Noncapillary Wave Dynamics due to Interfacial Coupling with Plasma Patterns at a Liquid Surface.

We identify a new class of surface waves that arise at a plasma-liquid interface due to resonant coupling between discrete plasma pattern modes and a continuum of interfacial liquid surface wave modes. A wave mode is selected due to localized excitation by the plasma, and standing waves result when waves excited from different locations interact. These waves propagate with a slower phase velocity than traditional capillary waves, but exhibit the same damping behavior with respect to liquid viscosity. Surface tension does not appear to play a significant role. We propose a curvature-dependent Maxwell pressure mechanism to explain these nondispersive interfacial waves in the presence of plasma.

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.

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.

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.

Liquid biopsy technologies based on membrane microfluidics: High-yield purification and selective quantification of biomarkers in nanocarriers.

Liquid biopsy, screening cancer non-invasively and frequently by detecting and quantifying molecular markers in physiological fluids, would significantly improve cancer survival rate but it remains a distant goal. The key obstacles presented by the highly heterogeneous samples are rapid/high-yield purification and precise/selective marker capture by their antibody and oligo probes. As irregular expressions of these molecular biomarkers are the key signals, quantifying only those from the cancer cells would greatly enhance the performance of the screening tests. The recent discovery that the biomarkers are carried by nanocarriers, such as exosomes, with cell-specific membrane proteins suggests that such selection may be possible, although a new suite of fractionation and quantification technologies would need to be developed. Although under-appreciated, membrane microfluidics has made considerable contributions to resolving these issues. We review the progress made so far, based on ion-selective, track-etched, and gel membranes and advanced electrophoretic and nano-filtration designs, in this perspective and suggest future directions.

Resistive amplitude fingerprints during translocation of linear molecules through charged solid-state nanopores.

We report the first analytical theory on the amplitude of resistive signals during molecular translocation through charged solid-state nanopores with variable cross-sectional area and piecewise-constant surface charge densities. By providing closed-form explicit algebraic expressions for the concentration profiles inside charged nanopores, this theory allows the prediction of baseline and translocation resistive signals without the need for numerical simulation of the electrokinetic phenomena. A transversely homogenized theory and an asymptotic expansion for weakly charged pores capture DC or quasi-static rectification due to field-induced intrapore concentration polarization (as a result of pore charge inhomogeneity or a translocating molecule). This theory, validated by simulations and experiments, is then used to explain why the amplitude of a single stranded DNA molecule can be twice as high as the amplitude of its double stranded counterpart. It also suggests designs for intrapore concentration polarization and volume exclusion effects that can produce biphasic and other amplitude fingerprints for high-throughput and yet discriminating molecular identification.

Simultaneous isolation and preconcentration of exosomes by ion concentration polarization.

Exosomes carry microRNA biomarkers, occur in higher abundance in cancerous patients than in healthy ones, and because they are present in most biofluids, including blood and urine, these can be obtained noninvasively. Standard laboratory techniques to isolate exosomes are expensive, time consuming, provide poor purity, and recover on the order of 25% of the available exosomes. We present a new microfluidic technique to simultaneously isolate exosomes and preconcentrate them by electrophoresis using a high transverse local electric field generated by ion-depleting ion-selective membrane. We use pressure-driven flow to deliver an exosome sample to a microfluidic chip such that the transverse electric field forces them out of the cross flow and into an agarose gel which filters out unwanted cellular debris while the ion-selective membrane concentrates the exosomes through an enrichment effect. We efficiently isolated exosomes from 1× PBS buffer, cell culture media, and blood serum. Using flow rates from 150 to 200 µL/h and field strengths of 100 V/cm, we consistently captured between 60 and 80% of exosomes from buffer, cell culture media, and blood serum as confirmed by both fluorescence spectroscopy and nanoparticle tracking analysis. Our microfluidic chip maintained this recovery rate for more than 20 min with a concentration factor of 15 for 10 min of isolation.

Acceleration of DNA melting kinetics using alternating electric fields.

We verify both theoretically and by simulation that an AC electric field, with a frequency much higher than the dissociation rate, can significantly accelerate the dissociation rate of biological molecules under isothermal conditions. The cumulative effect of the AC field is shown to break a key bottleneck by reducing the entropy (and increasing the free energy of the local minimum) via the alignment of the molecular dipole with the field. For frequencies below a resonant frequency which corresponds to the inverse Debye dipole relaxation time, the dissociation rate can be accelerated by a factor that scales as ω(ϵ'(ω)-1)E02 , where ω is the field frequency, E0 is the field amplitude, and ϵ'(ω) is the frequency-dependent real permittivity of the molecule. At large amplitudes, we find that the accelerated melting rate becomes universal, independent of duplex size and sequence, which is in drastic contrast to Ohmic thermal melting. We confirm our theory with isothermal all-atomic molecular dynamics simulation of short DNA duplexes with known melting rates, demonstrating several orders in enhancement with realistic fields.

Selectivity enhancements in gel-based DNA-nanoparticle assays by membrane-induced isotachophoresis: thermodynamics versus kinetics.

Selectivity against mutant nontargets with a few mismatches remains challenging in nucleic acid sensing. Sensitivity enhancement by analyte concentration does not improve selectivity because it affects targets and nontargets equally. Hydrodynamic or electrical shear enhanced selectivity is often accompanied by substantial losses in target signals, thereby leading to poor limits of detection. We introduce a platform based on depletion isotachophoresis in agarose gel generated by an ion-selective membrane that allows both selectivity and sensitivity enhancement with a two-step assay involving concentration polarization at an ion-selective membrane. By concentrating both the targets and probe-functionalized nanoparticles by ion enrichment at the membrane, the effective thermodynamic dissociation constant is lowered from 40 nM to below 500 pM, and the detection limit is 10 pM as reported previously. A dynamically optimized ion depletion front is then generated from the membrane with a high electrical shear force to selectively and irreversibly dehybridize nontargets. The optimized selectivity against a two-mismatch nontarget (in a 35-base pairing sequence) is shown to be better than the thermodynamic equilibrium selectivity by more than a hundred-fold, such that there is no detectable signal from the two-mismatch nontarget. We offer empirical evidence that irreversible cooperative dehybridization plays an important role in this kinetic selectivity enhancement and that mismatch location controls the optimum selectivity even when there is little change in the corresponding thermodynamic dissociation constant.

Kinetic theory for DNA melting with vibrational entropy.

By treating DNA as a vibrating nonlinear lattice, an activated kinetic theory for DNA melting is developed to capture the breakage of the hydrogen bonds and subsequent softening of torsional and bending vibration modes. With a coarse-grained lattice model, we identify a key bending mode with GHz frequency that replaces the hydrogen vibration modes as the dominant out-of-phase phonon vibration at the transition state. By associating its bending modulus to a universal in-phase bending vibration modulus at equilibrium, we can hence estimate the entropic change in the out-of-phase vibration from near-equilibrium all-atom simulations. This and estimates of torsional and bending entropy changes lead to the first predictive and sequence-dependent theory with good quantitative agreement with experimental data for the activation energy of melting of short DNA molecules without intermediate hairpin structures.

Universal Scaling of Robust Thermal Hot Spot and Ionic Current Enhancement by Focused Ohmic Heating in a Conic Nanopore.

A stable nanoscale thermal hot spot, with temperature approaching 100 °C, is shown to be sustained by localized Ohmic heating of a focused electric field at the tip of a slender conic nanopore. The self-similar (length-independent) conic geometry allows us to match the singular heat source at the tip to the singular radial heat loss from the slender cone to obtain a self-similar steady temperature profile along the cone and the resulting ionic current conductance enhancement due to viscosity reduction. The universal scaling, which depends only on a single dimensionless parameter Z, collapses the measured conductance data and computed temperature profiles in ion-track conic nanopores and conic nanopipettes. The collapsed numerical data reveal universal values for the hot-spot location and temperature in an aqueous electrolyte.

Fluidic-Based Ion Memristors and Ionic Latches.

The formation of a nanoscale anodic silicon oxide layer on silicon electrodes in an aqueous environment leads to fluidic-based ionic memristive devices and ionic latches for large integrated fluidic ion logic circuitry, which can enable massively multiplexed smart biosensor arrays and complex active chemical circuits.

Universal low-frequency asymptotes of dynamic conic nanopore rectification: An ionic nanofluidic inductor.

We report the first nanofluidic inductor (L) to complement the known nanofluidic capacitors (C), resistors (R), and diodes for ion currents. Under negative bias, the nanopore behaves like a parallel RC circuit at low frequencies; however, under positive bias, the asymptotic dynamics is that of a serial RL circuit. This new ionic circuit element can lead to nanofluidic RLC or diode-inductor oscillator circuits and new intrapore biosensing/rapid sequencing strategies. A universal theory, with explicit estimates for the capacitance and inductance at opposite biases, is derived to collapse the rectified dynamics of all conic nanopores to facilitate design of this new nanofluidic circuit.

Ion current rectification in funnel-shaped nanochannels: Hysteresis and inversion effects.

Ion current rectification inversion is observed in a funnel-shaped nanochannel above a threshold voltage roughly corresponding to the under-limiting to over-limiting current transition. Previous experimental studies have examined rectification at either low-voltages (under-limiting current region) for conical nanopores/funnel-shaped nanochannels or at high-voltages (over-limiting region) for straight nanochannels with asymmetric entrances or asymmetric interfacing microchannels. The observed rectification inversion occurs because the system resistance is shifted, beyond a threshold voltage, from being controlled by intra-channel ion concentration-polarization to that controlled by external concentration-polarization. Additionally, strong hysteresis effects, due to residual concentration-polarization, manifest themselves through the dependence of the transient current rectification on voltage scan rate.

Extracellular vesicle and lipoprotein diagnostics (ExoLP-Dx) with membrane sensor: A robust microfluidic platform to overcome heterogeneity.

The physiological origins and functions of extracellular vesicles (EVs) and lipoproteins (LPs) propel advancements in precision medicine by offering non-invasive diagnostic and therapeutic prospects for cancers, cardiovascular, and neurodegenerative diseases. However, EV/LP diagnostics (ExoLP-Dx) face considerable challenges. Their intrinsic heterogeneity, spanning biogenesis pathways, surface protein composition, and concentration metrics complicate traditional diagnostic approaches. Commonly used methods such as nanoparticle tracking analysis, enzyme-linked immunosorbent assay, and nuclear magnetic resonance do not provide any information about their proteomic subfractions, including active proteins/enzymes involved in essential pathways/functions. Size constraints limit the efficacy of flow cytometry for small EVs and LPs, while ultracentrifugation isolation is hampered by co-elution with non-target entities. In this perspective, we propose a charge-based electrokinetic membrane sensor, with silica nanoparticle reporters providing salient features, that can overcome the interference, long incubation time, sensitivity, and normalization issues of ExoLP-Dx from raw plasma without needing sample pretreatment/isolation. A universal EV/LP standard curve is obtained despite their heterogeneities.

A Mem-dELISA platform for dual color and ultrasensitive digital detection of colocalized proteins on extracellular vesicles.

Accurate, multiplex, and ultrasensitive measurement of different colocalized protein markers on individual tumor-derived extracellular vesicles (EVs) and dimerized proteins with multiple epitopes could provide insights into cancer heterogeneity, therapy management and early diagnostics that cannot be extracted from bulk methods. However, current digital protein assays lack certain features to enable robust colocalization, including multi-color detection capability, large dynamic range, and selectivity against background proteins. Here, we report a lithography-free, inexpensive (< $0.1) and ultrasensitive dual-color Membrane Digital ELISA (Mem-dELISA) platform by using track-etched polycarbonate (PCTE) membranes to overcome these shortcomings. Their through-pores remove air bubbles through wicking before they are sealed on one side by adhesion to form microwells. Immunomagnetic bead-analyte complexes and substrate solution are then loaded into the microwells from the opposite side, with >80% loading efficiency, before sealing with oil. This enables duplex digital protein colorimetric assay with beta galactosidase and alkaline phosphatase enzymes. The platform achieves 5 logs of dynamic range with a limit of detection of 10 aM for both Biotinylated ß-galactosidase (B-ßG) and Biotin Alkaline Phosphatase Conjugated (B-ALP) proteins. We demonstrate its potential by showing that a higher dosage of paclitaxel suppresses EpCAM-positive EVs but not GPC-1 positive EVs from breast cancer cells, a decline in chemo-resistance that cannot be detected with Western blot analysis of cell lysate. The Mem-dELISA is poised to empower researchers to conduct ultrasensitive, high throughput protein colocalization studies for disease diagnostics, treatment monitoring and biomarker discovery.

Immunojanus Particles for low-volume and isolation-free unlabeled characterization of small Extracellular Vesicle in biofluids: Characterization of disease type by surface marker profiling.

Small extracellular vesicles (sEVs) are vital for cellular communication and serve as critical biomarker carriers for diseases such as cancer. However, quantifying and profiling sEV surface markers presents significant challenges due to the low concentration of specific sEV-bound proteins and interference by more abundant dispersed proteins. This paper presents Immunojanus Particles (IJPs), a new method that enables the direct detection of sEVs in less than an hour without isolation. The design of IJPs incorporates fluorescent and non-fluorescent halves, utilizing rotational Brownian motion to detect captured sEVs through the change in the blinking rate, without interference from the smaller dispersed proteins. We demonstrate a detection limit of 2E5 sEVs/mL with low sample volumes and the capability to characterize sEVs directly from plasma, serum, cell culture media, and urine. In a small pilot study involving 87 subjects, including individuals with colorectal cancer, pancreatic ductal adenocarcinoma, glioblastoma, Alzheimer's disease, and healthy controls, our method accurately identified the type of disease with high 0.90-0.99 AUC in a blind setting. Compared with an orthogonal ultracentrifugation plus surface plasmon resonance (UC+SPR) method that requires about 24 hours, the sensitivity and dynamic range of IJP are better by 2 logs.

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.

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 15.7-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.

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 15.7-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.