Precise control of liposome size using characteristic time depends on solvent type and membrane properties.

Controlling the sizes of liposomes is critical in drug delivery systems because it directly influences their cellular uptake, transportation, and accumulation behavior. Although hydrodynamic focusing has frequently been employed when synthesizing nano-sized liposomes, little is known regarding how flow characteristics determine liposome formation. Here, various sizes of homogeneous liposomes (50-400 nm) were prepared according to flow rate ratios in two solvents, ethanol, and isopropyl alcohol (IPA). Relatively small liposomes formed in ethanol due to its low viscosity and high diffusivity, whereas larger, more poly-dispersed liposomes formed when using IPA as a solvent. This difference was investigated via numerical simulations using the characteristic time factor to predict the liposome size; this approach was also used to examine the flow characteristics inside the microfluidic channel. In case of the liposomes, the membrane rigidity also has a critical role in determining their size. The increased viscosity and packing density of the membrane by addition of cholesterol confirmed by fluorescence anisotropy and polarity lead to increase in liposome size (40-530 nm). However, the interposition of short-chain lipids de-aligned the bilayer membrane, leading to its degradation; this decreased the liposome size. Adding short-chain lipids linearly decreased the liposome size (130-230 nm), but at a shallower gradient than that of cholesterol. This analytical study expands the understanding of microfluidic environment in the liposome synthesis by offering design parameters and their relation to the size of liposomes.

A Simple Method for Continuous Synthesis of Bicelles in Microfluidic Systems.

Bicelle has great potential for drug delivery systems due to its small size and biocompatibility. The conventional method of bicelle preparation contains a long process and harsh conditions, which limit its feasibility and damage the biological substances. For these reasons, a continuous manufacturing method in mild conditions has been demanded. Here, we propose a novel method for DMPC/DHPC bicelle synthesis based on a microfluidic device without heating and freezing processes. Bicelles were successfully prepared using this continuous method, which was identified by the physicochemical properties and morphologies of the synthesized assemblies. Experimental and analytical studies confirm that there is critical lipid concentration and critical mixing time for bicelle synthesis in this microfluidic system. Furthermore, a linear relation between the actual composition of bicelle and initial lipid ratio is deduced, and this enables the size of bicelles to be controlled.

Precise Microfluidic Luminescent Sensor Platform with Controlled Injection System.

Efforts have been devoted to screening various prevalent diseases, such as severe acute respiratory syndrome (SARS) and coronavirus disease 2019 (COVID-19). Real-time polymerase chain reaction (RT-PCR), which is currently the most widely used, has high accuracy, but it requires several facilities and takes a relatively long time to check; so, new testing technology is necessary for a higher test efficiency. A chemiluminescence (CL) sensor is a relatively simple device and suitable as an alternative because it can detect very precise specimens. However, in measurements via CL, the quantitative formulation of reagents that cause color development is important. In the case of mixing using micropipettes, precise analysis is possible, but this technique is limited by uncontrollable errors or deviations in detection amounts. In addition, in using a microfluidic chip to increase field applicability, a syringe pump or other quantification injection tools are required, so problems must be overcome for practical use. Therefore, in this study, a microchip was designed and manufactured to supply a sample of a certain volume by simply blowing air and injecting a sample into the chamber. By utilizing the luminescence reaction of luminol, CuSO4 and H2O2 the performance of the prepared chip was confirmed, and the desired amount of the sample could be injected with a simple device with an error rate of 2% or less. For feasible applications, an experiment was performed to quantitatively analyze thrombin, a biomarker of heart disease. Results demonstrated that biomarkers could be more precisely detected using the proposed microchips than using micropipettes.

Hybrid Nanofiber Scaffold-Based Direct Conversion of Neural Precursor Cells/Dopamine Neurons.

The concept of cellular reprogramming was developed to generate induced neural precursor cells (iNPCs)/dopaminergic (iDA) neurons using diverse approaches. Here, we investigated the effects of various nanoscale scaffolds (fiber, dot, and line) on iNPC/iDA differentiation by direct reprogramming. The generation and maturation of iDA neurons (microtubule-associated protein 2-positive and tyrosine hydroxylase-positive) and iNPCs (NESTIN-positive and SOX2-positive) increased on fiber and dot scaffolds as compared to that of the flat (control) scaffold. This study demonstrates that nanotopographical environments are suitable for direct differentiation methods and may improve the differentiation efficiency.

Catalytic characteristics of a sn-1(3) regioselective lipase from Cordyceps militaris.

A total of 39 agricultural products were screened for natural sources of lipases with distinctive positional specificity. Based on this, Cordyceps militaris lipase (CML) was selected and subsequently purified by sequential chromatography involving anion-exchange, hydrophobic-interaction, and gel-permeation columns. As a result of the overall purification procedure, a remarkable increase in the specific activity of the CML (4.733 U/mg protein) was achieved, with a yield of 2.47% (purification fold of 94.54). The purified CML has a monomeric structure with a molecular mass of approximately 62 kDa. It was further identified as a putative extracellular lipase from C. militaris by the partial sequence analysis using ESI-Q-TOF MS. In a kinetic study of the CML-catalyzed hydrolysis, the values of Vmax , Km , and kcat were determined to be 4.86 µmol·min-1 ·mg-1 , 0.07 mM, and 0.29 min-1 , respectively. In particular, the relatively low Km value indicated that CML has a high affinity for its substrate. With regard to positional specificity, CML selectively cleaved triolein at the sn-1 or 3 positions of glycerol backbone, releasing 1,2(2,3)-diolein as the major products. Therefore, CML can be considered a distinctive biocatalyst with sn-1(3) regioselectivity. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2744, 2019.

Hydrophilic and lipophilic characteristics of non-fatty acid moieties: significant factors affecting antibacterial activity of lauric acid esters.

It has been proposed that the hydrophilic and/or lipophilic characteristics of fatty acid derivatives affect their antibacterial activities according to their ability to incorporate into the bacterial cell membrane. To verify this hypothesis, six kinds of lauric acid derivatives esterified with different non-fatty acid moieties were selected to confirm whether antibacterial activity from their precursor (i.e., lauric acid) is retained or lost. Three compounds, monolaurin, sucrose laurate, and erythorbyl laurate, exerted bacteriostatic and bactericidal effects against Gram-positive bacteria, while the others showed no inhibitory activity. Interestingly, the calculated log P (octanol-water partition coefficient) values of monolaurin, sucrose laurate, and erythorbyl laurate were - 4.122, - 0.686, and 3.670, respectively, relatively lower than those of the other compounds without antibacterial activity. Moreover, the hydrophilic-lipophilic balance values of the three compounds with antibacterial activity were higher than those of the other compounds, corresponding to the log P result.

Design of a simple paper-based colorimetric biosensor using polydiacetylene liposomes for neomycin detection.

We developed a paper-based analytical device (µPAD) combined with self-signaling polydiacetylene (PDA) liposomes for convenient visual neomycin detection. The simple dot array type of µPAD was fabricated by the wax printing technique, and the PDA liposomes in the aqueous solution were facilely immobilized onto the hydrophilic dot region of the paper substrate. We found that, when the PDA liposomes were inserted to the paper matrix, the stability of the PDA liposomes can be significantly enhanced by adding a hydrophilic reagent such as polyvinyl alcohol and glycerol to the liposome solution. In particular, polyvinyl alcohol (PVA) provides the best stabilization among the various hydrophilic reagents tested in this contribution, and the enhanced stability sharply increased the sensitivity of the PDA liposomes in the paper matrix. Based on the above results, we successfully detected neomycin through both naked-eye observation and fluorescence measurement of PDA signals. The detection limit was 1 ppm and was selective to non-aminoglycoside antibiotics.

Amperometric Detection of Conformational Change of Proteins Using Immobilized-Liposome Sensor System.

An immobilized liposome electrode (ILE)-based sensor was developed to quantify conformational changes of the proteins under various stress conditions. The ILE surface was characterized by using a tapping-mode atomic force microscopy (TM-AFM) to confirm surface immobilization of liposome. The uniform layer of liposome was formed on the electrode. The current deviations generated based on the status of the proteins under different stress were then measured. Bovine carbonic anhydrase (CAB) and lysozyme were tested with three different conditions: native, reduced and partially denatured. For both proteins, a linear dynamic range formed between denatured concentrations and output electric current signals was able to quantify conformational changes of the proteins. The pattern recognition (PARC) technique was integrated with ILE-based sensor to perform data analysis and provided an effective method to improve the prediction of protein structural changes. The ILE-based stress sensor showed potential of leveraging the amperometric technique to manifest activity of proteins based on various external conditions.

Mussel-Inspired Universal Bioconjugation of Polydiacetylene Liposome for Droplet-Array Biosensors.

Most solid-state biosensor platforms require a specific immobilization chemistry and a bioconjugation strategy separately to tether sensory molecules to a substrate and attach specific receptors to the sensory unit, respectively. We developed a mussel-inspired universal conjugation method that enables both surface immobilization and bioconjugation at the same time. By incorporating dopamine or catechol moiety into self-signaling polydiacetylene (PDA) liposomes, we demonstrated efficient immobilization of the PDA liposomes to a wide range of substrates, without any substrate modification. Moreover, receptor molecules having a specificity toward a target molecule can also be attached to the immobilized PDA liposome layer without any chemical modification. We applied our mussel-inspired conjugation method to a droplet-array biosensor by exploiting the hydrophilic nature of PDA liposomes coated on a hydrophobic polytetrafluoroethylene surface and demonstrated selective and sensitive detection of vascular endothelial growth factor down to 10 nM.

Selective production of 1-monocaprin by porcine liver carboxylesterase-catalyzed esterification: Its enzyme kinetics and catalytic performance.

Porcine liver carboxylesterase (PLE) belongs to carboxylesterase family (EC 3.1.1.1) as a serine-type esterase. The PLE-catalyzed esterification of capric acid with glycerol in reverse micelles was investigated on the catalytic performance and enzyme kinetics. The most suitable structure of reverse micelles was comprised of isooctane (reaction medium) and bis(2-ethylhexyl) sodium sulfosuccinate (AOT, anionic surfactant) with 0.1 of R-value ([water]/[surfactant]) and 3.0 of G/F-value ([glycerol]/[fatty acid]) for the PLE-catalyzed esterification. In the aspect of regio-selectivity, the PLE mainly produced 1-monocaprin without any other products (di- and/or tricaprins of subsequent reactions). Furthermore, the degree of esterification at equilibrium state (after 4 h from the initiation) was 62.7% under the optimum conditions at pH 7.0 and 60 °C. Based on Hanes-Woolf plot, the apparent Km and Vmax values were calculated to be 16.44 mM and 38.91 µM/min/mg protein, respectively.

AOT/isooctane reverse micelles with a microaqueous core act as protective shells for enhancing the thermal stability of Chromobacterium viscosum lipase.

According to the different environmental systems for lipase reactions, changes in thermal stability were investigated by employing the Chromobacterium viscosum lipase and a two-step series-type deactivation model. The half-life (6.81 h) of the lipase entrapped in reverse micelles at 70 °C was 9.87- and 14.80-fold longer than that in glycerol pool or in aqueous buffer. The deactivation constants for the first and second step (k1 and k2) at all temperatures drastically decreased when the lipase was entrapped in reverse micelles. In particular, k1 (3.84 h(-1)) at 70 °C in reverse micelles was 1.57-fold lower than that in aqueous buffer (6.03 h(-1)). Based on the fluorescence spectrometry, the amount of excited forms of tryptophan and tyrosine increased markedly during the thermal-treatment in aqueous buffer, whereas no significant fluctuation was noted in the reversed micellar system. These results indicated that the encapsulation in reverse micelles could be favorable for preventing the enzyme from heat-induced denaturation.

Janus-compartmental alginate microbeads having polydiacetylene liposomes and magnetic nanoparticles for visual lead(II) detection.

Janus-compartmental alginate microbeads having two divided phases of sensory polydiacetylene (PDA) liposomes and magnetic nanoparticles were fabricated for facile sensory applications. The sensory liposomes are composed of PDA for label-free signal generation and 1,2-dipalmitoyl-sn-glycero-3-galloyl (DPGG) lipids whose galloyl headgroup has specific interactions with lead(II). The second phase having magnetic nanoparticles is designed for convenient handling of the microbeads, such as washing, solvent exchange, stirring, and detection, by applying magnetic field. Selective and convenient colorimetric detection of lead(II) and efficient removal of lead(II) by alginate matrix at the same time are demonstrated.

A nanoscale ridge/groove pattern arrayed surface enhances adipogenic differentiation of human supernumerary tooth-derived dental pulp stem cells in vitro.

OBJECTIVE: The aim of this study was to establish human dental pulp stem cells (hDPSCs) from supernumerary teeth and determine the effects of a 350-nm nano-patterned surface on adipogenic and osteogenic differentiation of hDPSCs. DESIGN: Several surface markers were analysed by FACS to confirm the isolated cells as hDPSCs. To demonstrate the effects of a nano-patterned surface on the differentiation of hDPSCs, the cells were cultured on a nano-patterned surface with or without adipogenic or osteogenic induction factors. Cells were then stained with Oil red O or Alizarin red, and the lineage specific genes LPL and Runx-2 were analysed by real-time PCR at 3, 6 and 9 days after culture. RESULTS: The hDPSCs on a nano-patterned surface showed a linear arrangement compared to irregular cells on a conventional surface. During adipogenic differentiation, more Oil red O stained cells were found in the nano-patterned group than in the conventional group. On the other hand, there was no significant difference in Alizarin red staining between the nano-pattern and conventional surface groups after induction of osteogenic differentiation. Gene expression analyses revealed significantly higher expression of LPL in the nano-patterned group than in the conventional group, whereas Runx-2 expression was higher in the conventional group than in the nano-patterned group. CONCLUSION: This study showed that a nano-patterned surface may be able to enhance adipogenic differentiation of hDPSCs by altering their morphology and gene expression patterns, whereas the same surface may inhibit or suppress osteogenic differentiation of the cells.

Physiological Characteristics and Production of Folic Acid of Lactobacillus plantarum JA71 Isolated from Jeotgal, a Traditional Korean Fermented Seafood.

Folic acid, one of the B group of vitamins, is an essential substance for maintaining the functions of the nervous system, and is also known to decrease the level of homocysteine in plasma. Homocysteine influences the lowering of the cognitive function in humans, and especially in elderly people. In order to determine the strains with a strong capacity to produce folic acid, 190 bacteria were isolated from various kinds of jeotgal and chungkuk-jang. In our test experiment, JA71 was found to contain 9.03µg/mL of folic acid after 24 h of incubation in an MRS broth. This showed that JA71 has the highest folic acid production ability compared to the other lactic acid bacteria that were isolated. JA71 was identified as Lactobacillus plantarum by the result of API carbohydrate fermentation pattern and 16s rDNA sequence. JA71 was investigated for its physiological characteristics. The optimum growth temperature of JA71 was 37℃, and the cultures took 12 h to reach pH 4.4. JA71 proved more sensitive to bacitracin when compared with fifteen different antibiotics, and showed most resistance to neomycin and vancomycin. Moreover, it was comparatively tolerant of bile juice and acid, and displayed resistance to Escherichia coli, Salmonella Typhimurium, and Staphylococcus aureus with restraint rates of 60.4%, 96.7%, and 76.2%, respectively. These results demonstrate that JA71 could be an excellent strain for application to functional products.

Biomimetic detection of aminoglycosidic antibiotics using polydiacetylene-phospholipids supramolecules.

We rationally designed highly sensitive and selective polydiacetylene (PDA)-phospholipids liposomes for the facile detection of aminoglycosidic antibiotics. The detecting mechanism mimics the cellular membrane interactions between neomycin and phosphatidylinositol-4,5-bisphosphate (PIP(2)) phospholipids. The developed PDA-PIP(2) sensory system showed a detection limit of 61 ppb for neomycin and was very specific to aminoglycosidic antibodies only.

Design of polydiacetylene-phospholipid supramolecules for enhanced stability and sensitivity.

We present polydiacetylene (PDA) liposome assemblies with various phospholipids that have different headgroup charges and phase transition temperatures (T(m)). 10,12-Pentacosadiynoic acid (PCDA)-epoxy was used as a base PDA monomer and the insertion of highly charged phospholipids resulted in notable changes in the size of liposome and reduction of the aggregation of PDA liposome. Among the various phospholipids, the phospholipid with a moderate T(m) demonstrated enhanced stability and sensitivity, as measured by the size and zeta potential over storage time, thermochoromic response, and transmission electron microscopy images. By combining these results, we were able to detect immunologically an antibody of bovine viral diarrhea virus over a wide dynamic range of 0.001 to 100 µg/mL.

Sensitive Mie scattering immunoagglutination assay of porcine reproductive and respiratory syndrome virus (PRRSV) from lung tissue samples in a microfluidic chip.

A microfluidic immunosensor utilizing Mie scattering immunoaggultination assay was developed for rapid and sensitive detection of porcine reproductive and respiratory syndrome virus (PRRSV) from lung tissue samples of domesticated pigs. Antibodies against PRRSV were conjugated to the surface of highly carboxylated polystyrene microparticles (diameter=920nm) and mixed with the diluted PRRSV tissue samples in a Y-shaped microchannel. Antibody-antigen binding induced microparticle immunoagglutination, which was detected by measuring the forward 45° light scattering of 380nm incident beam using microcallipered, proximity fiber optics. For comparison, multi-well experiments were also performed using the same optical detection setup. The detection limit was determined to be 10(-3)TCID(50)ml(-1) for PRRSV dissolved in PBS, while those of previous RT-PCR studies for PRRSV were 10(1)TCID(50)ml(-1) (conventional assays) or <1TCID(50)ml(-1) (quantitative real-time assays). Mie scattering simulations were able to predict the shape of the PRRSV standard curve, indicating that any non-linearity of the standard curve can be interpreted purely as an optical phenomenon. Each assay took less than 5min. A strong correlation could be found between RT-PCR and this method for the lung tissue samples, even though their respective detection mechanisms are different fundamentally (nucleic acids for RT-PCR and virus antigens for light scattering immunoagglutination assay). Several different dilution factors were also tested for tissue samples, and 1/10 and 1/100 were found to be usable. If the microfluidic chips are used only once (i.e. without re-using them), both superior sensitivity and satisfactory specificity can be demonstrated. Specificity studies revealed the presence of Type II PRRSV and non-presence of Type I PRRSV and that the microfluidic chip assay could detect Type II North American strain of PRRSV for the animals tested. This work demonstrates the potential of the Mie scattering immunoassay on a microfluidic chip towards real-time detection system for viral pathogens in domesticated animals.

High-sensitivity detection of oxytetracycline using light scattering agglutination assay with aptasensor.

We present an aptamer-based biosensor (aptasensor) for rapid and high-sensitive detection of oxytetracycline (OTC) antibiotic in PBS inside a Y-channel PDMS microfluidic device. The detection was made by real-time monitoring of the agglutination assay of ssDNA aptamer-conjugated polystyrene latex microspheres with proximity optical fibers. The agglutination assay was performed with serially diluted OTC antibiotic solutions using highly carboxylated polystyrene particles of 920 nm diameter conjugated with OTC-binding ssDNA aptamer. Proximity optical fibers were used to measure the increase in 45° forward light scattering of the aggregated particles by fixing them around the viewing cell of the device with stable angle and distance to the detector. The detection limit was around 100 ppb for the current aptasensor system with the detection time less than 3 min.

Microfluidic immunosensor with integrated liquid core waveguides for sensitive Mie scattering detection of avian influenza antigens in a real biological matrix.

This work presents the use of integrated, liquid core, optical waveguides for measuring immunoagglutination-induced light scattering in a microfluidic device, towards rapid and sensitive detection of avian influenza (AI) viral antigens in a real biological matrix (chicken feces). Mie scattering simulations were performed and tested to optimize the scattering efficiency of the device through proper scatter angle waveguide geometry. The detection limit is demonstrated to be 1 pg mL(-1) in both clean buffer and real biological matrix. This low detection limit is made possible through on-chip diffusional mixing of AI target antigens and high acid content microparticle assay reagents, coupled with real-time monitoring of immunoagglutination-induced forward Mie scattering via high refractive index liquid core optical waveguides in close proximity (100 µm) to the sample chamber. The detection time for the assay is <2 min. This device could easily be modified to detect trace levels of any biological molecules that antibodies are available for, moving towards a robust platform for point-of-care disease diagnostics.