Label-free active single-cell encapsulation enabled by microvalve-based on-demand droplet generation and real-time image processing.

Droplet microfluidics-based single-cell encapsulation is a critical technology that enables large-scale parallel single-cell analysis by capturing and processing thousands of individual cells. As the efficiency of passive single-cell encapsulation is limited by Poisson distribution, active single-cell encapsulation has been developed to theoretically ensure that each droplet contains one cell. However, existing active single-cell encapsulation technologies still face issues related to fluorescence labeling and low throughput. Here, we present an active single-cell encapsulation technique by using microvalve-based drop-on-demand technology and real-time image processing to encapsulate single cells with high throughput in a label-free manner. Our experiments demonstrated that the single-cell encapsulation system can encapsulate individual polystyrene beads with 96.3 % efficiency and HeLa cells with 94.9 % efficiency. The flow speed of cells in this system can reach 150 mm/s, resulting in a corresponding theoretical encapsulation throughput of 150 Hz. This technology has significant potential in various biomedical applications, including single-cell omics, secretion detection, and drug screening.

Multicolor-Assay-on-a-Chip Processed by Robotic Operation (MACpro) with Improved Diagnostic Accuracy for Field-Deployable Detection.

The ability to deploy decentralized laboratories with autonomous and reliable disease diagnosis holds the potential to deliver accessible healthcare services for public safety. While microfluidic technologies provide precise manipulation of small fluid volumes with improved assay performance, their limited automation and versatility confine them to laboratories. Herein, we report the utility of multicolor assay-on-a-chip processed by robotic operation (MACpro), to address this unmet need. The MACpro platform comprises a robot-microfluidic interface and an eye-in-hand module that provides flexible yet stable actions to execute tasks in a programmable manner, such as the precise manipulation of the microfluidic chip along with different paths. Notably, MACpro shows improved detection performance by integrating the microbead-based antibody immobilization with enhanced target recognition and multicolor sensing via Cu2+-catalyzed plasmonic etching of gold nanorods for rapid and sensitive analyte quantification. Using interferon-gamma as an example, we demonstrate that MACpro completes a sample-to-answer immunoassay within 30 min and achieves a 10-fold broader dynamic range and a 10-fold lower detection limit compared to standard enzyme-linked immunosorbent assays (0.66 vs 5.2 pg/mL). MACpro extends the applications beyond traditional laboratories and presents an automated solution to expand diagnostic capacity in diverse settings.

Microfluidics in the eye: a review of glaucoma implants from an engineering perspective.

Glaucoma is a progressive optic neuropathy in the eye, which is a leading cause of irreversible blindness worldwide and currently affects over 70 million individuals. Clinically, intraocular pressure (IOP) reduction is the only proven treatment to halt the progression of glaucoma. Microfluidic devices such as glaucoma drainage devices (GDDs) and minimally invasive glaucoma surgery (MIGS) devices are routinely used by ophthalmologists to manage elevated IOP, by creating an artificial pathway for the over-accumulated aqueous humor (AH) in a glaucomatous eye, when the natural pathways are severely blocked. Herein, a detailed modelling and analysis of both the natural microfluidic pathways of the AH in the eye and artificial microfluidic pathways formed additionally by the various glaucoma implants are conducted to provide an insight into the causes of the IOP abnormality and the improvement schemes of current implant designs. The mechanisms of representative glaucoma implants have been critically reviewed from the perspective of microfluidics, and we have categorized the current implants into four groups according to the targeted drainage sites of the AH, namely Schlemm's canal, suprachoroidal space, subconjunctival space, and ocular surface. In addition, we propose to divide the development and evolution of glaucoma implant designs into three technological waves, which include microtube (1st), microvalve (2nd) and microsystem (3rd). With the emerging trends of minimal invasiveness and artificial intelligence in the development of medical implants, we envision that a comprehensive glaucoma treatment microsystem is on the horizon, which is featured with active and wireless control of IOP, real-time continuous monitoring of IOP and aqueous rate, etc. The current review could potentially cast light on the unmatched needs, challenges, and future directions of the microfluidic structural and functional designs of glaucoma implants, which would enable an enhanced safety profile, reduced complications, increased efficacy of lowering IOP and reduced IOP fluctuations, closed-loop and on-demand control of IOP, etc.

Structural Electronic Skin for Conformal Tactile Sensing.

The conformal integration of the electronic skin on the non-developable surface is in great demand for the comprehensive tactile sensing of robotics and prosthetics. However, the current techniques still encounter obstacles in achieving conformal integration of film-like electronic skin on non-developable surfaces with substantial curvatures for contact pressure detection and tactile mapping. In this paper, by utilizing the 3D printing technology to prepare the 3D electrode array in the structural component following its surface curvature, and covering it with a molded functional shell to form the pressure sensitive iontronic interface, a device is proposed to achieve high-sensitive pressure detection and high-fidelity tactile mapping on a complicated non-developable surface, called structural electronic skin (SES). The SES is prepared in a 3D printed fingertip with 46 tactile sensing units distributed on its curved surface, achieving the integration of both structural and tactile functions in a single component. By integrating the smart fingertip into a dexterous hand, a series of demonstrations are presented to show the dead-zone free pressure detection and tactile mapping with high sensitivity, for instance, 2D pulse wave monitoring and robotic injection in a medical robot, object recognition and compliant control in a smart prosthesis.

Real-time fluorescence imaging flow cytometry enabled by motion deblurring and deep learning algorithms.

Fluorescence imaging flow cytometry (IFC) has been demonstrated as a crucial biomedical technique for analyzing specific cell subpopulations from heterogeneous cellular populations. However, the high-speed flow of fluorescent cells leads to motion blur in cell images, making it challenging to identify cell types from the raw images. In this study, we present a real-time single-cell imaging and classification system based on a fluorescence microscope and deep learning algorithm, which is able to directly identify cell types from motion-blur images. To obtain annotated datasets of blurred images for deep learning model training, we developed a motion deblurring algorithm for the reconstruction of blur-free images. To demonstrate the ability of this system, deblurred images of HeLa cells with various fluorescent labels and HeLa cells at different cell cycle stages were acquired. The trained ResNet achieved a high accuracy of 96.6% for single-cell classification of HeLa cells in three different mitotic stages, with a short processing time of only 2 ms. This technology provides a simple way to realize single-cell fluorescence IFC and real-time cell classification, offering significant potential in various biological and medical applications.

Protocol for electrotaxis of large epithelial cell sheets.

Here, we present a protocol for electrotaxis of large epithelial cell sheets without compromising the integrity of cell epithelia in a high-throughput customized directed current electrotaxis chamber. We describe the fabrication and use of polydimethylsiloxane stencils to control the size and shape of human keratinocyte cell sheets. We detail cell tracking, cell sheet contour assay, and particle image velocimetry to reveal the spatial and temporal motility dynamics of cell sheets. This approach is applicable to other collective cell migration studies. For complete details on the use and execution of this protocol, please refer to Zhang et al. (2022).1.

Propagation dynamics of electrotactic motility in large epithelial cell sheets.

Directional migration initiated at the wound edge leads epithelia to migrate in wound healing. How such coherent migration is achieved is not well understood. Here, we used electric fields to induce robust migration of sheets of human keratinocytes and developed an in silico model to characterize initiation and propagation of epithelial collective migration. Electric fields initiate an increase in migration directionality and speed at the leading edge. The increases propagate across the epithelial sheets, resulting in directional migration of cell sheets as coherent units. Both the experimental and in silico models demonstrated vector-like integration of the electric and default directional cues at free edge in space and time. The resultant collective migration is consistent in experiments and modeling, both qualitatively and quantitatively. The keratinocyte model thus faithfully reflects key features of epithelial migration as a coherent tissue in vivo, e.g. that leading cells lead, and that epithelium maintains cell-cell junction.

Regional-Controlled Tissue Expanders Increase Skin Expansion and Thickness Compared to Standard Tissue Expanders in a Rat Model.

BACKGROUND: Skin and soft-tissue expansion are widely used. However, the existing methods cannot expand targeted areas on the top flap. Thus, the authors developed a new expander with a partially thickened top. The authors hypothesized that pressure differences would lead to higher growth near nonthickened regions and lower growth near thickened regions, allowing targeted expansion. METHODS: Eighteen male Sprague Dawley rats (8 weeks old) were used; 20-ml rectangular regional-controlled expanders ( n = 12) and ordinary expanders ( n = 6) were implanted. Flaps on regional-controlled expanders were divided into nonthickened and thickened regions and tattooed. Discontinuous inflation began 14 days postoperatively, 3 ml every 3 days, until the volume reached 50 ml. Tattooed skin area and thickness were measured. Immunofluorescence staining detected cell proliferation (proliferating cell nuclear antigen-positive) and vascular density (CD31 + ). Growth factors (transforming growth factor-ß, epidermal growth factor, vascular endothelial growth factor, and basic fibroblast growth factor) were assessed by enzyme-linked immunosorbent assay. RESULTS: The expanded skin area of regional-controlled expansion nonthickened regions (396.2 ± 41.4 mm 2 ) was 33.8 ± 10.0 percent larger than that of thickened regions (297.8 ± 38.9 mm 2 ). Thickened regions had a 28.9 ± 14.6 percent thicker dermal layer (942.4 ± 55.5 µm) than nonthickened regions (737.1 ± 64.5 µm). Nonthickened regions had 295.0 ± 145.0 percent more proliferating cell nuclear antigen-positive cells (92.4 ± 16.2/mm 2 ) than thickened regions (25.6 ± 7.4/mm 2 ). The vascular density was 133.0 ± 61.7 percent higher in thickened regions (24.8 ± 4.7/mm 2 ) than in nonthickened regions (11.1 ± 2.7/mm 2 ) (all above, p < 0.05). CONCLUSIONS: Regional-controlled expansion specifically expands only the targeted area, causing thicker skin flaps with abundant vessels for defect repair. Although this technique has great clinical potential, it should be further validated with large animals and humans. ( Plast. Reconstr. Surg. 150: 1273, 2022.). CLINICAL RELEVANCE STATEMENT: This study presents the newly developed regional-controlled expansion technique that realized the targeted expansion. It is suitable for repairing defects and would contribute to shortening the expansion process and reducing complication rates.

Microfluidic Printing-Based Method for the Multifactorial Study of Cell-Free Protein Networks.

Protein networks can be assembled in vitro for basic biochemistry research, drug screening, and the creation of artificial cells. Two standard methodologies are used: manual pipetting and pipetting robots. Manual pipetting has limited throughput in the number of input reagents and the combination of reagents in a single sample. While pipetting robots are evident in improving pipetting efficiency and saving hands-on time, their liquid handling volume usually ranges from a few to hundreds of microliters. Microfluidic methods have been developed to minimize the reagent consumption and speed up screening but are challenging in multifactorial protein studies due to their reliance on complex structures and labeling dyes. Here, we engineered a new impact-printing-based methodology to generate printed microdroplet arrays containing water-in-oil droplets. The printed droplet volume was linearly proportional (R2 = 0.9999) to the single droplet number, and each single droplet volume was around 59.2 nL (coefficient of variation = 93.8%). Our new methodology enables the study of protein networks in both membrane-unbound and -bound states, without and with anchor lipids DGS-NTA(Ni), respectively. The methodology is demonstrated using a subnetwork of mitogen-activated protein kinase (MAPK). It takes less than 10 min to prepare 100 different droplet-based reactions, using <1 µL reaction volume at each reaction site. We validate the kinase (ATPase) activity of MEK1 (R4F)* and ERK2 WT individually and together under different concentrations, without and with the selective membrane attachment. Our new methodology provides a reagent-saving, efficient, and flexible way for protein network research and related applications.

Wearable Iontronic FMG for Classification of Muscular Locomotion.

Human motion recognition with high accuracy and fast response speed has long been considered an essential component in human-machine interactive activities such as assistive robotics, medical prosthesis, and wearable electronics. The force myography (FMG) signal has been the focus of much investigation in the search for a reliable and efficient muscular locomotion recognition system. However, the effect of the sensing system on FMG-based locomotion classification accuracy has yet to be understood. This study proposed a novel FMG sensing strategy for human lower limb locomotion classification based on flexible supercapacitive iontronic sensors. Benefiting from the ultrahigh sensitivity (up to 1 nF/mmHg) and low activation pressure (less than 5 mmHg) of the supercapacitive iontronic pressure sensor, FMG signal can be acquired accurately from 5 iontronic sensors strapped to the thigh. In the experiment with 12 subjects, the real-time classification strategy based on sliding window and SVM model gave an average locomotion classification accuracy of 99% for seven categories, including sitting, standing, walking on level ground, ramp ascent, ramp descent, stair ascent, stair descent. Compared with traditional FSR sensors, the result showed that iontronic sensors improved the classification accuracy by up to 10 percentage points in the case of short time window. The implementation of the high sensitivity flexible iontronic sensors in the wearable system brings a valuable tool for detecting small human body pressure signals and has great potential to improve the performance of the human-machine interface in rehabilitation and medical applications.

Improving the Sensitivity of Nanofibrous Membrane-Based ELISA for On-Site Antibiotics Detection.

An ultrasensitive and portable colorimetric enzyme-linked immunosorbent assay (ELISA) sensor for antibiotics was fabricated by immobilizing antibodies inside the largely porous and highly hydrophilic nanofibrous membranes. Different from regular electrospun nanofibrous membranes where antibodies may frequently be blocked by the heterogeneous porous structure and sterically crowded loaded on the surface, the controlled microporous structure and increased hydrophilicity of nanofibrous membranes could improve the diffusion properties of antibodies, reduce the sterically crowding effect, and dramatically improve the sensitivity of the membrane-based ELISA. The limitation of detection (LOD) for chloramphenicol (CAP) reached 0.005 ng/mL, around 200 times lower than the conventional paper-based ELISA, making quantitative analysis and portable on-site detection achievable via the use of smartphones. The successful design and fabrication of the nanofibrous membrane-based ELISA with novel features overcome the structural drawbacks of regular electrospun nanofibrous membranes and provide new paths to develop highly sensitive on-site detection of hazardous chemical agents.

High-Throughput Experimentation Using Cell-Free Protein Synthesis Systems.

Cell-free protein synthesis can enable the combinatorial screening of many different components and concentrations. However, manual pipetting methods are unfit to handle many cell-free reactions. Here, we describe a microfluidic method that can generate hundreds of unique submicroliter scale reactions. The method is coupled with a high yield cell-free system that can be applied for broad protein screening assays.

Immunometabolism modulation, a new trick of edible and medicinal plants in cancer treatment.

The edible and medicinal plants (EMPs) are becoming an abundant source for cancer prevention and treatment since the natural and healthy trend for modern human beings. Currently, there are more than one hundred species of EMPs widely used and listed by the national health commission of China, and most of them indicate immune or metabolic regulation potential in cancer treatment with numerous studies over the past two decades. In the present review, we focused on the metabolic influence in immunocytes and tumor microenvironment, including immune response, immunosuppressive factors and cancer cells, discussing the immunometabolic potential of EMPs in cancer treatment. There are more than five hundred references collected and analyzed through retrieving pharmacological studies deposited in PubMed by medical subject headings and the corresponding names derived from pharmacopoeia of China as a sole criterion. Finally, the immunometabolism modulation of EMPs was sketch out implying an immunometabolic control in cancer treatment.

Label-free single-cell isolation enabled by microfluidic impact printing and real-time cellular recognition.

Analysis of cellular components at the single-cell level is important to reveal cellular heterogeneity. However, current technologies to isolate individual cells are either label-based or have low performance. Here, we present a novel technique by integrating real-time cellular recognition and microfluidic impact printing (MIP) to isolate single cells with high efficiency and high throughput in a label-free manner. Specifically, morphological characteristics of polystyrene beads and cells, computed by an efficient image processing algorithm, are utilized as selection criteria to identify target objects. Subsequently, each detected single-cell object in the suspension is ejected from the microfluidic channel by impact force. It has been demonstrated that the single-cell isolating system has the ability to encapsulate polystyrene beads in droplets with an efficiency of 95%, while for HeLa cells, this has been experimentally measured as 90.3%. Single-cell droplet arrays are generated at a throughput of 2 Hz and 96.6% of the cells remain alive after isolation. This technology has significant potential in various emerging applications, including single-cell omics, tissue engineering, and cell-line development.

Diffusion of Protein Molecules through Microporous Nanofibrous Polyacrylonitrile Membranes.

Porous nanofibrous membranes have ultrahigh specific surface areas and could be broadly employed in protein purification, enzyme immobilization, and biosensors with enhanced selectivity, sensitivity, and efficiency. However, large biomolecules, such as proteins, have hindered diffusion behavior in the micro-porous media, significantly reducing the benefits provided by the nanofibrous membranes. The study of protein diffusion in polyacrylonitrile (PAN) nanofibrous membranes produced under varied humidity and polymer concentration of electrospinning revealed that heterogeneous structures of the nanofibrous membranes possess much smaller effective pore sizes than the measured pore sizes, which significantly affects the diffusion of large molecules through the system though sizes of proteins and pH conditions also have great impacts. Only when the measured membrane pore size is at least 1000 times higher than the protein size, the diffusion behavior of the protein is predictable in the system. The results provide insights into the design and applications of proper nanofibrous materials for improved applications in protein purification and immobilizations.

DNA tetrahedron-mediated immune-sandwich assay for rapid and sensitive detection of PSA through a microfluidic electrochemical detection system.

Prostate-specific antigen (PSA) is the most widely used biomarker for the early diagnosis of prostate cancer. Existing methods for PSA detection are burdened with some limitations and require improvement. Herein, we developed a novel microfluidic-electrochemical (µFEC) detection system for PSA detection. First, we constructed an electrochemical biosensor based on screen-printed electrodes (SPEs) with modification of gold nanoflowers (Au NFs) and DNA tetrahedron structural probes (TSPs), which showed great detection performance. Second, we fabricated microfluidic chips by DNA TSP-Au NF-modified SPEs and a PDMS layer with designed dense meandering microchannels. Finally, the µFEC detection system was achieved based on microfluidic chips integrated with the liquid automatic conveying unit and electrochemical detection platform. The µFEC system we developed acquired great detection performance for PSA detection in PBS solution. For PSA assays in spiked serum samples of the µFEC system, we obtained a linear dynamic range of 1-100 ng/mL with a limit of detection of 0.2 ng/mL and a total reaction time <25 min. Real serum samples of prostate cancer patients presented a strong correlation between the "gold-standard" chemiluminescence assays and the µFEC system. In terms of operation procedure, cost, and reaction time, our method was superior to the current methods for PSA detection and shows great potential for practical clinical application in the future.

Sample-to-Answer Robotic ELISA.

Enzyme-linked immunosorbent assays (ELISA), as one of the most used immunoassays, have been conducted ubiquitously in hospitals, research laboratories, etc. However, the conventional ELISA procedure is usually laborious, occupies bulky instruments, consumes lengthy operation time, and relies considerably on the skills of technicians, and such limitations call for innovations to develop a fully automated ELISA platform. In this paper, we have presented a system incorporating a robotic-microfluidic interface (RoMI) and a modular hybrid microfluidic chip that embeds a highly sensitive nanofibrous membrane, referred to as the Robotic ELISA, to achieve human-free sample-to-answer ELISA tests in a fully programmable and automated manner. It carries out multiple bioanalytical procedures to replace the manual steps involved in classic ELISA operations, including the pneumatically driven high-precision pipetting, efficient mixing and enrichment enabled by back-and-forth flows, washing, and integrated machine vision for colorimetric readout. The Robotic ELISA platform has achieved a low limit of detection of 0.1 ng/mL in the detection of a low sample volume (15 µL) of chloramphenicol within 20 min without human intervention, which is significantly faster than that of the conventional ELISA procedure. Benefiting from its modular design and automated operations, the Robotic ELISA platform has great potential to be deployed for a broad range of detections in various resource-limited settings or high-risk environments, where human involvement needs to be minimized while the testing timeliness, consistency, and sensitivity are all desired.

Blink-sensing glasses: A flexible iontronic sensing wearable for continuous blink monitoring.

Blink reflex has long been considered closely related to physiological states, from which abundant information on ocular health and activities can be revealed. In this study, a smart glasses wearable has been developed, incorporating a flexible and sensitive pressure sensor, to monitor blink patterns by continuously detecting ocular muscular movements, referred to as blink-sensing glasses. By applying the emerging flexible iontronic sensing (FITS) sensor with the sensitivity of 340 pF/mmHg, the skin pressure variations induced by movements of the orbicularis oculi muscles can be monitored in real time. The blink-sensing glasses can successfully capture blink patterns with a high accuracy of 96.3% and have been used to differentiate the blink features from both dry-eye subjects and healthy controls. This device can be potentially used as a new clinical and research monitoring tool for continuous eye blink analysis, while providing patients with high comfortableness in long-term ambulatory and home settings.

Building protein networks in synthetic systems from the bottom-up.

The recent development of synthetic biology has expanded the capability to design and construct protein networks outside of living cells from the bottom-up. The new capability has enabled us to assemble protein networks for the basic study of cellular pathways, expression of proteins outside cells, and building tissue materials. Furthermore, the integration of natural and synthetic protein networks has enabled new functions of synthetic or artificial cells. Here, we review the underlying technologies for assembling protein networks in liposomes, water-in-oil droplets, and biomaterials from the bottom-up. We cover the recent applications of protein networks in biological transduction pathways, energy self-supplying systems, cellular environmental sensors, and cell-free protein scaffolds. We also review new technologies for assembling protein networks, including multiprotein purification methods, high-throughput assay screen platforms, and controllable fusion of liposomes. Finally, we present existing challenges towards building protein networks that rival the complexity and dynamic response akin to natural systems. This review addresses the gap in our understanding of synthetic and natural protein networks. It presents a vision towards developing smart and resilient protein networks for various biomedical applications.

iWRAP: A Theranostic Wearable Device With Real-Time Vital Monitoring and Auto-Adjustable Compression Level for Venous Thromboembolism.

OBJECTIVE: Venous Thromboembolism (VTE) is a commonly underdiagnosed disease with severe consequences and an exceedingly high mortality rate. Conventional compression wraps are devised for therapeutic purpose but lack diagnostic capacity. Recent advances in flexible electronics and wearable technologies offer many possibilities for chronic disease management. In particular, vital signs have been studied to show a strong correlation with the risk of VTE patients. In this study, we aim to develop an intelligent theranostic compression device, referred to as iWRAP, with the built-in capacity of real-time vital sign monitoring together with auto-adjustable compression level. METHODS: An instantaneous pneumatic feedback control with a high-resolution pressure sensor is integrated to provide a highly stabilized compression level at the prescribed interface pressure for an improved therapeutic outcome. Meanwhile, arterial pulse waveforms extracted from the pressure readings from the smart compression device can be utilized to derive the body vital signs, including heart rate (HR), respiratory rate (RR) and blood pressure (BP). RESULTS: A reliable delivery of the targeted compression level within ±5% accuracy in the range of 20-60 mmHg has been achieved through the feedback of the interface pressure. Both HR and RR have been measured within clinical-grade accuracies. Moreover, BP estimated using an ALA model has been achieved at low compression levels, which is also within a clinical-acceptable accuracy. The acquired vital information has been instantaneously fit into the clinically acceptable criteria for life-threatening PE risk with timely assessments. CONCLUSION: The iWRAP has shown the potential to become the first theranostic wearable device with both continuous delivery of accurate and effective compression therapy and real-time monitoring of life-threatening conditions for VTE patients.