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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.

To facilitate the interrogation of proteins at scale, we have developed High-throughput Insertion of Tags Across the Genome (HITAG). HITAG enables users to produce libraries of cells, each with a different protein of interest C-terminally tagged, to rapidly characterize protein function. To demonstrate the utility of HITAG, we fused mCherry to a set of 167 stress granule-associated proteins and characterized the factors which drive proteins to strongly accumulate within stress granules.

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. A two-cell setup containing tryptophanase, a flavin-dependent monooxygenase and a regiospecific halogenase (linked to a flavin reductase as a solubility tag) enables the production of 6,6'-dibromoindigo and other indigoid dyes in Escherichia coli.

Conventional 3D printing methods require the addition of a supporting layer in order to accurately and reliably fabricate the desired final product. However, the use of supporting material is not economically viable, and during the process of removing the supporting material, the shape or the properties of the final product may be distorted. In our previous work, we proposed and demonstrated the concept of a new 3D printing method that utilizes the in situ light as a guide for the fabrication of freestanding overhanging structures without the need for supporting material. In this study, the influence of the light intensity on the diameter of the structure and the thickness of the layer produced per droplet is analyzed in order to identify the geometric range of structures that can be fabricated by the new 3D printing method. As the intensity of the light increased, the diameter of the structure also increased and the thickness of the layer per droplet decreased. This result is determined by a combination of factors; (1) the rebound motion of the photocurable droplet and (2) the surface area of the structure that needs to be covered.

In this paper, a design method to develop a bending-insensitive capacitive-type touch sensor is reported. To achieve bending-insensitive properties and a shielding effect to block the parasitic cap...

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.

Several regiospecific enantiomers of hydroxy-(

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

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.

In this talk, we will discuss an effective strategy to design and fabricate 3D vascular replicas composed of elastomer - hydrogel skin multilayers mimicking the geometries and mechanical properties of blood vessels, enabling a realistic simulation of endovascular intervention and more.

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., Carbon sources can modulate biofilm formation and host colonization in Vibrio cholerae. Here, Heo et al. show that this process is mediated by a component of the PEP:carbohydrate phosphotransferase system (PTS), which regulates c-di-GMP hydrolysis by interacting with a c-di-GMP phosphodiesterase.

We propose a microarray-based microfluidic platform that uses a continuous single-phase flow-assisted isolation (CSFI) method to conduct multiplex analysis of reactions between co-located heteroparticles in a single-phase aqueous solution. The CSFI method is performed by consistently and continuously introducing an aqueous-phase fluid through microchannels. Hence, the unit elements of a particle pair array can be isolated without interelement cross-contamination, as they exist in independent air curtains. The formation of particle pairs with different sizes is accomplished by sequentially loading microparticles without changing the flow directions, which achieves a particle pairing efficiency up to 91%. The immunoreaction between the receptor-coated beads and the fluorescence-labeled analytes encapsulated within the hydrogels was used as a proof of concept. The analytes were diffused from the hydrogels and only treated the paired receptor-coated beads under continuous single-phase flow. Parallel monitoring of reactions on the receptor-coated beads was conducted using the CSFI method. By characterizing the operation flow rate of the CSFI method, we demonstrated the prevention of interelement cross-contamination and the enhancement of binding efficiency on receptor-coated beads. [2019-0114]

Indirubin is a biologically active compound found in Danggui Longhui Wan, which is a traditional Chinese medicine for chronic myelocytic leukemia. In the biosynthesis of indirubin, the formation of...

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.

In this study, the biosynthesis of various indigoids with novel spectral features and antibacterial activities was investigated. First, 12 indole derivatives as substrates were biotransformed into functional indigoid dyes by E. coli cells expressing CYP102G4 hydroxylase. The indole derivatives included chloro (Cl-), nitro (NO2-), hydroxy (HO-), methoxy (CH3O-), methyl (CH3-), carboxy (COOH-), amino (NH3-), and cyano (CN-) indoles at the C4 to C7 positions. Interestingly, dramatic color shifts were observed from blue to red, green, purple, and even pink depending on the functional groups and their positions. Next, the biological and physical properties, antibacterial effects, and dying fastness of the prepared compounds were investigated and visually measured. Among the synthesized indigoid dyes, 6,6’-dichloroindigo and 5,5’-dichloroindigo showed the relatively higher cell growth inhibitory activity in the liquid phase. Finally, a one-pot producing strain which produced 7,7’-dichloroindigo from l -tryptophan using tryptophan-7-halogenase (PrnA) and CYP102G4 simultaneously was developed to overcome the disadvantages of uneconomical semi-synthesis through indole precursor feedstocks. The developed producing strain produced approximately 15.4 ± 1.4 mg/L of 7,7’-dichloroindigo in 24 h. To the best of our knowledge, this is the first report of the production of 7,7’-dichloroindigo in E. coli via a one-pot process.

Tragacanth, a highly branched carbohydrate polymer isolated from Astragalus, is one of the most commonly used gums in food industry. The primary structure of tragacanth is composed of galacturonic acid monomers connected with α 1-4 links, and it is very similar to the pectin. Tragacanth degradation by microorganisms is significant in two aspects: first, food preservation and microbial growth control due to too much use of tragacanth in the food industry, second, therapeutic and pharmaceutical potential of obtained oligosaccharides. In the present study, we report three new strains of bacteria, Acinetobacter guillouiae strain TD1, Kosakonia sacchari strain TD2, and Bacillus vallismortis strain PD1 with the capability of growing in tragacanth as an only source of carbon and energy. The evolutionary history of the isolated strains was analyzed based on 16S rRNA gene sequences in MEGA7 using the neighbor-joining method. The production of di and tri galacturonic acid due to pectinase activities of the strains were detected by thin layer chromatography (TLC) and liquid chromatography/Mass spectroscopy (LC/MS) analysis. Here is the first report of the ability to grow in tragacanth and pectinase activity monitoring in bacteria. Our results revealed that all of the isolated strains are capable of degrading pectin and tragacanth to oligo-galacturonic acids. The obtained products, which have different structures depending on the tragacanth structures and types of pectinolytic enzymes, would show therapeutic and pharmaceutical potentials.

Transcriptomics is now recognized as a primary tool for metabolic engineering as it can be used for identifying new strain designs by diagnosing current states of microbial cells. This review summarizes current application of transcriptomic data for strain design. Along with a few successful examples, limitations of conventionally used differentially expressed gene-based strain design approaches have been discussed, which have been major reasons why transcriptomic data are considerably underutilized. Recently, integrative network-based approaches interpreting transcriptomic data in the context of biological networks were invented to provide complimentary solutions for metabolic engineering by overcoming the limitations of conventional approaches. Here, we highlight recent pioneering studies in which integrative network-based methods have been used for providing novel strain designs.

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

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.

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%).

In this study, the spring constant of an accelerometer with a liquid-type proof mass was analyzed. Unlike a general solid-type microelectromechanical system accelerometer, the Laplace pressure is considered a restoring force in the analyzed accelerometer. Using a base excitation mathematical model, the sensor output could be estimated for a specific spring constant. Although the estimated sensor output data fit well with the experimental results, the spring constant of the device could also be determined dynamically (for oscillations below 5 Hz). Moreover, the damping constants could be inferred depending on whether sandblasting treatment was performed. Finally, the effects of the oscillation, surface condition, and volume of liquid metal droplets on the spring constant were analyzed.

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 FADH

Although several biomarkers can be used to distinguish cholangiocarcinoma (CCA) from healthy controls, differentiating the disease from benign biliary disease (BBD) or pancreatic cancer (PC) is a challenge. CCA biomarkers are associated with low specificity or have not been validated in relation to the biological effects of CCA. In this study, we quantitatively analyzed 15 biliary bile acids in CCA (n = 30), BBD (n = 57) and PC (n = 17) patients and discovered glycocholic acid (GCA) and taurochenodeoxycholic acid (TCDCA) as specific CCA biomarkers. Firstly, we showed that the average concentration of total biliary bile acids in CCA patients was quantitatively less than in other patient groups. In addition, the average composition ratio of primary bile acids and conjugated bile acids in CCA patients was the highest in all patient groups. The average composition ratio of GCA (35.6%) in CCA patients was significantly higher than in other patient groups. Conversely, the average composition ratio of TCDCA (13.8%) in CCA patients was significantly lower in all patient groups. To verify the biological effects of GCA and TCDCA, we analyzed the gene expression of bile acid receptors associated with the development of CCA in a CCA cell line. The gene expression of transmembrane G protein coupled receptor (TGR5) and sphingosine 1-phosphate receptor 2 (S1PR2) in CCA cells treated with GCA was 8.6-fold and 3.4-fold higher compared with control (untreated with bile acids), respectively. Gene expression of TGR5 and S1PR2 in TCDCA-treated cells was not significantly different from the control. Taken together, our study identified GCA and TCDCA as phenotype-specific biomarkers for CCA.

A potent phytoestrogen, (S)-equol, is a promising isoflavone derivative drawing our great attention owing to its various biological and clinical benefits. Through selective activation of the estrogen receptor ERβ or androgen receptor, (S)-equol reduces menopausal symptoms, osteoporosis, skin aging, hair loss, and incidence of prostate or ovarian cancers without adverse effects. Traditional biosynthesis of (S)-equol exploited non-productive natural equol-producing anaerobic bacteria that mainly belong to Coriobacteriaceae isolated from human intestine. Recently, we developed a recombinant Escherichia coli strain which could convert daidzein into (S)-equol effectively under an aerobic condition. However, the yield was limited up to about the 200 mg/L level due to unknown reasons. In this study, we identified that the bottleneck of the limited production was the low solubility of isoflavone (i.e., 2.4 mg/L) in the reaction medium. In order to solve the solubility problem without harmful effect to the whole-cell catalyst, we applied commercial hydrophilic polymers (HPs) and a polar aprotic co-solvent in the reaction medium. Among the examined water-soluble polymers, polyvinylpyrrolidone (PVP)-40k was verified as the most promising supplement which increased daidzein solubility by 40 times and (S)-equol yield up to 1.22 g/L, the highest ever reported and the first g/L level biotransformation. Furthermore, PVP-40k was verified to significantly increase the solubilities of other water-insoluble natural polyphenols in aqueous solution. We suggest that addition of both HP and polar aprotic solvent in the reaction mixture is a powerful alternative to enhance production of polyphenolic chemicals rather than screening appropriate organic solvents for whole-cell catalysis of polyphenols.

Surface repellency to high and low surface tension (LST) liquids has a wide range of commercial applications such as non-fouling, self-cleaning and separation of liquids. However, little research has been achieved to control surface repellency to LST liquids due to difficulty of developing re-entrant surface curvatures. Here, we report an effective method to control surface repellency of stainless steel meshes for liquids that have LST. We used electrochemical etching followed by hydrophobic coating where adjusting the voltage and duration of electrochemical etching controllably cover the mesh surfaces with nanoscale re-entrant structures. Variation in re-entrant surface curvatures determines the mesh's sensitivity to liquid-vapor surface tension, and changed the apparent contact angles and sliding angles for LST liquids. The difference in sensitivity of surface tension of mesh was exploited to rapidly separate liquids in binary mixtures of general solvents. The meshes achieve fast separation, and can be reused. This method to provide omniphobicity is compatible with large-scale production.

Prevention of biofouling and microbial contamination of implanted biomedical devices is essential to maintain their functionality and biocompatibility. For this purpose, polypept(o)ide block copolymers have been developed, in which a protein-resistant polysarcosine (pSar) block is combined with a dopamine-modified poly(glutamic acid) block for surface coating and silver nanoparticles (Ag NPs) formation. In the development of a novel, versatile, and biocompatible antibacterial surface coating, block lengths pSar were varied to derive structure-property relationships. Notably, the catechol moiety performs two important tasks in parallel; primarily it acts as an efficient anchoring group to metal oxide surfaces, while it furthermore induces the formation of Ag NPs. Attributing to the dual function of catechol moieties, antifouling pSar brush and antimicrobial Ag NPs can not only adhere stably on metal oxide surfaces, but also display passive antifouling and active antimicrobial activity, showing good biocompatibility simultaneously. The developed strategy seems to provide a promising platform for functional modification of biomaterials surface to preserve their performance while reducing the risk of bacterial infections.

This study presents a finger-triggered portable polydimethylsiloxane suction cup that enables equipment-free microfluidic pumping. The key feature of this method is that its operation only involves a “pressing-and-releasing” action for the cup placed at the outlet of a microfluidic device, which transports the fluid at the inlet toward the outlet through a microchannel. This method is simple, but effective and powerful. The cup is portable and can easily be fabricated from a three-dimensional printed mold, used without any pre-treatment, reversibly bonded to microfluidic devices without leakage, and applied to various material-based microfluidic devices. The effect of the suction cup geometry and fabrication conditions on the pumping performance was investigated. Furthermore, we demonstrated the practical applications of the suction cup by conducting an equipment-free pumping of thermoplastic-based microfluidic devices and water-in-oil droplet generation.

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.

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.