Tae-Joon Jeon

Electrowetting on dielectric-based microfluidics for integrated lipid bilayer formation and measurement

We present a microfluidic platform for the formation and electrical measurement of lipid bilayer membranes. Using electrowetting on dielectric (EWOD), two or more aqueous droplets surrounded by a lipid-containing organic phase were manipulated into contact to form a lipid bilayer at their interface. Thin-film Ag/AgCl electrodes integrated into the device enabled electrical measurement of membrane formation and the incorporation of gramicidin channels of two bilayers in parallel.

Ion channel and toxin measurement using a high throughput lipid membrane platform

Measurements of ion channels are important for scientific, sensing and pharmaceutical applications. Reconstitution of ion channels into lipid vesicles and planar lipid bilayers for measurement at the single molecule level is a laborious and slow process incompatible with the high throughput methods and equipment used for sensing and drug discovery. A recently published method of lipid bilayer formation mechanically combines lipid monolayers self-assembled at the interfaces of aqueous and apolar phases. We have expanded on this method by vertically orienting these phases and using gravity as the driving force to combine the monolayers. As this method only requires fluid dispensation, it is trivially integrated with high throughput automated liquid-handling robotics. In a proof-of-concept demonstration, we created over 2200 lipid bilayers in 3h. We show single molecule measurements of technologically and physiologically relevant ion channels incorporated into lipid bilayers formed with this method.

Single molecule measurements of channel proteins incorporated into biomimetic polymer membranes

Lipid bilayer membranes have been extensively utilized to examine membrane channel and pore proteins and are the subjects of study in their own right. There has been considerable recent interest in developing technologies to substitute or strengthen lipid bilayer membranes for a number of applications, including sensing or drug delivery. In particular, biomimetic amphiphilic block co-polymers have been shown to have the capacity to form membrane structures and to contain membrane proteins within them. In this work, we describe the creation of biomimetic membranes from a 5.7 nm thick tri-block co-polymer and the investigation of the effects of the polymer environment on incorporated channel proteins (α-haemolysin, OmpG, and alamethicin) with single molecule transport measurements. We found that the polymer membranes consistently have seal resistances of tens of GΩ and greater, and that the conductance of single channels is reduced by approximately 10% from that measured in diphytanoyl phosphatidylcholine lipid membranes, possibly as a result of increased cohesion of the polymer compared to lipid. The voltage gating ability and threshold voltages of voltage gated channels were also found to be very similar in the lipid and polymer environments.

A nanoporous membrane-based impedimetric immunosensor for label-free detection of pathogenic bacteria in whole milk

We introduce a nanoporous membrane based impedimetric immunosensor for the label-free detection of bacterial pathogens in whole milk. A simple and rapid method to modify a commercially available alumina nanoporous membrane with hyaluronic acid (HA) effectively reduced the non-specific binding of biomolecules and other cells, and permitted successful immobilization of antibodies. Escherichia coli O157:H7, one of the most harmful food-borne pathogenic bacteria, was tested as a model pathogen in this study. The ionic impedance of electrolytes through nanopores, due to antibody-pathogen interactions, was monitored by impedance spectra and analyzed by normalized impedance change (NIC). The regression equation for the NIC at 1 kHz versus concentration of E. coli O157:H7 (10-10(5)cfu/ml) was obtained, and the detection limit found to be as low as 10 cfu/ml. In addition, the proposed immunosensor was successfully used for the detection of E. coli O157:H7 in whole milk samples with the detection limit as low as 83.7 cfu/ml with 95% probability. The specificity of the immunosensor was also demonstrated using non-target bacteria, including Staphylococcus aureus, Bacillus cereus, and non pathogenic E. coli DH5α. This study shows that a HA-functionalized nanoporous membrane-based impedimetric sensor is capable of detecting pathogenic bacteria in whole milk without any pretreatment. This is a significant step for evaluating the safety of food and environmental samples and other medical diagnostics.

Synthetic Biomimetic Membranes and Their Sensor Applications

Synthetic biomimetic membranes provide biological environments to membrane proteins. By exploiting the central roles of biological membranes, it is possible to devise biosensors, drug delivery systems, and nanocontainers using a biomimetic membrane system integrated with functional proteins. Biomimetic membranes can be created with synthetic lipids or block copolymers. These amphiphilic lipids and polymers self-assemble in an aqueous solution either into planar membranes or into vesicles. Using various techniques developed to date, both planar membranes and vesicles can provide versatile and robust platforms for a number of applications. In particular, biomimetic membranes with modified lipids or functional proteins are promising platforms for biosensors. We review recent technologies used to create synthetic biomimetic membranes and their engineered sensors applications.

Elucidation of molecular interactions between lipid membranes and ionic liquids using model cell membranes

Ionic liquids (ILs) are often considered to be green solvents based on their unusual stability, although their toxicity to living organisms has become an emerging issue based on a number of recent studies. We assume that one of the main reasons for this high level of cell toxicity is the molecular interactions between ILs and cell membranes. In this study, we used model cells to demonstrate that ILs can incorporate into lipid membranes, resulting in the perturbation of membrane structure. We employed various methods to elucidate the molecular interactions between cell membranes and ILs. Our results demonstrate that the stability of cell membranes is inversely related to the alkyl chain length and concentration of ILs, providing important information for the design of greener and safer ILs.

Black lipid membranes stabilized through substrate conjugation to a hydrogel

Recent research in stabilizing lipid bilayer membranes has been directed toward tethering the membrane to a solid surface or contacting the membrane with a solid support such as a gel. It is also known that the solvent annulus plays an important role in lipid bilayer stability. In this work, the authors set out to stabilize the solvent annulus. Glass substrates with ∼500 μm apertures were functionalized with 3-methacryloxypropyltrimethoxysilane to allow cross-linking with a surrounding polyethyleneglycol dimethacrylate hydrogel. The hydrogel makes a conformal mold around both the lipid bilayer and the solvent reservoir. Since the hydrogel is covalently conjugated with the glass substrate via vinyl groups, the solvent annulus is prevented from leaving the aperture boundary. Measurements of a membrane created with this approach showed that it remained a stable bilayer with a resistance greater than 1 GΩ( for 12 days. Measurements of the ion channel gramicidin A, α-hemolysin, and alamethicin incorporated into these membranes showed the same conductance behavior as conventional membranes.

Chromatic biosensor for detection of phosphinothricin acetyltransferase by use of polydiacetylene vesicles encapsulated within automatically generated immunohydrogel beads.

We developed a simple and sensitive colorimetric biosensor in the form of microparticles by using polydiacetylene (PDA) vesicles encapsulated within a hydrogel matrix for the detection of phosphinothricin acetyltransferase (PAT) protein, which is one of the most important marker proteins in genetically modified (GM) crops. Although PDA is commonly used as a sensing material due to its unique colorimetric properties, existing PDA biosensors are ineffective due to their low sensitivity as well as their lack of robustness. To overcome these disadvantages, we devised immunohydrogel beads made of anti-PAT-conjugated PDA vesicles embedded at high density within a poly(ethylene glycol) diacrylate (PEG-DA) hydrogel matrix. In addition, the construction of immunohydrogel beads was automated by use of a microfluidic device. In the immunoreaction, the sensitivity of antibody-conjugated PDA vesicles was significantly amplified, as monitored by the unaided eye. The limit of detection for target molecules reached as low as 20 nM, which is sufficiently low enough to detect target materials in GM organisms. Collectively, the results show that immunohydrogel beads constitute a promising colorimetric sensing platform for onsite testing in a number of fields, such as the food and medical industries, as well as warfare situations.

Self-assembled graphene oxide with organo-building blocks of Fe-aminoclay for heterogeneous Fenton-like reaction at near-neutral pH: A batch experiment

Respective water-soluble graphene oxide (GO) and Fe-aminoclay were investigated for decoloration of recalcitrant organic dyes with different charges in the presence of hydrogen peroxide (H2O2). This oxidation process was updated in order to enable development of heterogeneous catalysts by self-assembled (precipitated) GO with organo-building blocks of Fe-aminoclay in aqueous solution. Due to electrostatic attraction between the negatively charged GO and the positively charged Fe-aminoclay, the obtained catalysts decomposed H2O2 at pH 6.0 to generate •OH radicals capable of breaking aromatic carbon raphene oxide (GO) e-aminoclay elf-assembly enton-like reaction eutral pH structures. These novel catalysts were tested for cationic methylene blue (MB) and anionic orange II (OII) as model pollutants. At 1.0 wt% of H2O2 treatment, the apparent rate constants of MB and OII by the optimal loading of catalyst (0.61 mg/mL) were 0.35288 h−1 (r2 = 0.9721) and 0.57930 (r2 = 0.9581), respectively. Furthermore, in the two-dye mixture system, simultaneous decoloration of both dyes, to near-complete ∼100% removal, was achieved after 5 h. Thus, it can be concluded that our developed catalysts for heterogeneous Fenton-like reaction at neutral pH are suitable for practical application.

Automated formation of multicomponent-encapuslating vesosomes using continuous flow microcentrifugation

Vesosomes - hierarchical assemblies consisting of membrane-bound vesicles of various scales - are potentially powerful models of cellular compartmentalization. Current methods of vesosome fabrication are labor intensive, and offer little control over the size and uniformity of the final product. In this article, we report the development of an automated vesosome formation platform using a microfluidic device and a continuous flow microcentrifuge. In the microfluidic device, water-in-oil droplets containing nanoscale vesicles in the water phase were formed using T-junction geometry, in which a lipid monolayer is formed at the oil/water interface. These water-in-oil droplets were then immediately transferred to the continuous flow microcentrifuge. When a water-in-oil droplet passed through a second lipid monolayer formed in the continuous flow microcentrifuge, a bilayer-encapsulated vesosome was created, which contained all of the contents of the aqueous phase encapsulated within the vesosome. Encapsulation of nanoscale liposomes within the outer vesosome membrane was confirmed by fluorescence microscopy. Laser diffraction analysis showed that the vesosomes we fabricated were uniform (coefficient of variation of 0.029). The yield of the continuous flow microcentrifuge is high, with over 60% of impinging water droplets being converted to vesosomes. Our system provides a fully automatable route for the generation of vesosomes encapsulating arbitrary contents. The method employed in this work is simple and can be readily applied to a variety of systems, providing a facile platform for fabricating multicomponent carriers and model cells.