Stimulative piezoelectric nanofibrous scaffolds for enhanced small extracellular vesicle production in 3D cultures.

Small extracellular vesicles (sEVs) have great promise as effective carriers for drug delivery. However, the challenges associated with the efficient production of sEVs hinder their clinical applications. Herein, we report a stimulative 3D culture platform for enhanced sEV production. The proposed platform consists of a piezoelectric nanofibrous scaffold (PES) coupled with acoustic stimulation to enhance sEV production of cells in a 3D biomimetic microenvironment. Combining cell stimulation with a 3D culture platform in this stimulative PES enables a 49 fold increase in the production rate per cell with minimal deviations in particle size and protein composition compared with standard 2D cultures. We find that the enhanced sEV production is attributable to the activation and upregulation of crucial sEV production steps through the synergistic effect of stimulation and the 3D microenvironment. Moreover, changes in cell morphology lead to cytoskeleton redistribution through cell matrix interactions in the 3D cultures. This in turn facilitates intracellular EV trafficking, which impacts the production rate. Overall, our work provides a promising 3D cell culture platform based on piezoelectric biomaterials for enhanced sEV production. This platform is expected to accelerate the potential use of sEVs for drug delivery and broad biomedical applications.

RNA-based detection of genetically modified plants via current-voltage characteristic measurement.

The widespread adoption of genetically modified (GM) crops has escalated concerns about their safety and ethical implications, underscoring the need for efficient GM crop detection methods. Conventional detection methods, such as polymerase chain reaction, can be costly, lab-bound, and time-consuming. To overcome these challenges, we have developed RapiSense, a cost-effective, portable, and sensitive biosensor platform. This sensor generates a measurable voltage shift (0.1-1 V) in the system's current-voltage characteristics, triggered by an increase in membrane's negative charge upon hybridization of DNA/RNA targets with a specific DNA probe. Probes designed to identify the herbicide resistance gene hygromycin phosphotransferase show a detection range from ∼1 nM to ∼10 µM and can discriminate between complementary, non-specific, and mismatched nucleotide targets. The incorporation of a small membrane sensor to detect fragmented RNA samples substantially improve the platform's sensitivity. In this study, RapiSense has been effectively used to detect specific DNA and fragmented RNA in transgenic variants of Arabidopsis, sweet potato, and rice, showcasing its potential for rapid, on-site GM crop screening.

Detection of EGFR and its Activity State in Plasma CD63-EVs from Glioblastoma Patients: Rapid Profiling using an Anion Exchange Membrane Sensor.

We present a novel quantitative immunoassay for CD63 EVs (extracellular vesicles) and a constituent surface cargo, EGFR and its activity state, that provides a sensitive, selective, fluorophore-free and rapid alternative to current EV-based diagnostic methods. Our sensing design utilizes a charge-gating strategy, with a hydrophilic anion exchange membrane and a charged silica nanoparticle reporter. With sensitivity and robustness enhancement by the ion-depletion action of the membrane, this hydrophilic design with charged reporters minimizes interference from dispersed proteins and fluorophore degradation, thus enabling direct plasma analysis. With a limit of detection of 30 EVs/µL and a high relative sensitivity of 0.01% for targeted proteomic subfractions, our assay enables accurate quantification of the EV marker, CD63, with colocalized EGFR by an operator/sample insensitive universal normalized calibration. Glioblastoma necessitates improved non-invasive diagnostic approaches for early detection and monitoring. Notably, we target both total and "active" EGFR on EVs; with a monoclonal antibody mAb806 that recognizes a normally hidden epitope on overexpressed or mutant variant III EGFR. This approach offers direct glioblastoma detection from untreated human patient samples. Analysis of glioblastoma clinical samples yielded an area-under-the-curve (AUC) value of 0.99 and low p-value of 0.000033, significantly surpassing the performance of existing assays and markers.

Chiral Graphene Quantum Dots Enhanced Drug Loading into Small Extracellular Vesicles.

As nanoscale extracellular vesicles secreted by cells, small extracellular vesicles (sEVs) have enormous potential as safe and effective vehicles to deliver drugs into lesion locations. Despite promising advances with sEV-based drug delivery systems, there are still challenges to drug loading into sEVs, which hinder the clinical applications of sEVs. Herein, we report an exogenous drug-agnostic chiral graphene quantum dots (GQDs) sEV-loading platform, based on chirality matching with the sEV lipid bilayer. Both hydrophobic and hydrophilic chemical and biological drugs can be functionalized or adsorbed onto GQDs by π-π stacking and van der Waals interactions. By tuning the ligands and GQD size to optimize its chirality, we demonstrate drug loading efficiency of 66.3% and 64.1% for doxorubicin and siRNA, which is significantly higher than other reported sEV loading techniques.

Fractal dimension to characterize interactions between blood and lymphatic endothelial cells.

Spatial patterning of different cell types is crucial for tissue engineering and is characterized by the formation of sharp boundary between segregated groups of cells of different lineages. The cell-cell boundary layers, depending on the relative adhesion forces, can result in kinks in the border, similar to fingering patterns between two viscous partially miscible fluids which can be characterized by its fractal dimension. This suggests that mathematical models used to analyze the fingering patterns can be applied to cell migration data as a metric for intercellular adhesion forces. In this study, we develop a novel computational analysis method to characterize the interactions between blood endothelial cells (BECs) and lymphatic endothelial cells (LECs), which form segregated vasculature by recognizing each other through podoplanin. We observed indiscriminate mixing with LEC-LEC and BEC-BEC pairs and a sharp boundary between LEC-BEC pair, and fingering-like patterns with pseudo-LEC-BEC pairs. We found that the box counting method yields fractal dimension between 1 for sharp boundaries and 1.3 for indiscriminate mixing, and intermediate values for fingering-like boundaries. We further verify that these results are due to differential affinity by performing random walk simulations with differential attraction to nearby cells and generate similar migration pattern, confirming that higher differential attraction between different cell types result in lower fractal dimensions. We estimate the characteristic velocity and interfacial tension for our simulated and experimental data to show that the fractal dimension negatively correlates with capillary number (Ca), further indicating that the mathematical models used to study viscous fingering pattern can be used to characterize cell-cell mixing. Taken together, these results indicate that the fractal analysis of segregation boundaries can be used as a simple metric to estimate relative cell-cell adhesion forces between different cell types.

A Scalable High-Throughput Isoelectric Fractionation Platform for Extracellular Nanocarriers: Comprehensive and Bias-Free Isolation of Ribonucleoproteins from Plasma, Urine, and Saliva.

Extracellular nanocarriers (extracellular vesicles (EVs), lipoproteins, and ribonucleoproteins) of protein and nucleic acids mediate intercellular communication and are clinically adaptable as distinct circulating biomarkers. However, the overlapping size and density of the nanocarriers have so far prevented their efficient physical fractionation, thus impeding independent downstream molecular assays. Here, we report a bias-free high-throughput and high-yield continuous isoelectric fractionation nanocarrier fractionation technique based on their distinct isoelectric points. This nanocarrier fractionation platform is enabled by a robust and tunable linear pH profile provided by water-splitting at a bipolar membrane and stabilized by flow without ampholytes. The linear pH profile that allows easy tuning is a result of rapid equilibration of the water dissociation reaction and stabilization by flow. The platform is automated with a machine learning procedure to allow recalibration for different physiological fluids and nanocarriers. The optimized technique has a resolution of 0.3 ΔpI, sufficient to separate all nanocarriers and even subclasses of nanocarriers. Its performance is then evaluated with several biofluids, including plasma, urine, and saliva samples. Comprehensive, high-purity (plasma: >93%, urine: >95% and saliva: >97%), high-yield (plasma: >78%, urine: >87% and saliva: >96%), and probe-free isolation of ribonucleoproteins in 0.75 mL samples of various biofluids in 30 min is demonstrated, significantly outperforming affinity-based and highly biased gold standards having low yield and day-long protocols. Binary fractionation of EVs and different lipoproteins is also achieved with similar performance.

Quantifying PON1 on HDL with nanoparticle-gated electrokinetic membrane sensor for accurate cardiovascular risk assessment.

Cardiovascular disease-related deaths (one-third of global deaths) can be reduced with a simple screening test for better biomarkers than the current lipid and lipoprotein profiles. We propose using a highly atheroprotective subset of HDL with colocalized PON1 (PON1-HDL) for superior cardiovascular risk assessment. However, direct quantification of HDL proteomic subclasses are complicated by the peroxides/antioxidants associated with HDL interfering with redox reactions in enzymatic calorimetric and electrochemical immunoassays. Hence, we developed an enzyme-free Nanoparticle-Gated Electrokinetic Membrane Sensor (NGEMS) platform for quantification of PON1-HDL in plasma within 60 min, with a sub-picomolar limit of detection, 3-4 log dynamic range and without needing sample pretreatment or individual-sample calibration. Using NGEMS, we report our study on human plasma PON1-HDL as a cardiovascular risk marker with AUC~0.99 significantly outperforming others (AUC~0.6-0.8), including cholesterol/triglycerides tests. Validation for a larger cohort can establish PON1-HDL as a biomarker that can potentially reshape cardiovascular landscape.

A home-made pipette droplet microfluidics rapid prototyping and training kit for digital PCR, microorganism/cell encapsulation and controlled microgel synthesis.

Droplet microfluidics offers a platform from which new digital molecular assay, disease screening, wound healing and material synthesis technologies have been proposed. However, the current commercial droplet generation, assembly and imaging technologies are too expensive and rigid to permit rapid and broad-range tuning of droplet features/cargoes. This rapid prototyping bottleneck has limited further expansion of its application. Herein, an inexpensive home-made pipette droplet microfluidics kit is introduced. This kit includes elliptical pipette tips that can be fabricated with a simple DIY (Do-It-Yourself) tool, a unique tape-based or 3D printed shallow-center imaging chip that allows rapid monolayer droplet assembly/immobilization and imaging with a smart-phone camera or miniature microscope. The droplets are generated by manual or automatic pipetting without expensive and lab-bound microfluidic pumps. The droplet size and fluid viscosity/surface tension can be varied significantly because of our particular droplet generation, assembly and imaging designs. The versatility of this rapid prototyping kit is demonstrated with three representative applications that can benefit from a droplet microfluidic platform: (1) Droplets as microreactors for PCR reaction with reverse transcription to detect and quantify target RNAs. (2) Droplets as microcompartments for spirulina culturing and the optical color/turbidity changes in droplets with spirulina confirm successful photosynthetic culturing. (3) Droplets as templates/molds for controlled synthesis of gold-capped polyacrylamide/gold composite Janus microgels. The easily fabricated and user-friendly portable kit is hence ideally suited for design, training and educational labs.

Chiral Graphene Quantum Dots Enhanced Drug Loading into Exosomes.

As nanoscale extracellular vesicles secreted by cells, exosomes have enormous potential as safe and effective vehicles to deliver drugs into lesion locations. Despite promising advances with exosome-based drug delivery systems, there are still challenges to drug loading into exosome, which hinder the clinical applications of exosomes. Herein, we report an exogenous drug-agnostic chiral graphene quantum dots (GQDs) exosome-loading platform, based on chirality matching with the exosome lipid bilayer. Both hydrophobic and hydrophilic chemical and biological drugs can be functionalized or adsorbed onto GQDs by π-π stacking and van der Waals interactions. By tuning the ligands and GQD size to optimize its chirality, we demonstrate drug loading efficiency of 66.3% and 64.1% for Doxorubicin and siRNA, which is significantly higher than other reported exosome loading techniques.

An integrated ion-exchange membrane-based microfluidic device for irreversible dissociation and quantification of miRNA from ribonucleoproteins.

Ribonucleoproteins (RNPs), particularly microRNA-induced silencing complex (miRISC), have been associated with cancer-related gene regulation. Specific RNA-protein associations in miRISC complexes or those found in let-7 lin28A complexes can downregulate tumor-suppressing genes and can be directly linked to cancer. The high protein-RNA electrostatic binding affinity is a particular challenge for the quantification of the associated microRNAs (miRNAs). We report here the first microfluidic point-of-care assay that allows direct quantification of RNP-associated RNAs, which has the potential to greatly advance RNP profiling for liquid biopsy. Key to the technology is an integrated cation-anion exchange membrane (CEM/AEM) platform for rapid and irreversible dissociation (k = 0.0025 s-1) of the RNP (Cas9-miR-21) complex and quantification of its associated miR-21 in 40 minutes. The CEM-induced depletion front is used to concentrate the RNP at the depletion front such that the high electric field (>100 V cm-1) within the concentration boundary layer induces irreversible dissociation of the low KD (∼0.5 nM) complex, with ∼100% dissociation even though the association rate (kon = 6.1 s-1) is 1000 times higher. The high field also electrophoretically drives the dissociated RNA out of the concentrated zone without reassociation. A detection limit of 1.1 nM is achieved for Cy3 labelled miR-21.

Electrodeposited magnetic nanoporous membrane for high-yield and high-throughput immunocapture of extracellular vesicles and lipoproteins.

Superparamagnetic nanobeads offer several advantages over microbeads for immunocapture of nanocarriers (extracellular vesicles, lipoproteins, and viruses) in a bioassay: high-yield capture, reduction in incubation time, and higher capture capacity. However, nanobeads are difficult to "pull-down" because their superparamagnetic feature requires high nanoscale magnetic field gradients. Here, an electrodeposited track-etched membrane is shown to produce a unique superparamagnetic nano-edge ring with multiple edges around nanopores. With a uniform external magnetic field, the induced monopole and dipole of this nano edge junction combine to produce a 10× higher nanobead trapping force. A dense nanobead suspension can be filtered through the magnetic nanoporous membrane (MNM) at high throughput with a 99% bead capture rate. The yield of specific nanocarriers in heterogeneous media by nanobeads/MNM exceeds 80%. Reproducibility, low loss, and concentration-independent capture rates are also demonstrated. This MNM material hence expands the application of nanobead immunocapture to physiological samples.

Phase 2 of extracellular RNA communication consortium charts next-generation approaches for extracellular RNA research.

The extracellular RNA communication consortium (ERCC) is an NIH-funded program aiming to promote the development of new technologies, resources, and knowledge about exRNAs and their carriers. After Phase 1 (2013-2018), Phase 2 of the program (ERCC2, 2019-2023) aims to fill critical gaps in knowledge and technology to enable rigorous and reproducible methods for separation and characterization of both bulk populations of exRNA carriers and single EVs. ERCC2 investigators are also developing new bioinformatic pipelines to promote data integration through the exRNA atlas database. ERCC2 has established several Working Groups (Resource Sharing, Reagent Development, Data Analysis and Coordination, Technology Development, nomenclature, and Scientific Outreach) to promote collaboration between ERCC2 members and the broader scientific community. We expect that ERCC2's current and future achievements will significantly improve our understanding of exRNA biology and the development of accurate and efficient exRNA-based diagnostic, prognostic, and theranostic biomarker assays.

Human Heart Anoxia and Reperfusion Tissue (HEART) Model for the Rapid Study of Exosome Bound miRNA Expression As Biomarkers for Myocardial Infarction.

Current biomarkers for myocardial infarction (MI) diagnosis are typically late markers released upon cell death, incapable of distinguishing between ischemic and reperfusion injury and can be symptoms of other pathologies. Circulating microRNAs (miRNAs) have recently been proposed as alternative biomarkers for MI diagnosis; however, detecting the changes in the human cardiac miRNA profile during MI is extremely difficult. Here, to study the changes in miRNA levels during acute MI, a heart-on-chip model with a cardiac channel, containing human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes in human heart decellularized matrix and collagen, and a vascular channel, containing hiPSC-derived endothelial cells, is developed. This model is exposed to anoxia followed by normoxia to mimic ischemia and reperfusion, respectively. Using a highly sensitive miRNA biosensor that the authors developed, the exact same increase in miR-1, miR-208b, and miR-499 levels in the MI-on-chip and the time-matched human blood plasma samples collected before and after ischemia and reperfusion, is shown. That the surface marker profile of exosomes in the engineered model changes in response to ischemic and reperfusion injury, which can be used as biomarkers to detect MI, is also shown. Hence, the MI-on-chip model developed here can be used in biomarker discovery.

Development of a Multi-target Protein Biomarker Assay for Circulating Tumor Cells.

We report a highly sensitive and selective CNT-switch liquid biopsy platform that detects and quantifies protein biomarker expressions from circulating tumor cells in blood for early detection of metastatic breast cancer and its relapse. This platform first isolates and enriches more than 99% of tumor cells with an off-chip micro-size membrane filtration technique and then conducts on-chip detection of the membrane and internal protein biomarkers of the tumor cells with high sensitivity and selectivity. High sensitivity is achieved with complete association of the antibody-antigen-antibody (Ab-Ag-Ab) complex by precisely and rapidly assembling carbon nanotubes (CNTs) across two parallel electrodes via sequential DC electrophoresis and dielectrophoresis (DEP) deposition. Each bridged CNT acts as a switch that connects the electrodes and closes the circuit to generate an electrical signal. The high selectivity is achieved with a critical hydrodynamic shear rate that irreversibly removes non-target linkers of the aligned CNTs. At present, we are able to detect the protein biomarkers from 5 spiked breast cancer tumor cells of different types within 7.5 ml of human blood samples. This demonstrates the potential of this platform as an inexpensive and noninvasive alternative to MRI scans and tissue biopsies currently used to detect early metastatic breast cancer and its relapse.

Nanoparticle-assisted detection of nucleic acids in a polymeric nanopore with a large pore size.

Rapid and accurate detection of nucleic acids is of paramount importance in many fields, including medical diagnosis, gene therapy and virus identification. In this work, by taking advantage of two DNA hybridization probes, one of which was immobilized on the surface of gold nanoparticles, while the other was free in solution, detection of short length nucleic acids was successfully achieved using a large size (20 nm tip diameter) polyethylene terephythalate (PET) nanopore. The sensor was sensitive and selective: DNA samples with concentrations at as low as 0.5 nM could be detected within minutes and the number of mismatches can be discerned from the translocation frequency. Furthermore, the nanopore can be repeatedly used many times. Our developed large-size nanopore sensing platform offers the potential for fieldable/point-of-care diagnostic applications.

Ion-Depleting Action of Perm-Selective Membranes for Enhancing Electrical Communication and Gated Ion Channel Activity in Cell Cultures.

Ion-depletion action of an ion-selective membrane produces a moat channel that electrically insulates a cell colony and elevates the cell medium potential uniformly to synchronously activate and deactivate the voltage-gated ion channels of all cells. The result is robust synchronization with strong intercellular electrical communication and the discovery of ion channel deactivation that is only possible when the cells are in communication. The study suggests that the collective response of a cell colony to external stimuli is distinct from that of a single cell. Cell proliferation must hence be guided with strong intercellular communication and proper exogenous stimuli.

A multiplexed ion-exchange membrane-based miRNA (MIX·miR) detection platform for rapid diagnosis of myocardial infarction.

Micro RNAs (miRNAs) have shown great potential as rapid and discriminating biomarkers for acute myocardial infarction (AMI) diagnosis. We have developed a multiplexed ion-exchange membrane-based miRNA (MIX·miR) preconcentration/sensing amplification-free platform for quantifying in parallel a panel of miRNAs, including miR-1, miR-208b, and miR-499, from the same plasma samples from: 1) reference subjects with no evident coronary artery disease (NCAD); 2) subjects with stable coronary artery disease (CAD); and 3) subjects experiencing ST-elevation myocardial infarction (STEMI) prior to (STEMI-pre) and following (STEMI-PCI) percutaneous coronary intervention. The picomolar limit of detection from raw plasma and 3-decade dynamic range of MIX·miR permits detection of the miRNA panel in untreated samples from disease patients and its precise standard curve, provided by large 0.1 to 1 V signals and eliminates individual sensor calibration. The use of molecular concentration feature reduces the assay time to less than 30 minutes and increases the detection sensitivity by bringing all targets close to the sensors. miR-1 was low for NCAD patients but more than one order of magnitude above the normal value for all samples from three categories (CAD, STEMI-pre, and STEMI-PCI) of patients with CAD. In fact, miR-1 expression levels of stable CAD, STEMI-pre and STEMI-PCI are each more than 10-fold higher than the previous class, in that order, well above the 95% confidence level of MIX·miR. Its overexpression estimate is significantly higher than the PCR benchmark. This suggests that, in contrast to protein biomarkers of myocardial injury, miR-1 appears to differentiate ischemia from both reperfusion injury and non-AMI CAD patients. The battery-operated MIX·miR can be a portable and low-cost AMI diagnostic device, particularly useful in settings where cardiac catheterization is not readily available to determine the status of coronary reperfusion.

Correction: Constant-potential environment for activating and synchronizing cardiomyocyte colonies with on-chip ion-depleting perm-selective membranes.

Correction for 'Constant-potential environment for activating and synchronizing cardiomyocyte colonies with on-chip ion-depleting perm-selective membranes' by Vivek Yadav et al., Lab Chip, 2020, 20, 4273-4284, DOI: 10.1039/D0LC00809E.

Conformal single cell hydrogel coating with electrically induced tip streaming of an AC cone.

Encapsulation of single cells in a thin hydrogel provides a more precise control of stem cell niches and better molecular transport. Despite the recent advances in microfluidic technologies to allow encapsulation of single cells, existing methods rely on special crosslinking agents that are pre-coated on the cell surface and subject to the variation of the cell membrane, which limits their widespread adoption. This work reports a high-throughput single-cell encapsulation method based on the "tip streaming" mode of alternating current (AC) electrospray, with encapsulation efficiencies over 80% after tuned centrifugation. Dripping with multiple cells is curtailed due to gating by the sharp conic meniscus of the tip streaming mode that only allows one cell to be ejected at a time. Moreover, the method can be universally applied to both natural and synthetic hydrogels, as well as various cell types, including human multipotent mesenchymal stromal cells (hMSCs). Encapsulated hMSCs maintain good cell viability over an extended culture period and exhibit robust differentiation potential into osteoblasts and adipocytes. Collectively, electrically induced tip streaming enables high-throughput encapsulation of single cells with high efficiency and universality, which is applicable for various applications in cell therapy, pharmacokinetic studies, and regenerative medicine.

Elliptical Pipette Generated Large Microdroplets for POC Visual ddPCR Quantification of Low Viral Load.

Rapid point-of-care (POC) quantification of low virus RNA load would significantly reduce the turn-around time for the PCR test and help contain a fast-spreading epidemic. Herein, we report a droplet digital PCR (ddPCR) platform that can achieve this sensitivity and rapidity without bulky lab-bound equipment. The key technology is a flattened pipette tip with an elliptical cross-section, which extends a high aspect-ratio microfluidic chip design to pipette scale, for rapid (<5 min) generation of several thousand monodispersed droplets ∼150 to 350 µm in size with a CV of ∼2.3%. A block copolymer surfactant (polyoxyalkylene F127) is used to stabilize these large droplets in oil during thermal cycling. At this droplet size and number, positive droplets can be counted by eye or imaged by a smartphone with appropriate illumination/filtering to accurately quantify up to 100 target copies. We demonstrate with 2019 nCoV-PCR assay LODs of 3.8 copies per 20 µL of sample and a dynamic range of 4-100 copies. The ddPCR platform is shown to be inhibitor resistant with spiked saliva samples, suggesting RNA extraction may not be necessary. It represents a rapid 1.5-h POC quantitative PCR test that requires just a pipette equipped with elliptical pipette tip, a commercial portable thermal cycler, a smartphone, and a portable trans-illuminator, without bulky and expensive micropumps and optical detectors that prevent POC application.