High-throughput fabrication of monodisperse spherical supraparticles through a reliable thin oil film and rapid water diffusion.

A supraparticle is a spherical superstructure composed of fine building blocks, typically synthesized through colloidal assembly from evaporating and contracting suspension droplets. Microfluidic emulsification is known to be effective in producing large amounts of water-in-oil droplets. However, the process of supraparticle self-assembly has been limited by the evaporation of the oil that supports it and the sluggish shrinkage of water droplets. These are caused by the high volatility and low diffusion rates of water in the bulk oil layer, making the process last hours or even days. To address these challenges, we introduce a new system in this paper: the supraparticle reliable fabrication (SURF) system. This microfluidic-based system can quickly and reliably assemble spherical supraparticles in 20 min. The SURF system combines a conventional flow focusing device with a thinly layered low-volatile/water-soluble oil, and an open-microfluidic droplet evaporator. This setup facilitates the creation of uniform supraparticles with various materials and diameters (coefficient of variation: <3.5%). As a proof-of-concept for potential biochemical applications, we demonstrate a sensitive chemical reaction on the fabricated supraparticles, emphasizing the effectiveness of the SURF system as an alternative to traditional supraparticle synthesis and particle-based applications.

Conformal Hydrogel-Skin Coating on a Microfluidic Channel through Microstamping Transfer of the Masking Layer.

Poly(dimethylsiloxane) (PDMS) is used in microfluidics owing to its biocompatibility and simple fabrication. However, its intrinsic hydrophobicity and biofouling inhibit its microfluidic applications. Conformal hydrogel-skin coating for PDMS microchannels, involving the microstamping transfer of the masking layer, is reported herein. A selective uniform hydrogel layer with a thickness of ∼1 µm was coated in diverse PDMS microchannels with a resolution of ∼3 µm, maintaining its structure and hydrophilicity after 180 days (6 months). The wettability transition of PDMS was demonstrated through the switched emulsification in a flow-focusing device (water-in-oil [pristine PDMS] to oil-in-water [hydrophilic PDMS]). A one-step bead-based immunoassay was performed to detect the anti-severe acute respiratory syndrome coronavirus 2 IgG using a hydrogel-skin-coated point-of-care platform.

Frictional force analysis of stent retriever devices using a realistic vascular model: Pilot study.

Objective: To date, no vascular model to analyze frictional forces between stent retriever devices and vessel walls has been designed to be similar to the real human vasculature. We developed a novel in vitro intracranial cerebrovascular model and analyzed frictional forces of three stent retriever devices. Methods: A vascular mold was created based on digital subtraction angiography of a patient's cerebral vessels. The vascular model was constructed using polydimethylsiloxane (PDMS, Dow Corning, Inc.) as a silicone elastomer. The vascular model was coated on its inner surface with a lubricating layer to create a low coefficient of friction (~0.037) to closely approximate the intima. A pulsatile blood pump was used to produce blood flow inside the model to approximate real vascular conditions. The frictional forces of Trevo XP, Solitaire 2, and Eric 4 were analyzed for initial and maximal friction retrieval forces using this vascular model. The total pulling energy generated during the 3 cm movement was also obtained. Results: Results for initial retrieval force were as follows: Trevo, 0.09 ± 0.04 N; Solitaire, 0.25 ± 0.07 N; and Eric, 0.33 ± 0.21 N. Results for maximal retrieval force were as follows: Trevo, 0.36 ± 0.07 N; Solitaire, 0.54 ± 0.06 N; and Eric, 0.80 ± 0.13 N. Total pulling energy (N·cm) was 0.40 ± 0.10 in Trevo, 0.65 ± 0.10 in Solitaire, and 0.87 ± 0.14 in Eric, respectively. Conclusions: Using a realistic vascular model, different stent retriever devices were shown to have statistically different frictional forces. Future studies using a realistic vascular model are warranted to assess SRT devices.

Constructing multi-enzymatic cascade reactions for selective production of 6-bromoindirubin from tryptophan in Escherichia coli.

6-Bromoindirubin (6BrIR), found in Murex sea snails, is a precursor of indirubin-derivatives anticancer drugs. However, its synthesis remains limited due to uncharacterized biosynthetic pathways and difficulties in site-specific bromination and oxidation at the indole ring. Here, we present an efficient 6BrIR production strategy in Escherichia coli by using four enzymes, that is, tryptophan 6-halogenase fused with flavin reductase Fre (Fre-L3-SttH), tryptophanase (TnaA), toluene 4-monooxygenase (PmT4MO), and flavin-containing monooxygenase (MaFMO). Although most indole oxygenases preferentially oxygenate the electronically active C3 position of indole, PmT4MO was newly characterized to perform C2 oxygenation of 6-bromoindole with 45% yield to produce 6-bromo-2-oxindole. In addition, 6BrIR was selectively generated without indigo and indirubin byproducts by controlling the reducing power of cysteine and oxygen supply during the MaFMO reaction. These approaches led to 34.1 mg/L 6BrIR productions, making it possible to produce the critical precursor of the anticancer drugs only from natural ingredients such as tryptophan, NaBr, and oxygen.

Cytocompatible asymmetrical coating for Janus carrier synthesis through capillary wetting and ascending.

Despite the successful implementation of elegant strategies for the fabrication of Janus microstructures, two critical factors have limited the applicability of most techniques for the partial modification of living cell surfaces: harsh conditions that could disintegrate cells, and the lack of an effective route to accomplish a mild modification for living cells. In this study, an expeditious synthesis, named lower-half occupation by capillary ascended liquids (LOCAL), is proposed for the fabrication of asymmetrical structures surrounding not only microbeads but also both living adherent and buoyant mammalian cells. The microbeads or living cells are safely supported and trapped on the apical sides of a micropillar array, which prevents them from contacting the bottom substrate. As the coating agents further transfer and contact the trapped particles through interpillar capillary flow, the autonomous capillary ascending coats the free bottom surfaces of the target particles within 2 min, with significantly small quantities of coating agents. The self-assembled architectures of the cells demonstrate thoroughly maintained cell viability, highlighting the potential of the LOCAL method as a desirable alternative to the widely applied state-of-art methods for developing Janus beads and Janus cells.

Regioselective One-Pot Synthesis of Hydroxy-(S)-Equols Using Isoflavonoid Reductases and Monooxygenases and Evaluation of the Hydroxyequol Derivatives as Selective Estrogen Receptor Modulators and Antioxidants.

Several regiospecific enantiomers of hydroxy-(S)-equol (HE) were enzymatically synthesized from daidzein and genistein using consecutive reduction (four daidzein-to-equol-converting reductases) and oxidation (4-hydroxyphenylacetate 3-monooxygenase, HpaBC). Despite the natural occurrence of several HEs, most of them had not been studied owing to the lack of their preparation methods. Herein, the one-pot synthesis pathway of 6-hydroxyequol (6HE) was developed using HpaBC (EcHpaB) from Escherichia coli and (S)-equol-producing E. coli, previously developed by our group. Based on docking analysis of the substrate or products, a potential active site and several key residues for substrate binding were predicted to interpret the (S)-equol hydroxylation regioselectivity of EcHpaB. Through investigating mutations on the key residues, the T292A variant was verified to display specific mono-ortho-hydroxylation activity at C6 without further 3'-hydroxylation. In the consecutive oxidoreductive bioconversion using T292A, 0.95 mM 6HE could be synthesized from 1 mM daidzein, while 5HE and 3'HE were also prepared from genistein and 3'-hydroxydaidzein (3'HD or 3'-ODI), respectively. In the following efficacy tests, 3'HE and 6HE showed about 30∼200-fold higher EC50 than (S)-equol in both ERα and ERß, and they did not have significant SERM efficacy except 6HE showing 10% lower ß/α ratio response than that of 17ß-estradiol. In DPPH radical scavenging assay, 3'HE showed the highest antioxidative activity among the examined isoflavone derivatives: more than 40% higher than the well-known 3'HD. In conclusion, we demonstrated that HEs could be produced efficiently and regioselectively through the one-pot bioconversion platform and evaluated estrogenic and antioxidative activities of each HE regio-isomer for the first time.

Hydroprinting Technology to Transfer Ultrathin, Transparent, and Double-Sided Conductive Nanomembranes for Multiscale 3D Conformal Electronics.

Transparent multiscale 3D conformal electronics using hydroprinting with polyvinyl alcohol (PVA) as a sacrificial layer to transfer networks of silver nanowires (AgNWs) without a carrier layer is developed. However, AgNWs are known to disperse on water surfaces during the transfer process. Therefore, a functional film is developed by simultaneously welding and embedding AgNWs in the PVA through a simple one-step thermal pressing, demonstrating that ultrathin, transparent, and double-sided conductive/patterned nanomembranes with welded AgNWs can float on water without dispersion. The nanomembrane with an excellent figure of merit of 1200, a low sheet resistance of 16.2 Ω sq-1 , and a high transmittance of 98.17% achieves conformal contact with excellent step surface coverage of complex macro- and microstructures because of its nanoscale thickness (54.39 nm) and numerous deformable micro- and nanopores. Furthermore, the double-sided conductive nanomembranes facilitate wiring and layer-by-layer assembly, regardless of the transfer direction of the surface. As a proof-of-concept demonstration, a nanomembrane-based aneurysm sensor is developed. Its high transparency enables coil embolization, and the sensor can measure the pushing force of the coil within an aneurysm in an endovascular simulator. Moreover, this newly developed hydroprinting technology provides a new method for the fabrication of transparent multiscale 3D conformal electronics.

High-Resolution and Facile Patterning of Silver Nanowire Electrodes by Solvent-Free Photolithographic Technique Using UV-Curable Pressure Sensitive Adhesive Film.

Patterning of silver nanowires (AgNWs) used in fabricating flexible and transparent electrodes (FTEs) is essential for constructing a variety of optoelectronic devices. However, patterning AgNW electrodes using a simple, inexpensive, high-resolution, designable, and scalable process remains a challenge. Therefore, herein a novel solvent-free photolithographic technique using a UV-curable pressure sensitive adhesive (PSA) film for patterning AgNWs is introduced. The UV-curable PSA film can be selectively patterned by photopolymerization under UV exposure through a photomask. The AgNWs embedded in the non-photocured adhesive areas of the film are firmly held by a crosslinked network of photocurable resin when the patterned film is attached to the AgNW-coated substrate and additionally irradiated by UV light. After peeling off the film, the positive pattern of AgNW electrodes remains on the substrate, while the negative pattern is transferred to the film. This solvent-free photolithographic technique, which does not use toxic solvents, provides superior pattern features, such as fine line widths and spacings, sharp line edges, and low roughness. Therefore, the developed technique could be successfully applied in the development of flexible and transparent optoelectronic devices, such as a self-cleaning electro-wetting-on-dielectric (EWOD) devices, transparent heaters, and FTEs.

Polyphenol-Hydroxylating Tyrosinase Activity under Acidic pH Enables Efficient Synthesis of Plant Catechols and Gallols.

Tyrosinase is generally known as a melanin-forming enzyme, facilitating monooxygenation of phenols, oxidation of catechols into quinones, and finally generating biological melanin. As a homologous form of tyrosinase in plants, plant polyphenol oxidases perform the same oxidation reactions specifically toward plant polyphenols. Recent studies reported synthetic strategies for large scale preparation of hydroxylated plant polyphenols, using bacterial tyrosinases rather than plant polyphenol oxidase or other monooxygenases, by leveraging its robust monophenolase activity and broad substrate specificity. Herein, we report a novel synthesis of functional plant polyphenols, especially quercetin and myricetin from kaempferol, using screened bacterial tyrosinases. The critical bottleneck of the biocatalysis was identified as instability of the catechol and gallol under neutral and basic conditions. To overcome such instability of the products, the tyrosinase reaction proceeded under acidic conditions. Under mild acidic conditions supplemented with reducing agents, a bacterial tyrosinase from Bacillus megaterium (BmTy) displayed efficient consecutive two-step monophenolase activities producing quercetin and myricetin from kaempferol. Furthermore, the broad substrate specificity of BmTy toward diverse polyphenols enabled us to achieve the first biosynthesis of tricetin and 3'-hydroxyeriodictyol from apigenin and naringenin, respectively. These results suggest that microbial tyrosinase is a useful biocatalyst to prepare plant polyphenolic catechols and gallols with high productivity, which were hardly achieved by using other monooxygenases such as cytochrome P450s.

A single snapshot multiplex immunoassay platform utilizing dense test lines based on engineered beads.

Co-circulation of coronavirus disease 2019 (COVID-19) and dengue fever has been reported. Accurate and timely multiplex diagnosis is required to prevent future pandemics. Here, we developed an innovative microfluidic chip that enables a snapshot multiplex immunoassay for timely on-site response and offers unprecedented multiplexing capability with an operating procedure similar to that of lateral flow assays. An open microchannel assembly of individually engineered microbeads was developed to construct nine high-density test lines, which can be imaged in a 1 mm2 field-of-view. Thus, simultaneous detection of multiple antibodies would be achievable in a single high-resolution snapshot. Next, we developed a novel pixel intensity-based imaging process to distinguish effective and non-specific fluorescence signals, thereby improving the reliability of this fluorescence-based immunoassay. Finally, the chip specifically identified and classified random combinations of arbovirus (Zika, dengue, and chikungunya viruses) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies within 30 min. Therefore, we believe that this snapshot multiplex immunoassay chip is a powerful diagnostic tool to control current and future pandemics.

Enhancement of Steel Sandwich Sheet Adhesion Using Mechanical Interlocking Structures Formed by Electrochemical Etching.

Steel sandwich sheets (steel-polymer-steel), which are composed of lightweight polymers bonded on both sides with rigid steel sheets, have recently been developed as functional lightweight materials. In this study, a steel sandwich sheet (electrogalvanized (EG) steel sheet-polypropylene (PP)-EG steel sheet) with improved normal adhesion is fabricated without adhesives. Instead, adhesion is achieved via mechanical interlocking between the etched EG steel sheets and PP. Hierarchical structures were formed on the EG steel sheet surface by electrochemical etching to attain effective mechanical interlocking for improving normal adhesion without any adhesives. In the case of the EG steel sheet etched at 6 V for 7 s, a high fraction (∼35%) of holes (size: <1 µm2) with nanoscale scalloped structures was formed on the EG steel sheet surface. The normal adhesion test result of the fabricated steel sandwich sheet showed that the adhesion strength increased from virtually 0 (bare) to 559.6 kPa as a result of mechanical interlocking. The results of the focused ion beam-scanning electron microscopy and energy-dispersive spectrometry analyses confirmed the cohesive failure of PP resulting from the successful mechanical interlocking of PP with the holes formed on the etched EG steel sheet. To examine the effect of hierarchical structures on the normal adhesion of the steel sandwich sheet, finite element analysis was implemented.

High-yield production of (R)-acetoin in Saccharomyces cerevisiae by deleting genes for NAD(P)H-dependent ketone reductases producing meso-2,3-butanediol and 2,3-dimethylglycerate.

Acetoin is widely used in food and cosmetics industries as a taste and fragrance enhancer. To produce (R)-acetoin in Saccharomyces cerevisiae, acetoin biosynthetic genes encoding α-acetolactate synthase (AlsS) and α-acetolactate decarboxylase (AlsD) from Bacillus subtilis and water-forming NADH oxidase (NoxE) from Lactococcus lactis were integrated into delta-sequences in JHY605 strain, where the production of ethanol, glycerol, and (R,R)-2,3-butanediol (BDO) was largely eliminated. We further improved acetoin production by increasing acetoin tolerance by adaptive laboratory evolution, and eliminating other byproducts including meso-2,3-BDO and 2,3-dimethylglycerate, a newly identified byproduct. Ara1, Ypr1, and Ymr226c (named Ora1) were identified as (S)-alcohol-forming reductases, which can reduce (R)-acetoin to meso-2,3-BDO in vitro. However, only Ara1 and Ypr1 contributed to meso-2,3-BDO production in vivo. We elucidate that Ora1, having a substrate preference for (S)-acetoin, reduces (S)-α-acetolactate to 2,3-dimethylglycerate, thus competing with AlsD-mediated (R)-acetoin production. By deleting ARA1, YPR1, and ORA1, 101.3 g/L of (R)-acetoin was produced with a high yield (96% of the maximum theoretical yield) and high stereospecificity (98.2%).

Application of Random Mutagenesis and Synthetic FadR Promoter for de novo Production of ω-Hydroxy Fatty Acid in Yarrowia lipolytica.

As a means to develop oleaginous biorefinery, Yarrowia lipolytica was utilized to produce ω-hydroxy palmitic acid from glucose using evolutionary metabolic engineering and synthetic FadR promoters for cytochrome P450 (CYP) expression. First, a base strain was constructed to produce free fatty acids (FFAs) from glucose using metabolic engineering strategies. Subsequently, through ethyl methanesulfonate (EMS)-induced random mutagenesis and fluorescence-activated cell sorting (FACS) screening, improved FFA overproducers were screened. Additionally, synthetic promoters containing bacterial FadR binding sequences for CYP expression were designed to respond to the surge of the concentration of FFAs to activate the ω-hydroxylating pathway, resulting in increased transcriptional activity by 14 times from the third day of culture compared to the first day. Then, endogenous alk5 was screened and expressed using the synthetic FadR promoter in the developed strain for the production of ω-hydroxy palmitic acid. By implementing the synthetic FadR promoter, cell growth and production phases could be efficiently decoupled. Finally, in batch fermentation, we demonstrated de novo production of 160 mg/L of ω-hydroxy palmitic acid using FmeN3-TR1-alk5 in nitrogen-limited media. This study presents an excellent example of the production of ω-hydroxy fatty acids using synthetic promoters with bacterial transcriptional regulator (i.e., FadR) binding sequences in oleaginous yeasts.

Embolization of Vascular Malformations via In Situ Photocrosslinking of Mechanically Reinforced Alginate Microfibers using an Optical-Fiber-Integrated Microfluidic Device.

Embolization, which is a minimally invasive endovascular treatment, is a safe and effective procedure for treating vascular malformations (e.g., aneurysms). Hydrogel microfibers obtained via spatiotemporally controllable in situ photocrosslinking exhibit great potential for embolizing aneurysms. However, this process is challenging because of the absence of biocompatible and morphologically stable hydrogels and the difficulty in continuously spinning the microfibers via in situ photocrosslinking in extreme endovascular environments such as those involving a tortuous geometry and high absorbance. A double-crosslinked alginate-based hydrogel with tantalum nanopowder (DAT) that exploits the synergistic effect of covalent crosslinking by visible-light irradiation and ionic crosslinking using Ca2+ , which is present in the blood, is developed in this study. Furthermore, an effective strategy to design and produce an optical-fiber-integrated microfluidic device (OFI-MD) that can continuously spin hydrogel microfibers via in situ photocrosslinking in extreme endovascular environments is proposed. As an embolic material, DAT exhibits promising characteristics such as radiopacity, nondissociation, nonswelling, and constant mechanical strength in blood, in addition to excellent cyto- and hemo-compatibilities. Using OFI-MD to spin DAT microfibers continuously can help fill aneurysms safely, uniformly, and completely within the endovascular simulator without generating microscopic fragments, which demonstrates its potential as an effective embolization strategy.

Production of Tyrian purple indigoid dye from tryptophan in Escherichia coli.

Tyrian purple, mainly composed of 6,6'-dibromoindigo (6BrIG), is an ancient dye extracted from sea snails and was recently demonstrated as a biocompatible semiconductor material. However, its synthesis remains limited due to uncharacterized biosynthetic pathways and the difficulty of regiospecific bromination. Here, we introduce an effective 6BrIG production strategy in Escherichia coli using tryptophan 6-halogenase SttH, tryptophanase TnaA and flavin-containing monooxygenase MaFMO. Since tryptophan halogenases are expressed in highly insoluble forms in E. coli, a flavin reductase (Fre) that regenerates FADH2 for the halogenase reaction was used as an N-terminal soluble tag of SttH. A consecutive two-cell reaction system was designed to overproduce regiospecifically brominated precursors of 6BrIG by spatiotemporal separation of bromination and bromotryptophan degradation. These approaches led to 315.0 mg l-1 6BrIG production from tryptophan and successful synthesis of regiospecifically dihalogenated indigos. Furthermore, it was demonstrated that 6BrIG overproducing cells can be directly used as a bacterial dye.

Characterization of a Tryptophan 6-Halogenase from Streptomyces albus and Its Regioselectivity Determinants.

Tryptophan halogenases are found in diverse organisms and catalyze regiospecific halogenation. They play an important role in the biosynthesis of halogenated indole alkaloids, which are biologically active and of therapeutic importance. Here, a tryptophan 6-halogenase (SatH) from Streptomyces albus was characterized by using a whole-cell reaction system in Escherichia coli. SatH showed substrate specificity for chloride and bromide ions, leading to regiospecific halogenation at the C6-position of l-tryptophan. In addition, SatH exhibited higher performance in bromination than that of previously reported tryptophan halogenases in the whole-cell reaction system. Through structure-based protein mutagenesis, it has been revealed that two consecutive residues, A78/V79 in SatH and G77/I78 in PyrH, are key determinants in the regioselectivity difference between tryptophan 6- and 5-halogenases. Substituting the AV with GI residues switched the regioselectivity of SatH by moving the orientation of tryptophan. These data contribute to an understanding of the key residues that determine the regioselectivity of tryptophan halogenases.

Sugar-mediated regulation of a c-di-GMP phosphodiesterase in Vibrio cholerae.

Biofilm formation protects bacteria from stresses including antibiotics and host immune responses. Carbon sources can modulate biofilm formation and host colonization in Vibrio cholerae, but the underlying mechanisms remain unclear. Here, we show that EIIAGlc, a component of the phosphoenolpyruvate (PEP):carbohydrate phosphotransferase system (PTS), regulates the intracellular concentration of the cyclic dinucleotide c-di-GMP, and thus biofilm formation. The availability of preferred sugars such as glucose affects EIIAGlc phosphorylation state, which in turn modulates the interaction of EIIAGlc with a c-di-GMP phosphodiesterase (hereafter referred to as PdeS). In a Drosophila model of V. cholerae infection, sugars in the host diet regulate gut colonization in a manner dependent on the PdeS-EIIAGlc interaction. Our results shed light into the mechanisms by which some nutrients regulate biofilm formation and host colonization.

Production of 12-hydroxy dodecanoic acid methyl ester using a signal peptide sequence-optimized transporter AlkL and a novel monooxygenase.

In this study, a signal peptide of AlkL was replaced with other signal peptides to improve the soluble expression and thereby facilitate the transport of dodecanoic acid methyl ester (DAME) substrate into the E. coli. Consequently, AlkL with signal peptide FadL (AlkLf) showed higher transport activity toward DAME. Furthermore, the promoter optimization for the efficient heterologous expression of the transporter AlkLf and alkane monooxygenase (AlkBGT) system was conducted and resulted in increased ω-oxygenation activity of AlkBGT system. Moreover, bioinformatic studies led to the identification of novel monooxygenase from Pseudomonas pelagia (Pel), which exhibited 20% higher activity towards DAME as substrate compared to AlkB. Finally, the construction of a chimeric transporter and the expression of newly identified monooxygenase enabled the production of 44.8 ±â€¯7.5 mM of 12-hydroxy dodecanoic acid methyl ester (HADME) and 31.8 ±â€¯1.7 mM of dodecanedioic acid monomethyl ester (DDAME) in a two-phase reaction system.

In silico identification of metabolic engineering strategies for improved lipid production in Yarrowia lipolytica by genome-scale metabolic modeling.

BACKGROUND: Yarrowia lipolytica, an oleaginous yeast, is a promising platform strain for production of biofuels and oleochemicals as it can accumulate a high level of lipids in response to nitrogen limitation. Accordingly, many metabolic engineering efforts have been made to develop engineered strains of Y. lipolytica with higher lipid yields. Genome-scale model of metabolism (GEM) is a powerful tool for identifying novel genetic designs for metabolic engineering. Several GEMs for Y. lipolytica have recently been developed; however, not many applications of the GEMs have been reported for actual metabolic engineering of Y. lipolytica. The major obstacle impeding the application of Y. lipolytica GEMs is the lack of proper methods for predicting phenotypes of the cells in the nitrogen-limited condition, or more specifically in the stationary phase of a batch culture. RESULTS: In this study, we showed that environmental version of minimization of metabolic adjustment (eMOMA) can be used for predicting metabolic flux distribution of Y. lipolytica under the nitrogen-limited condition and identifying metabolic engineering strategies to improve lipid production in Y. lipolytica. Several well-characterized overexpression targets, such as diglyceride acyltransferase, acetyl-CoA carboxylase, and stearoyl-CoA desaturase, were successfully rediscovered by our eMOMA-based design method, showing the relevance of prediction results. Interestingly, the eMOMA-based design method also suggested non-intuitive knockout targets, and we experimentally validated the prediction with a mutant lacking YALI0F30745g, one of the predicted targets involved in one-carbon/methionine metabolism. The mutant accumulated 45% more lipids compared to the wild-type. CONCLUSION: This study demonstrated that eMOMA is a powerful computational method for understanding and engineering the metabolism of Y. lipolytica and potentially other oleaginous microorganisms.

Oscillatory flow-assisted efficient target enrichment with small volumes of sample by using a particle-based microarray device.

In the study, we describe an oscillatory flow-assisted efficient target enrichment method by using a particle-based microarray device. Periodic oscillating flow effectively increased the mixing and binding performance between the target molecules in the sample solution and surface functionalized microparticles. Particles were trapped, secured, and released with an elastic microvalve structure operated via differences in the flow conditions. Single particle (20-µm diameter) trapping efficiency exceeded 95%. Secured particles can freely move inside each array element based on oscillating sample flow. Furthermore, the particles can be released from the array and collected at the outlet of the device, and this provides an opportunity for further off-chip analysis. As a proof-of-concept, we used the interaction between streptavidin-coated microparticles and fluorescence labeled biotin solution and demonstrated that target enrichment and detection based on oscillatory flow were significantly more efficient than that based on unidirectional or static flow. The applicability of the method was further examined by conducting an on-chip immunoassay to detect the presence of anti-Zika nonstructural protein 1 (NS1) monoclonal antibody. The limit of detection (LOD) was as low as 1 ng/mL with an assay time of only 10 min and less than 10 µL of sample consumption.