Charge inversion, water splitting, and vortex suppression due to DNA sorption on ion-selective membranes and their ion-current signatures.

The physisorption of negatively charged single-stranded DNA (ssDNA) of different lengths onto the surface of anion-exchange membranes is sensitively shown to alter the anion flux through the membrane. At low surface concentrations, the physisorbed DNAs act to suppress an electroconvection vortex instability that drives the anion flux into the membrane and hence reduce the overlimiting current through the membrane. Beyond a critical surface concentration, determined by the total number of phosphate charges on the DNA, the DNA layer becomes a cation-selective membrane, and the combined bipolar membrane has a lower net ion flux, at low voltages, than the original membrane as a result of ion depletion at the junction between the cation- (DNA) and anion-selective membranes. However, beyond a critical voltage that is dependent on the ssDNA coverage, water splitting occurs at the junction to produce a larger overlimiting current than that of the original membrane. These two large opposite effects of polyelectrolyte counterion sorption onto membrane surfaces may be used to eliminate limiting current constraints of ion-selective membranes for liquid fuel cells, dialysis, and desalination as well as to suggest a new low-cost membrane surface assay that can detect and quantify the number of large biomolecules captured by probes functionalized on the membrane surface.

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.

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.

Slowing down DNA translocation through solid-state nanopores by edge-field leakage.

Solid-state nanopores allow high-throughput single-molecule detection but identifying and even registering all translocating small molecules remain key challenges due to their high translocation speeds. We show here the same electric field that drives the molecules into the pore can be redirected to selectively pin and delay their transport. A thin high-permittivity dielectric coating on bullet-shaped polymer nanopores permits electric field leakage at the pore tip to produce a voltage-dependent surface field on the entry side that can reversibly edge-pin molecules. This mechanism renders molecular entry an activated process with sensitive exponential dependence on the bias voltage and molecular rigidity. This sensitivity allows us to selectively prolong the translocation time of short single-stranded DNA molecules by up to 5 orders of magnitude, to as long as minutes, allowing discrimination against their double-stranded duplexes with 97% confidence.

Novel Homogeneous Anion Exchange Membranes for Reproducible and Sensitive Nucleic Acid Detection via Current-Voltage Characteristic Measurement.

One-pot synthesis of novel hydrogel-based anion exchange membranes (AEMs), with only a single-phase monomer mixture, was used to eliminate surface heterogeneity and generate reproducible electroconvective microvortices in the over-limiting region of the current-voltage characteristic (CVC) curves. Diallyldimethylammonium chloride (DDA) was used as the main component to provide the cation charge groups, and 2-hydroxyethyl methacrylate (HEMA) and ethylene glycol dimethyl acrylate (EGDMA) were used as the auxiliary structure monomers. The uniform membrane structure allowed reproducible and sensitive DNA detection and quantification, as probe-target surface complexes can gate the ion flux and produce large voltage shifts in the over-limiting region. Suppressed membrane curvature due to controlled swelling is a crucial part to avoid the reduction of depletion region for maintaining the influence of target gene hybridization. Fourier-transform infrared (FTIR) spectroscopy verified the synthesized membrane structure, with a residual vinyl group that allows easy carboxylation via additional photografting reaction. Consequently, a significantly higher DNA probe functionalization efficiency is obtained on the homogeneous AEMs, evidenced by the increasing nitrogen element content and bonding via X-ray photoelectron spectroscopy (XPS). The DDA content was optimized to provide a sufficient coulomb force between AEM and nucleic acid backbone to promote the specific binding efficiency but without high dimensional swelling which might change the surface geometry and restrict the voltage shifting for sensing in the over-limiting region, and the optimal DDA/HEMA ratio was found to be 4/10. The synthesized AEM sensor for recombinant 35S promoter sequence identification exhibited a reproducible calibration standard curve with dynamic range between 30 fM and 1 µM and high selectivity with only 0.01 V shift for 1 µM nontarget oligo.

High-flux ionic diodes, ionic transistors and ionic amplifiers based on external ion concentration polarization by an ion exchange membrane: a new scalable ionic circuit platform.

A microfluidic ion exchange membrane hybrid chip is fabricated using polymer-based, lithography-free methods to achieve ionic diode, transistor and amplifier functionalities with the same four-terminal design. The high ionic flux (>100 µA) feature of the chip can enable a scalable integrated ionic circuit platform for micro-total-analytical systems.

Induced nanoparticle aggregation for short nucleic acid quantification by depletion isotachophoresis.

A rapid (<20min) gel-membrane biochip platform for the detection and quantification of short nucleic acids is presented based on a sandwich assay with probe-functionalized gold nanoparticles and their separation into concentrated bands by depletion-generated gel isotachophoresis. The platform sequentially exploits the enrichment and depletion phenomena of an ion-selective cation-exchange membrane created under an applied electric field. Enrichment is used to concentrate the nanoparticles and targets at a localized position at the gel-membrane interface for rapid hybridization. The depletion generates an isotachophoretic zone without the need for different conductivity buffers, and is used to separate linked nanoparticles from isolated ones in the gel medium and then by field-enhanced aggregation of only the linked particles at the depletion front. The selective field-induced aggregation of the linked nanoparticles during the subsequent depletion step produces two lateral-flow like bands within 1cm for easy visualization and quantification as the aggregates have negligible electrophoretic mobility in the gel and the isolated nanoparticles are isotachophoretically packed against the migrating depletion front. The detection limit for 69-base single-stranded DNA targets is 10 pM (about 10 million copies for our sample volume) with high selectivity against nontargets and a three decade linear range for quantification. The selectivity and signal intensity are maintained in heterogeneous mixtures where the nontargets outnumber the targets 10,000 to 1. The selective field-induced aggregation of DNA-linked nanoparticles at the ion depletion front is attributed to their trailing position at the isotachophoretic front with a large field gradient.

Microfluidic systems with ion-selective membranes.

When integrated into microfluidic chips, ion-selective nanoporous polymer and solid-state membranes can be used for on-chip pumping, pH actuation, analyte concentration, molecular separation, reactive mixing, and molecular sensing. They offer numerous functionalities and are hence superior to paper-based devices for point-of-care biochips, with only slightly more investment in fabrication and material costs required. In this review, we first discuss the fundamentals of several nonequilibrium ion current phenomena associated with ion-selective membranes, many of them revealed by studies with fabricated single nanochannels/nanopores. We then focus on how the plethora of phenomena has been applied for transport, separation, concentration, and detection of biomolecules on biochips.

An ion-exchange nanomembrane sensor for detection of nucleic acids using a surface charge inversion phenomenon.

We present a novel low-cost biosensor for rapid, sensitive and selective detection of nucleic acids based on an ionic diode feature of an anion exchange nanoporous membrane under DC bias. The ionic diode feature is associated with external surface charge inversion on the positively charged anion exchange nanomembrane upon hybridization of negatively charged nucleic acid molecules to single-stranded oligoprobes functionalized on the membrane surface resulting in the formation of a cation selective monolayer. The resulting bipolar membrane causes a transition from electroconvection-controlled to water-splitting controlled ion conductance, with a large ion current signature that can be used to accurately quantify the hybridized nucleic acids. The platform is capable of distinguishing two base-pair mismatches in a 22-base pairing segment of microRNAs associated with oral cancer, as well as serotype-specific detection of dengue virus. We also show the sensor' capability to selectively capture target nucleic acids from a heterogeneous mixture. The limit of detection is 1 pM for short 27 base target molecules in a 15-min assay. Similar hybridization results are shown for short DNA molecules as well as RNAs from Brucella and Escherichia coli. The versatility and simplicity of this low-cost biosensor should enable point-of-care diagnostics in food, medical and environmental safety markets.