can provide conveniences for spheroid assays such as high-throughput, simplifying manual operation and saving reagent. Here, we propose a concentration gradient generator based on microfluidic technology for cell spheroid culture and assay. The chip consists of upper microchannels and lower microwells. After partitioning HepG2 suspension into the microwells with concave and non-adhesive bottoms, spheroids can spontaneously form. By controlling the fluid replacement and flow in microchannels, the doxorubicin solution is diluted automatically into a series of concentration gradients, which spanning more than one order of magnitude. And then the effect of doxorubicin on spheroids is measured in situ by fluorescent staining. This chip provides a very promising approach to achieve the high-throughput and standardized anti-cancer drug screening in future.

Microfluidic Based Organ-on-Chips and Biomedical Application.

Organ-on-a-Chip is a microfluidic cell culture device manufactured via microchip fabrication methods [...].

Bionic Magnetic Sensor Based on the MagR/Cry4 Complex-Configured Graphene Transistor with an Integrated On-Chip Gate.

Magnetic-sensitive proteins are regarded as key factors in animals' precise perception of the geomagnetic field. Accurate feedback on the response of these tiny proteins to magnetic fields remains a challenge. Here, we first propose a real-time accurate magnetic sensor based on the MagR/Cry4 complex-configured graphene transistor with an integrated on-chip gate. A nanometer-thick denatured bovine serum albumin film was used as the bio-interface of graphene electrolyte-gated transistors (EGTs) to immobilize the MagR/Cry4 complex. With the optimization and characterization of this bionic graphene EGT, it could detect magnetic fields in real time with a sensitivity of 1 mT, which is far lower than in earlier research. It was concluded that our MagR/Cry4 complex-configured graphene EGTs with a side-gate held great promise in terms of geomagnetic field detection. Furthermore, the constructed approach in this paper could also be utilized as a general solution for recording the response of magnetically sensitive biomolecules to magnetic fields in real time.

Combining sensors and actuators with electrowetting-on-dielectric (EWOD): advanced digital microfluidic systems for biomedical applications.

The concept of digital microfluidics (DMF) enables highly flexible and precise droplet manipulation at a picoliter scale, making DMF a promising approach to realize integrated, miniaturized "lab-on-a-chip" (LOC) systems for research and clinical purposes. Owing to its simplicity and effectiveness, electrowetting-on-dielectric (EWOD) is one of the most commonly studied and applied effects to implement DMF. However, complex biomedical assays usually require more sophisticated sample handling and detection capabilities than basic EWOD manipulation. Alternatively, combined systems integrating EWOD actuators and other fluidic handling techniques are essential for bringing DMF into practical use. In this paper, we briefly review the main approaches for the integration/combination of EWOD with other microfluidic manipulation methods or additional external fields for specified biomedical applications. The form of integration ranges from independently operating sub-systems to fully coupled hybrid actuators. The corresponding biomedical applications of these works are also summarized to illustrate the significance of these innovative combination attempts.

Stem Cell-Derived Exosomes: A New Method for Reversing Skin Aging.

Senescence is an inevitable natural life process that involves structural and functional degeneration of tissues and organs. Recently, the process of skin aging has attracted much attention. Determining a means to delay or even reverse skin aging has become a research hotspot in medical cosmetology and anti-aging. Dysfunction in the epidermis and fibroblasts and changes in the composition and content of the extracellular matrix are common pathophysiological manifestations of skin aging. Reactive oxygen species and matrix metalloproteinases play essential roles in this process. Stem cells are pluripotent cells that possess self-replication abilities and can differentiate into multiple functional cells under certain conditions. These cells also possess a strong ability to facilitate tissue repair and regeneration. Stem cell transplantation has the potential for application in anti-aging therapy. Increasing studies have demonstrated that stem cells perform functions through paracrine processes, particularly those involving exosomes. Exosomes are nano-vesicular substances secreted by stem cells that participate in cell-to-cell communication by transporting their contents into target cells. In this chapter, the biological characteristics of exosomes were reviewed, including their effects on extracellular matrix formation, epidermal cell function, fibroblast function and antioxidation. Exosomes derived from stem cells may provide a new means to reverse skin aging.

Microfluidic Organ-on-a-Chip System for Disease Modeling and Drug Development.

An organ-on-a-chip is a device that combines micro-manufacturing and tissue engineering to replicate the critical physiological environment and functions of the human organs. Therefore, it can be used to predict drug responses and environmental effects on organs. Microfluidic technology can control micro-scale reagents with high precision. Hence, microfluidics have been widely applied in organ-on-chip systems to mimic specific organ or multiple organs in vivo. These models integrated with various sensors show great potential in simulating the human environment. In this review, we mainly introduce the typical structures and recent research achievements of several organ-on-a-chip platforms. We also discuss innovations in models applied to the fields of pharmacokinetics/pharmacodynamics, nano-medicine, continuous dynamic monitoring in disease modeling, and their further applications in other fields.

Analyzing Human Periodontal Soft Tissue Inflammation and Drug Responses In Vitro Using Epithelium-Capillary Interface On-a-Chip.

The gingival epithelium-capillary interface is a unique feature of periodontal soft tissue, preserving periodontal tissue homeostasis and preventing microorganism and toxic substances from entering the subepithelial tissue. However, the function of the interface is disturbed in periodontitis, and mechanisms of the breakdown of the interface are incompletely understood. To address these limitations, we developed a microfluidic epithelium-capillary barrier with a thin culture membrane (10 µm) that closely mimics the in vivo gingival epithelial barrier with an immune micro-environment. To test the validity of the fabricated gingival epithelial barrier model, epithelium-capillary interface-on-a-chip was cultured with human gingival epithelial cells (HGECs) and human vascular endothelial cells (HUVEC). Their key properties were tested using optical microscope, transepithelial/transendothelial electrical resistance (TEER), and permeability assays. The clear expression of VE-cadherin revealed the tight junctions in endothelial cells. Live/dead assays indicated a high cell viability, and the astrocytic morphology of HGE cells was confirmed by F-actin immunostaining. By the third day of cell culture, TEER levels typically exceeded in co-cultures. The resultant permeability coefficients showed a significant difference between 70 kDa and 40 kDa FITC-dextran. The expression of protein intercellular cell adhesion molecule (ICAM-1) and human beta defensin-2 (HBD2) decreased when exposed to TNF-α and LPS, but recovered with the NF-κB inhibitor treatment- Pyrrolidinedithiocarbamic acid (PDTC), indicating the stability of the fabricated chip. These results demonstrate that the developed epithelium-capillary interface system is a valid model for studying periodontal soft tissue function and drug delivery.

Micro-PCR chip-based multifunctional ultrafast SARS-CoV-2 detection platform.

When dealing with infectious pathogens, the point-of-care screening and diagnosis strategy should be low-cost, simple, rapid and accurate. Here, we report a multifunctional rapid PCR platform allowing both simultaneous screening of suspected cases and accurate identification and quantification of the virus. Based on the platform, samples suspected of being infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are screened first, after which subsequent precise quantification of the virus (SARS-CoV-2) can be performed if necessary. This fast screening technique offers a detection limit of 10 nucleic acid copies per test during the entire running time of 15 minutes, with a throughput of 9 samples at a time. Besides, depending on a droplet microfluidic chip, this platform could also provide assays of nucleic acids across four orders of magnitude of concentration within less than 15 minutes. Additionally, we successfully use the platform to quickly distinguish between positive and negative cases in clinical samples and rapidly quantify the viral load in each sample, which is consistent with standard RT-qPCR tests. As such, we demonstrate a promising and versatile rapid PCR platform for point-of-care diagnosis of infectious diseases.

Integrated microfluidic system for isolating exosome and analyzing protein marker PD-L1.

Exosomes are lipid-bilayered nanovesicles secreted by cells to mediate intercellular communication. Various kinds of biomolecules involved in exosomes offer non-invasive approaches for detecting or monitoring disease and developing targeted therapeutics. Here, we present an integrated microfluidic exosome isolation and detection system (EXID system) to analyze the abundance of the exosomal PD-L1 protein marker, which is a transmembrane protein expressed by tumors to suppress immune activation of T cells. By incorporating exosome isolation and biomarker labelling and quantification within a single microfluidic chip, our system reduced the total analysis time below 2 h. Using the EXID system, 7 categories of cell lines including cancer cell lines and control samples were profiled, where significant differences in the fluorescence intensity were observed with the limit of detection (LOD) down to 10.76 per microliter. Such noticeable variations in PD-L1 abundance among cancer cell lines highlighted the need of personalized treatments. Furthermore, 16 clinical samples from 7 post-treated cancer patients, 3 prior-treatment patients and 6 healthy controls, are tested, among which differences in sensitivity toward immune response were subsistent. Because the abundance of PD-L1 reflects the sensibility for immune response, our results provide useful guides to design immunotherapy strategies for different types of tumors.

A Microfluidic Cell Co-Culture Chip for the Monitoring of Interactions between Macrophages and Fibroblasts.

Macrophages and fibroblasts are two types of important cells in wound healing. The development of novel platforms for studying the interrelationship between these two cells is crucial for the exploration of wound-healing mechanisms and drug development. In this study, a microfluidic chip composed of two layers was designed for the co-culturing of these two cells. An air valve was employed to isolate fibroblasts to simulate the wound-healing microenvironment. The confluence rate of fibroblasts in the co-culture system with different macrophages was explored to reflect the role of different macrophages in wound healing. It was demonstrated that M2-type macrophages could promote the activation and migration of fibroblasts and it can be inferred that they could promote the wound-healing process. The proposed microfluidic co-culture system was designed for non-contact cell-cell interactions, which has potential significance for the study of cell-cell interactions in biological processes such as wound healing, tumor microenvironment, and embryonic development.

Microfluidic integrated capacitive biosensor for C-reactive protein label-free and real-time detection.

A microfluidic chip has been integrated with a capacitive biosensor based on mass-producible three-dimensional (3D) interdigital electrode arrays. To achieve the monitoring of biosensor preparation and cardiac- and periodontitis-related biomarkers, all the processes were detected in a continuously on-site way. Fabrication steps for the microfluidic chip-bonded 3D interdigital capacitor biosensor include gold thiol modification, the activation of EDC/sulfo-NHS, and the bioconjugation of antibodies. Fluorescent characterization and X-ray photoelectron spectroscopy analysis were applied to assess the successful immobilization of the C-reactive protein (CRP) antibody. The experimental results indicate the good specificity and high sensitivity of the microfluidic integrated 3D capacitive biosensor. The limit of detection of the 3D capacitive biosensor for CRP label-free detection was about 1 pg mL-1. This 3D capacitive biosensor with integrated microfluidics is mass-producible and has achieved the on-site continuous detection of cardiac- and periodontitis-related biomarkers with high performance.

Nanopore Technology and Its Applications in Gene Sequencing.

In recent years, nanopore technology has become increasingly important in the field of life science and biomedical research. By embedding a nano-scale hole in a thin membrane and measuring the electrochemical signal, nanopore technology can be used to investigate the nucleic acids and other biomacromolecules. One of the most successful applications of nanopore technology, the Oxford Nanopore Technology, marks the beginning of the fourth generation of gene sequencing technology. In this review, the operational principle and the technology for signal processing of the nanopore gene sequencing are documented. Moreover, this review focuses on the applications using nanopore gene sequencing technology, including the diagnosis of cancer, detection of viruses and other microbes, and the assembly of genomes. These applications show that nanopore technology is promising in the field of biological and biomedical sensing.

Ultrafast multiplexed detection of SARS-CoV-2 RNA using a rapid droplet digital PCR system.

We report the first combination of droplet digital and rapid PCR techniques for efficient, accurate, and quantitative detection of SARS-CoV-2 RNA. The presented rapid digital PCR system simultaneously detects two specific targets (ORF1ab and N genes) and one reference gene (RNase P) with a single PCR thermal cycling period around 7 s and the total running time less than 5 min. A clear positive signal could be identified within 115 s via the rapid digital RT-PCR, suggesting its efficiency for the end-point detection. In addition, benchmark tests with serial diluted reference samples of SARS-CoV-2 RNA reveal the excellent accuracy of our system (R2>0.99). More importantly, the rapid digital PCR system gives consistent and accurate detection of low-concentration reference samples, whereas qPCR yields Ct values with significant variations that could lead to false-negative results. Finally, we apply the rapid digital PCR system to analyze clinical samples with both positive and control cases, where results are consistent with qPCR test outcomes. By providing similar accuracy with qPCR while minimizing the detection time-consuming and the false-negative tendency, the presented rapid digital PCR system represents a promising improvement on the rapid diagnosis of COVID-19.

On-site analysis of COVID-19 on the surfaces in wards.

SARS-Cov-2 has erupted across the globe, and confirmed cases of COVID-19 pose a high infection risk. Infected patients typically receive their treatment in specific isolation wards, where they are confined for at least 14 days. The virus may contaminate any surface of the room, especially frequently touched surfaces. Therefore, surface contamination in wards should be monitored for disease control and hygiene purposes. Herein, surface contamination in the ward was detected on-site using an RNA extraction-free rapid method. The whole detection process, from surface sample collection to readout of the detection results, was finished within 45 min. The nucleic acid extraction-free method requires minimal labor. More importantly, the tests were performed on-site and the results were obtained almost in real-time. The test confirmed that 31 patients contaminated seven individual sites. Among the sampled surfaces, the electrocardiogram fingertip presented a 72.7% positive rate, indicating that this surface is an important hygiene site. Meanwhile, the bedrails showed the highest correlation with other surfaces, so should be detected daily. Another surface with high contamination risk was the door handle in the bathroom. To our knowledge, we present the first on-site analysis of COVID-19 surface contamination in wards. The results and applied technique provide a potential further reference for disease control and hygiene suggestions.