Efficient, High-Quality Engineering of Therapeutic Extracellular Vesicles on an Integrated Nanoplatform.

Engineered extracellular vesicles (EVs) have been recognized as important therapeutics for gene and cell therapy. To achieve clinically desired therapy, technologies for EV engineering have high demands on the efficacy in producing EVs and their qualities, which, however, remain challenging to conventional routes due to their limited control on therapeutic payload delivery, EV secretion, and extracellular microenvironments. Here, we report a nanoplatform (denoted as PURE) that enables efficient electro-transfection while stimulating cells to produce high-quality EVs carrying functional RNAs. PURE further employs an ammonium removal zone to maintain the physiological conditions of the extracellular microenvironment and an EV uptake zone that efficiently (87.1%) captures EVs in situ with porous hydrogels. The platform achieved about a 12-fold higher yield of engineered EVs and a 146-fold abundance of desired therapeutics compared to those naturally secreted from cells. The PURE-engineered miR-130a-EVs were validated for effectively upregulating the mTOR signaling pathway in both in vitro and in vivo. Their therapeutic capability was then verified by enhancing the in vitro activation of primordial follicles. In vivo applications further highlighted the therapeutic effects of miR-130a-EVs in restoring ovary function in aged mice. The PURE platform represents a strategy for the clinical translation of EV-mediated therapy.

Microdroplet-enhanced chip platform for high-throughput immunotherapy marker screening from extracellular vesicle RNAs and membrane proteins.

Extracellular vesicles (EVs) are considered as promising candidates for predicting patients who respond to immunotherapy. Nevertheless, simultaneous detection of multiple EVs markers still presents significant technical challenges. In this work, we developed a high-throughput microdroplet-enhanced chip (MEC) platform, which utilizes thousands of individual microchambers (∼pL) as reactors, accelerating the detection efficiency of the CRISPR/Cas systems and increasing the sensitivity by up to 100-fold (aM level). Ten biomarkers (including 5 RNAs and 5 proteins) from patients' EVs are successfully detected on one chip, and the comprehensive markers show increased accuracy (AUC 0.911) than the individual marker for the efficacy prediction of immunotherapy. This platform provides a high-throughput yet sensitive strategy for screening immunotherapy markers in clinical.

Recent Progress in DNA Biosensors for Detecting Biomarkers in Living Cells.

Analysis of biomarkers in living cells is crucial for deciphering the dynamics of cells as well as for precise diagnosis of diseases. DNA biosensors employ DNA sequences as probes to offer insights into living cells, and drive progress in disease diagnosis and drug development. In this review, we present recent advances in DNA biosensors for detecting biomarkers in living cells. The basic structural components of DNA biosensors and the signal output method are presented. The strategies of DNA biosensors crossing the cell membrane are also described, including coincubation, nanocarriers, and nanoelectroporation techniques. Based on biomarker categorization, we detail recent applications of DNA biosensors for detecting small molecules, RNAs, proteins, and integrated targets in living cells. Furthermore, the future development directions of DNA biosensors are summarized to encourage further research in this growing field.

A Degradable Bioelectronic Scaffold for Localized Cell Transfection toward Enhancing Wound Healing in a 3D Space.

Large skin wounds, with extensive surface area and deep vertical full-thickness involvement, can pose significant challenges in clinical settings. Traditional routes for repairing skin wounds encompass three hallmarks: 1) scab formation for hemostasis; 2) proliferation and migration of epidermal cells for wound closure; 3) proliferation, migration, and functionalization of fibroblasts and endothelial cells for dermal remodeling. However, this route face remarkable challenges to healing large wounds, usually leading to disordered structures and loss of functions in the regenerated skin, due to limited control on the transition among the three stages. In this work, an implantable bioelectronics is developed that enables the synchronization of the three stages, offering accelerated and high-quality healing of large skin wounds. The system efficiently electro-transfect local cells near the wounds, forcing cellular proliferation, while providing a 3D porous environments for synchronized migration of epidermal and dermal cells. In vivo experiments demonstrated that the system achieved synchronous progression of multiple layers within the wounds, leading to the reconstruction of a complete skin structure similar to healthy skin, which presents a new avenue for the clinical translation of large wound healing.

Temporal compressive edge imaging enabled by a lensless diffuser camera.

Lensless imagers based on diffusers or encoding masks enable high-dimensional imaging from a single-shot measurement and have been applied in various applications. However, to further extract image information such as edge detection, conventional post-processing filtering operations are needed after the reconstruction of the original object images in the diffuser imaging systems. Here, we present the concept of a temporal compressive edge detection method based on a lensless diffuser camera, which can directly recover a time sequence of edge images of a moving object from a single-shot measurement, without further post-processing steps. Our approach provides higher image quality during edge detection, compared with the "conventional post-processing method." We demonstrate the effectiveness of this approach by both numerical simulation and experiments. The proof-of-concept approach can be further developed with other image post-processing operations or versatile computer vision assignments toward task-oriented intelligent lensless imaging systems.

Advanced micro/nano-electroporation for gene therapy: recent advances and future outlook.

Gene therapy is a promising disease treatment approach by editing target genes, and thus plays a fundamental role in precision medicine. To ensure gene therapy efficacy, the effective delivery of therapeutic genes into specific cells is a key challenge. Electroporation utilizes short electric pulses to physically break the cell membrane barrier, allowing gene transfer into the cells. It dodges the off-target risks associated with viral vectors, and also stands out from other physical-based gene delivery methods with its high-throughput and cargo-accelerating features. In recent years, with the help of advanced micro/nanotechnology, micro/nanostructure-integrated electroporation (micro/nano-electroporation) techniques and devices have significantly improved cell viability, transfection efficiency and dose controllability of the electroporation strategy, enhancing its application practicality especially in vivo. This technical advancement makes micro/nano-electroporation an effective and versatile tool for gene therapy. In this review, we first introduce the evolution of electroporation technique with a brief explanation of the perforation mechanism, and then provide an overview of the recent advancements and prospects of micro/nano-electroporation technology in the field of gene therapy. To comprehensively showcase the latest developments of micro/nano-electroporation technology in gene therapy, we focus on discussing micro/nano-electroporation devices and current applications at both in vitro and in vivo levels. Additionally, we outline the ongoing clinical studies of gene electrotransfer (GET), revealing the tremendous potential of electroporation-based gene delivery in disease treatment and healthcare. Lastly, the challenges and future directions in this field are discussed.

Genetic and phenotypic profiling of single living circulating tumor cells from patients with microfluidics.

Accurate prediction of the efficacy of immunotherapy for cancer patients through the characterization of both genetic and phenotypic heterogeneity in individual patient cells holds great promise in informing targeted treatments, and ultimately in improving care pathways and clinical outcomes. Here, we describe the nanoplatform for interrogating living cell host-gene and (micro-)environment (NICHE) relationships, that integrates micro- and nanofluidics to enable highly efficient capture of circulating tumor cells (CTCs) from blood samples. The platform uses a unique nanopore-enhanced electrodelivery system that efficiently and rapidly integrates stable multichannel fluorescence probes into living CTCs for in situ quantification of target gene expression, while on-chip coculturing of CTCs with immune cells allows for the real-time correlative quantification of their phenotypic heterogeneities in response to immune checkpoint inhibitors (ICI). The NICHE microfluidic device provides a unique ability to perform both gene expression and phenotypic analysis on the same single cells in situ, allowing us to generate a predictive index for screening patients who could benefit from ICI. This index, which simultaneously integrates the heterogeneity of single cellular responses for both gene expression and phenotype, was validated by clinically tracing 80 non-small cell lung cancer patients, demonstrating significantly higher AUC (area under the curve) (0.906) than current clinical reference for immunotherapy prediction.

Combined Amniotic Membrane and Self-Powered Electrical Stimulator Bioelectronic Dress Promotes Wound Healing.

Human amniotic membranes (hAMs) are widely used as wound management biomaterials, especially as grafts for corneal reconstruction due to the structure of the extracellular matrix and excellent biological properties. However, their fragile nature and rapid degradation rate hinder widespread clinical use. In this work, we engineered a novel self-powered electronic dress (E-dress), combining the beneficial properties of an amniotic membrane and a flexible electrical electrode to enhance wound healing. The E-dress displayed a sustained discharge capacity, leading to increased epidermal growth factor (EGF) release from amniotic mesenchymal interstitial stem cells. Live/dead staining, CCK-8, and scratch-wound-closure assays were performed in vitro. Compared with amniotic membrane treatment alone, the E-dress promoted cell proliferation and migration of mouse fibroblast cells and lower cytotoxicity. In a mouse full-skin defect model, the E-dress achieved significantly accelerated wound closure. Histological analysis revealed that E-dress treatment promoted epithelialization and neovascularization in mouse skin. The E-dress exhibited a desirable flexibility that aligned with tissue organization and displayed maximum bioactivity within a short period to overcome rapid degradation, implying great potential for clinical applications.

Rapid Cryptococcus electroporated-lysis and sensitive detection on a miniaturized platform.

Fast and accurate detection of Cryptococcus and precise differentiation of its subtypes is of great significance in protecting people from cryptococcal disease and preventing its spread in populations. However, traditional Cryptococcus identification and detection techniques still face significant challenges in achieving high analysis speed as well as high sensitivity. In this work, we report an electric microfluidic biochip. Compared to conventional methods that take several hours or even a day, this chip can detect Cryptococcus within 20 min, and achieve its maximum detection limit within 1 h, with the ability to differentiate between the Cryptococcus neoformans (NEO) and rare Cryptococcus gattii (GAT) efficiently, which accounts for nearly 100%. This device integrated two functional zones of an electroporation lysis (EL) zone for rapid cell lysis (<30 s) and an electrochemical detection (ED) zone for sensitive analysis of the released nucleic acids. The EL zone adopted a design of microelectrode arrays, which obtains a large electric field intensity at the constriction of the microchannel, addressing the safety concerns associated with high-voltage lysis. The device enables a limit of detection (LOD) of 60 pg/mL for NEO and 100 pg/mL for GAT through the modification of nanocomposites and specific probes. In terms of the detection time and sensitivity, the integrated microfluidic biochip demonstrates broad potential in Cryptococcus diagnosis and disease prevention.

Battery-free, wireless, and electricity-driven soft swimmer for water quality and virus monitoring.

Miniaturized mobile electronic system is an effective candidate for in situ exploration of confined spaces. However, realizing such system still faces challenges in powering issue, untethered mobility, wireless data acquisition, sensing versatility, and integration in small scales. Here, we report a battery-free, wireless, and miniaturized soft electromagnetic swimmer (SES) electronic system that achieves multiple monitoring capability in confined water environments. Through radio frequency powering, the battery-free SES system demonstrates untethered motions in confined spaces with considerable moving speed under resonance. This system adopts soft electronic technologies to integrate thin multifunctional bio/chemical sensors and wireless data acquisition module, and performs real-time water quality and virus contamination detection with demonstrated promising limits of detection and high sensitivity. All sensing data are transmitted synchronously and displayed on a smartphone graphical user interface via near-field communication. Overall, this wireless smart system demonstrates broad potential for confined space exploration, ranging from pathogen detection to pollution investigation.

An Ion Concentration Polarization Microplatform for Efficient Enrichment and Analysis of ctDNA.

Strategies for rapid, effective nucleic acid processing hold tremendous significance to the clinical analysis of circulating tumor DNA (ctDNA), a family of important markers indicating tumorigenesis and metastasis. However, traditional techniques remain challenging to achieve efficient DNA enrichment, further bringing about complicated operation and limited detection sensitivity. Here, we developed an ion concentration polarization microplatform that enabled highly rapid, efficient enrichment and purification of ctDNA from a variety of clinical samples, including serum, urine, and feces. The platform demonstrated efficiently separating and enriching ctDNA within 30 s, with a 100-fold improvement over traditional methods. Integrating an on-chip isothermal amplification module, the platform further achieved 100-fold enhanced sensitivity in ctDNA detection, which significantly eliminated false-negative results in the serum or urine samples due to the low abundance of ctDNA. Such a simple-designed platform offers a user-friendly yet powerful diagnosis technique with a wide applicability, ranging from early tumor diagnosis to infection screening.

Recent Advances in Bioelectronics for Localized Drug Delivery.

The last decade has witnessed remarkable advancements in bioelectronics, ushering in a new era of wearable and implantable devices for drug delivery. By utilizing miniaturized system design and/or flexible materials, bioelectronics illustrates ideal integration with target organs and tissues, making them ideal platforms for localized drug delivery. Furthermore, the development of electrically assisted drug delivery systems has enhanced the efficiency and safety of therapeutic administration, particularly for the macromolecules that encounter additional challenges in penetrating biological barriers. In this review, a concise overview of recent progress in bioelectronic devices for in vivo localized drug delivery, with highlights on the latest trends in device design, working principles, and their corresponding functionalities, is provided. The reported systems based on their targeted delivery locations as wearable systems, ingestible systems, and implantable systems are categorized. Each category is introduced in detail by highlighting the special requirements for devices and the corresponding solutions. The remaining challenges in this field and future directions are also discussed.

A Nano-Electroporation-DNA Tensioner Platform Enhances Intracellular Delivery and Mechanical Analysis Toward Rapid Drug Assessment.

In vitro, drug assessment holds tremendous potential to success in novel drug development and precision medicine. Traditional techniques for drug assessment, however, face remarkable challenges to achieve high speed, as limited by incubation-based drug delivery (>several hours) and cell viability measurements (>1 d), which significantly compromise the efficacy in clinical trials. In this work, a nano-electroporation-DNA tensioner platform is reported that shortens the time of drug delivery to less than 3 s, and that of cellular mechanical force analysis to 30 min. The platform adopts a nanochannel structure to localize a safe electric field for cell perforation, while enhancing delivery speed by 103 times for intracellular delivery, as compared to molecular diffusion in coculture methods. The platform is further equipped with a DNA tensioner to detect cellular mechanical force for quantifying cell viability after drug treatment. Systematic head-to-head comparison, by analyzing FDA (food and drug administration)-approved drugs (paclitaxel, doxorubicin), demonstrated the platform with high speed, efficiency, and safety, showing a simple yet powerful tool for clinical drug screening and development.

A portable, integrated microfluidics for rapid and sensitive diagnosis of Streptococcus agalactiae in resource-limited environments.

Streptococcus agalactiae (Group B Streptococcus, GBS) has been the leading cause of infections in newborns. Rapid and accurate diagnosis of GBS in pregnant women is a deterministic strategy to prevent newborn infection. Conventional detection methods based on nucleic acid amplification assay have been applied in GBS diagnosis in central laboratories, with demonstrated high sensitivity. However, their heavy dependence on instrumentation and trained technicians forms remarkable obstacles to GBS detection in wide scenarios, including self-testing, and bedside-/community-screening. Furthermore, the structures of GBS bring about extra challenges to the nucleic acid extraction and purification. Novel GBS diagnosis platforms integrating sample processing, amplification, and read-out, are highly desired in clinical. Here, we report a portable, integrated microfluidics that enables rapid extraction of DNA from sampling swabs (<10 min), power-free DNA amplification (<30 min), and simple read-out in GBS detection. The platform works without an external pump, achieving rapid and highly efficient DNA extraction from clinical samples, with a significantly reduced time from 6 h to less than 50 min. Systematic clinical tests based on 47 patient samples validated the high performance of the platform, highlighted with a low limit of detection (LOD, 103 copies/ml), high sensitivity (100%), and specificity (100%). Head-to-head comparisons showed that the device improved the LOD by an order of magnitude than the traditional PCR method, showing a simple yet powerful POCT platform for home-/community-based testing towards GBS (and other pathogens) prevention in remote areas.

Intranasal mask for protecting the respiratory tract against viral aerosols.

The spread of many infectious diseases relies on aerosol transmission to the respiratory tract. Here we design an intranasal mask comprising a positively-charged thermosensitive hydrogel and cell-derived micro-sized vesicles with a specific viral receptor. We show that the positively charged hydrogel intercepts negatively charged viral aerosols, while the viral receptor on vesicles mediates the entrapment of viruses for inactivation. We demonstrate that when displaying matched viral receptors, the intranasal masks protect the nasal cavity and lung of mice from either severe acute respiratory syndrome coronavirus 2 or influenza A virus. With computerized tomography images of human nasal cavity, we further conduct computational fluid dynamics simulation and three-dimensional printing of an anatomically accurate human nasal cavity, which is connected to human lung organoids to generate a human respiratory tract model. Both simulative and experimental results support the suitability of intranasal masks in humans, as the likelihood of viral respiratory infections induced by different variant strains is dramatically reduced.

Wireless, battery-free, multifunctional integrated bioelectronics for respiratory pathogens monitoring and severity evaluation.

The rapid diagnosis of respiratory virus infection through breath and blow remains challenging. Here we develop a wireless, battery-free, multifunctional pathogenic infection diagnosis system (PIDS) for diagnosing SARS-CoV-2 infection and symptom severity by blow and breath within 110 s and 350 s, respectively. The accuracies reach to 100% and 92% for evaluating the infection and symptom severity of 42 participants, respectively. PIDS realizes simultaneous gaseous sample collection, biomarker identification, abnormal physical signs recording and machine learning analysis. We transform PIDS into other miniaturized wearable or portable electronic platforms that may widen the diagnostic modes at home, outdoors and public places. Collectively, we demonstrate a general-purpose technology for rapidly diagnosing respiratory pathogenic infection by breath and blow, alleviating the technical bottleneck of saliva and nasopharyngeal secretions. PIDS may serve as a complementary diagnostic tool for other point-of-care techniques and guide the symptomatic treatment of viral infections.

Sensitive, rapid detection of NCOA4-m6A towards precisely quantifying radiation dosage on a Cas13a-Microdroplet platform.

Precise quantification of low-dose ionizing radiation is of great significance in protecting people from damage caused by clinical radiotherapy or environmental radiation. Traditional techniques for detecting radiation, however, remain extreme challenges to achieve high sensitivity and speed in quantifying radiation dosage. In this work, we report a Cas13a-Microdroplet platform that enables sensitive detection of ultra-low doses of radiation (0.5 Gy vs. 1 Gy traditional) within 1 h. The micro-platform adopts an ideal, specific radiation-sensitive marker, m6A on NCOA4 gene (NCOA4-m6A) that was first reported in our recent work. Microfluidics of the platform generate uniform microdroplets that encapsulate a CRISPR/Cas13a detection system and NCOA4-m6A target from the whole RNA extraction, achieving 10-fold enhancement in sensitivity and significantly reduced limit of detection (LOD). Systematic mouse models and clinical patient samples demonstrated its superior sensitivity and LOD (0.5 Gy) than traditional qPCR, which show wide potentials in radiation tracking and damage protection.

Current research status of tumor cell biomarker detection.

With the annual increases in the morbidity and mortality rates of tumors, the use of biomarkers for early diagnosis and real-time monitoring of tumor cells is of great importance. Biomarkers used for tumor cell detection in body fluids include circulating tumor cells, nucleic acids, protein markers, and extracellular vesicles. Among them, circulating tumor cells, circulating tumor DNA, and exosomes have high potential for the prediction, diagnosis, and prognosis of tumor diseases due to the large amount of valuable information on tumor characteristics and evolution; in addition, in situ monitoring of telomerase and miRNA in living cells has been the topic of extensive research to understand tumor development in real time. Various techniques, such as enzyme-linked immunosorbent assays, immunoblotting, and mass spectrometry, have been widely used for the detection of these markers. Among them, the detection of tumor cell markers in body fluids based on electrochemical biosensors and fluorescence signal analysis is highly preferred because of its high sensitivity, rapid detection and portable operation. Herein, we summarize recent research progress in the detection of tumor cell biomarkers in body fluids using electrochemical and fluorescence biosensors, outline the current research status of in situ fluorescence monitoring and the analysis of tumor markers in living cells, and discuss the technical challenges for their practical clinical application to provide a reference for the development of new tumor marker detection methods.

Single-molecule RNA capture-assisted droplet digital loop-mediated isothermal amplification for ultrasensitive and rapid detection of infectious pathogens.

To minimize and control the transmission of infectious diseases, a sensitive, accurate, rapid, and robust assay strategy for application on-site screening is critical. Here, we report single-molecule RNA capture-assisted digital RT-LAMP (SCADL) for point-of-care testing of infectious diseases. Target RNA was captured and enriched by specific capture probes and oligonucleotide probes conjugated to magnetic beads, replacing laborious RNA extraction. Droplet generation, amplification, and the recording of results are all integrated on a microfluidic chip. In assaying commercial standard samples, quantitative results precisely corresponded to the actual concentration of samples. This method provides a limit of detection of 10 copies mL-1 for the N gene within 1 h, greatly reducing the need for skilled personnel and precision instruments. The ultrasensitivity, specificity, portability, rapidity and user-friendliness make SCADL a competitive candidate for the on-site screening of infectious diseases.

Sensitive Interrogation of Enhancer Activity in Living Cells on a Nanoelectroporation-Probing Platform.

Enhancers involved in the upregulation of multiple oncogenes play a fundamental role in tumorigenesis and immortalization. Exploring the activity of enhancers in living cells has emerged as a critical path to a deep understanding of cancer properties, further providing important clues to targeted therapy. However, identifying enhancer activity in living cells is challenging due to the double biological barriers of a cell cytoplasmic membrane and a nuclear membrane, limiting the sensitivity and responsiveness of conventional probing methods. In this work, we developed a nanoelectroporation-probing (NP) platform, which enables intranuclear probe delivery for sensitive interrogation of enhancer activity in living cells. The nanoelectroporation biochip achieved highly focused perforation of the cell cytoplasmic membrane and brought about additional driving force to expedite the delivery of probes into the nucleus. The probes targeting enhancer activity (named "PH probe") are programmed with a cyclic amplification strategy and enable an increase in the fluorescence signals over 100-fold within 1 h. The platform was leveraged to detect the activity of CCAT1 enhancers (CCAT1, colon cancer-associated transcript-1, a long noncoding RNA that functions in tumor invasion and metastasis) in cell samples from clinical lung cancer patients, as well as reveal the heterogeneity of enhancers among different patients. The observations may extend the linkages between enhancers and cancer cells while validating the robustness and reliability of the platform for the assay of enhancer activity. This platform will be a promising toolbox with wide applicable potential for the intranuclear study of living cells.