All-in-one platform: Versatile, Easy, and User-friendly System (VEUS) based on automated and expert-independent antibody immobilization and immunoassay by utilizing customized movement of magnetic particles.

The ELISA is the most worldwide method for immunoassay. However, the ELISA is losing ground due to low reproducibility of manual experimental processes in both R&D and IVD areas. An automated platform is a good solution, but there are still limitations owning to extremely high cost and requiring large space to set up especially for a small size laboratory. Here, we present a novel all-in-one platform called "VEUS" settable on the laboratory table that offers comprehensive automation of the entire multiplex immunoassay process by exploiting antibody conjugated magnetic particles, quality control and then immunoanalytical reaction, thereby enhancing detection sensitivity and high reproducibility. As a proof of concept, the system exhibits a sensitive LOD of 0.6 and 3.1 pg mL-1 within 1 h run, comparable precision that of molecular diagnostic systems based on PCR method, enabling rapid multiplex diagnosis of Influenza A, Influenza B, and COVID-19 viruses with similar symptoms. Through automation by the all-in-one system, it can be used by novice users, something innovative for immunoassays, relying heavily on user experience. Furthermore, it can contribute to streamline entire immunoassay processes of diverse biomarkers with high reproducibility and convenience in laboratories.

Bacterial Isolation Microwell-Plug (µWELLplug) for Rapid Antibiotic Susceptibility Testing Using Morphology Analysis.

The rapid and accurate diagnosis of infectious diseases with high morbidity rates is crucial because it can minimize the misuse and overuse of antibiotics and increase survival rates in dreadful conditions. The conventional antibiotic susceptibility test (AST) systems used to choose appropriate antibiotics require long wait times to obtain results and cannot prevent the misuse or overuse of antibiotics by clinicians who need to quickly treat patients and cannot wait to identify the underlying cause of their symptoms. Therefore, several rapid AST (rAST) methods have been developed to provide quick test results, but they are complicated to operate, require additional equipment or materials, and give less accurate results than the conventional AST methods. In this study, we propose an rAST method that can obtain precise outcomes from a simple process with a short running time using a bacterial isolation microwell-plug (µWELLplug) in a conventional 96-well plate. The specifically designed hydrogel component of the µWELLplug provides a simple process for cell isolation and the observation of bacterial growth and morphological changes induced by a variety of antibiotic concentrations. The µWELLplug is placed over each well of the 96-well plate, and then bacterial or eukaryotic cells are isolated in the microwells and treated with different antibiotic concentrations to observe their effects. Saccharomyces cerevisiae (yeast, eukaryote), Streptomyces atratus (actinomycetes, prokaryote), Escherichia coli, Staphylococcus aureus, and methicillin-resistant S. aureus were cultivated and tested using the µWELLplug. The minimum inhibitory concentration values from this system were obtained in 3-4 h and correlated well with those from the conventional AST methods whose running time is 18-24 h.

Marker Pen Device with Concentration Gradient Nib for Antibiotic Susceptibility Testing.

BACKGROUND: Pen-based devices have emerged as useful tools for measuring pH and glucose, and for fabricating microchannels and microarrays. Pen-based devices take advantage of flexible patterning, inexpensive costs, and small volumes, thereby saving time and increasing efficiency. We have developed a gradient nib marker pen device that generated simultaneously different antibiotic concentrations in bacteria antibiotic susceptibility testing (AST). METHODS: The device can deposit on the target surface with the antibiotic gradient. The designed polyester fiber nibs are a highly uniform porosity with unidirectional orientation and produce a visible gradient pattern. RESULTS: We have demonstrated and quantitatively analyzed bacterial growth after antibiotic marking. The antibiotic marking produces an inhibition zone of bacterial growth. The inhibition zones of bacterial growth are captured and converted to 8-bit grayscale images, and then quantified by gray values using the Image J program. A profile of the inhibition zone showed different gray values in response to bacterial viability. CONCLUSION: The gradient nib marker pen device can be used to determine the quantitative antibiotic concentration based on the relationship between gray values and bacterial density conveniently without requiring a series of dilution tubes, including nutrient medium, and diversely diluted antibiotics.

A rapid culture system uninfluenced by an inoculum effect increases reliability and convenience for drug susceptibility testing of Mycobacterium tuberculosis.

The Disc Agarose Channel (DAC) system utilizes microfluidics and imaging technologies and is fully automated and capable of tracking single cell growth to produce Mycobacterium tuberculosis (MTB) drug susceptibility testing (DST) results within 3~7 days. In particular, this system can be easily used to perform DSTs without the fastidious preparation of the inoculum of MTB cells. Inoculum effect is one of the major problems that causes DST errors. The DAC system was not influenced by the inoculum effect and produced reliable DST results. In this system, the minimum inhibitory concentration (MIC) values of the first-line drugs were consistent regardless of inoculum sizes ranging from ~103 to ~108 CFU/mL. The consistent MIC results enabled us to determine the critical concentrations for 12 anti-tuberculosis drugs. Based on the determined critical concentrations, further DSTs were performed with 254 MTB clinical isolates without measuring an inoculum size. There were high agreement rates (96.3%) between the DAC system and the absolute concentration method using Löwenstein-Jensen medium. According to these results, the DAC system is the first DST system that is not affected by the inoculum effect. It can thus increase reliability and convenience for DST of MTB. We expect that this system will be a potential substitute for conventional DST systems.

Bedaquiline susceptibility test for totally drug-resistant tuberculosis Mycobacterium tuberculosis.

This study aimed to provide information that bedaquilline is significantly effective for treatment of totally drug resistant (TDR) Mycobacterium tuberculosis that shows resistant to all first- and second-line drugs-using an innovative disc agarose channel (DAC) system. Time-lapse images of single bacterial cells under culture conditions with different concentrations of bedaquiline were analysed by image processing software to determine minimum inhibitory concentrations (MICs). Bedaquiline inhibited the growth of TDR M. tuberculosis strains, with MIC values ranging from 0.125 to 0.5 mg/L. The results of the present study demonstrate that bedaquiline, newly approved by the United States Food and Drug Administration (FDA), may offer therapeutic solutions for TDR-TB.

Rapid drug susceptibility test of Mycobacterium tuberculosis using microscopic time-lapse imaging in an agarose matrix.

Tuberculosis (TB) is a major global health problem, and multi-drug-resistant TB (MDR-TB) and extensively drug-resistant TB (XDR-TB) are spreading throughout the world. However, conventional drug susceptibility test (DST) methods, which rely on the detection of the colony formation on a solid medium, require 1-2 months to the result. A rapid and accurate DST is necessary to identify patients with drug-resistant TB and treat them with appropriate drugs. Here, we used microscopic imaging of Mycobacterium tuberculosis (MTB) immobilized in an agarose matrix for a rapid DST. The agarose matrix, which was molded in a microfluidic chip, was inoculated with MTB, and TB drugs in liquid culture medium diffused throughout the agarose to reach the MTB immobilized in the agarose matrix. After the responses of MTB to drugs were tracked with an automated microscopic system, an image-processing program automatically determined the susceptibility and resistance of MTB to specific doses of TB drugs. The automatic DST system was able to assess the drug susceptibility of various drug-resistant clinical TB strains within 9 days with an accuracy comparable to that of conventional method. Our rapid DST method based on microscopic time-lapse imaging greatly reduces the time required for a DST and can be used to rapidly and accurately treat TB patients.

Embedded biofilm, a new biofilm model based on the embedded growth of bacteria.

A variety of systems have been developed to study biofilm formation. However, most systems are based on the surface-attached growth of microbes under shear stress. In this study, we designed a microfluidic channel device, called a microfluidic agarose channel (MAC), and found that microbial cells in the MAC system formed an embedded cell aggregative structure (ECAS). ECASs were generated from the embedded growth of bacterial cells in an agarose matrix and better mimicked the clinical environment of biofilms formed within mucus or host tissue under shear-free conditions. ECASs were developed with the production of extracellular polymeric substances (EPS), the most important feature of biofilms, and eventually burst to release planktonic cells, which resembles the full developmental cycle of biofilms. Chemical and genetic effects have also confirmed that ECASs are a type of biofilm. Unlike the conventional biofilms formed in the flow cell model system, this embedded-type biofilm completes the developmental cycle in only 9 to 12 h and can easily be observed with ordinary microscopes. We suggest that ECASs are a type of biofilm and that the MAC is a system for observing biofilm formation.

A rapid antimicrobial susceptibility test based on single-cell morphological analysis.

A rapid antibiotic susceptibility test (AST) is desperately needed in clinical settings for fast and appropriate antibiotic administration. Traditional ASTs, which rely on cell culture, are not suitable for urgent cases of bacterial infection and antibiotic resistance owing to their relatively long test times. We describe a novel AST called single-cell morphological analysis (SCMA) that can determine antimicrobial susceptibility by automatically analyzing and categorizing morphological changes in single bacterial cells under various antimicrobial conditions. The SCMA was tested with four Clinical and Laboratory Standards Institute standard bacterial strains and 189 clinical samples, including extended-spectrum ß-lactamase-positive Escherichia coli and Klebsiella pneumoniae, imipenem-resistant Pseudomonas aeruginosa, methicillin-resistant Staphylococcus aureus, and vancomycin-resistant Enterococci from hospitals. The results were compared with the gold standard broth microdilution test. The SCMA results were obtained in less than 4 hours, with 91.5% categorical agreement and 6.51% minor, 2.56% major, and 1.49% very major discrepancies. Thus, SCMA provides rapid and accurate antimicrobial susceptibility data that satisfy the recommended performance of the U.S. Food and Drug Administration.

Rotated domains in chemical vapor deposition-grown monolayer graphene on Cu(111): an angle-resolved photoemission study.

Copper is considered to be the most promising substrate for the growth of high-quality and large area graphene by chemical vapor deposition (CVD), in particular, on the (111) facet. Because the interactions between graphene and Cu substrates influence the orientation, quality, and properties of the synthesized graphene, we studied the interactions using angle-resolved photoemission spectroscopy. The evolution of both the Shockley surface state of the Cu(111) and the π band of the graphene was measured from the initial stage of CVD growth to the formation of a monolayer. Graphene growth was initiated along the Cu(111) lattice, where the Dirac band crossed the Fermi energy (EF) at the K point without hybridization with the d-band of Cu. Then two rotated domains were additionally grown as the area covered with graphene became wider. The Dirac energy was about -0.4 eV and the energy of the Shockley surface state of Cu(111) shifted toward the EF by ~0.15 eV upon graphene formation. These results indicate weak interactions between graphene and Cu, and that the electron transfer is limited to that between the Shockley surface state of Cu(111) and the π band of graphene. This weak interaction and slight lattice mismatch between graphene and Cu resulted in the growth of rotated graphene domains (9.6° and 8.4°), which showed no significant differences in the Dirac band with respect to different orientations. These rotated graphene domains resulted in grain boundaries which would hinder a large-sized single monolayer growth on Cu substrates.

Rapid antibiotic susceptibility testing by tracking single cell growth in a microfluidic agarose channel system.

Sepsis is one of the major causes of death in the US, necessitating rapid treatment with proper antibiotics. Conventional systems for antibiotic susceptibility testing (AST) take far too long (16-24 h) for the timely treatment of sepsis. This is because they rely on measuring optical density, which relates to bacterial growth, to determine the minimal inhibitory concentrations (MICs) of relevant antibiotics. Thus, there is a desperate need for more improved and rapid AST (RAST) systems. The RAST system can also reduce the growing number of clinical problems that are associated with antibiotic resistance caused by methicillin-resistant Staphylococcus aureus, vancomycin-resistant Staphylococcus aureus, and vancomycin-resistant enterococci. In this study, we demonstrate a microfluidic agarose channel (MAC) system that reduces the AST assay time for determining MICs by single bacterial time lapse imaging. The MAC system immobilizes bacteria by using agarose in a microfluidic culture chamber so that single cell growth can be tracked by microscopy. Time lapse images of single bacterial cells under different antibiotic culture conditions were analyzed by image processing to determine MICs. Three standard bacteria from the Clinical and Laboratory Standard Institute (CLSI) were tested with several kinds of antibiotics. MIC values that were well matched with those of the CLSI were obtained within only 3-4 h. We expect that the MAC system can offer rapid diagnosis of sepsis and thus, more efficient and proper medication in the clinical setting.

The Ser/Thr protein kinase AfsK regulates polar growth and hyphal branching in the filamentous bacteria Streptomyces.

In cells that exhibit apical growth, mechanisms that regulate cell polarity are crucial for determination of cellular shape and for the adaptation of growth to intrinsic and extrinsic cues. Broadly conserved pathways control cell polarity in eukaryotes, but less is known about polarly growing prokaryotes. An evolutionarily ancient form of apical growth is found in the filamentous bacteria Streptomyces, and is directed by a polarisome-like complex involving the essential protein DivIVA. We report here that this bacterial polarization machinery is regulated by a eukaryotic-type Ser/Thr protein kinase, AfsK, which localizes to hyphal tips and phosphorylates DivIVA. During normal growth, AfsK regulates hyphal branching by modulating branch-site selection and some aspect of the underlying polarisome-splitting mechanism that controls branching of Streptomyces hyphae. Further, AfsK is activated by signals generated by the arrest of cell wall synthesis and directly communicates this to the polarisome by hyperphosphorylating DivIVA. Induction of high levels of DivIVA phosphorylation by using a constitutively active mutant AfsK causes disassembly of apical polarisomes, followed by establishment of multiple hyphal branches elsewhere in the cell, revealing a profound impact of this kinase on growth polarity. The function of AfsK is reminiscent of the phoshorylation of polarity proteins and polarisome components by Ser/Thr protein kinases in eukaryotes.

Determinants of redox sensitivity in RsrA, a zinc-containing anti-sigma factor for regulating thiol oxidative stress response.

Various environmental oxidative stresses are sensed by redox-sensitive regulators through cysteine thiol oxidation or modification. A few zinc-containing anti-sigma (ZAS) factors in actinomycetes have been reported to respond sensitively to thiol oxidation, among which RsrA from Streptomyces coelicolor is best characterized. It forms disulfide bonds upon oxidation and releases bound SigR to activate thiol oxidative stress response genes. Even though numerous ZAS proteins exist in bacteria, features that confer redox sensitivity to a subset of these have been uncharacterized. In this study, we identified seven additional redox-sensitive ZAS factors from actinomycetes. Comparison with redox-insensitive ZAS revealed characteristic sequence patterns. Domain swapping demonstrated the significance of the region K(33)FEHH(37)FEEC(41)SPC(44)LEK(47) that encompass the conserved HX(3)CX(2)C (HCC) motif. Mutational effect of each residue on diamide responsive induction of SigR target genes in vivo demonstrated that several residues, especially those that flank two cysteines (E39, E40, L45, E46), contribute to redox sensitivity. These residues are well conserved among redox-sensitive ZAS factors, and hence are proposed as redox-determinants in sensitive ZAS. H37A, C41A, C44A and F38A mutations, in contrast, compromised SigR-binding activity significantly, apparently affecting structural integrity of RsrA. The residue pattern around HCC motif could therefore serve as an indicator to predict redox-sensitive ZAS factors from sequence information.

Capillary based patterning of cellular communities in laterally open channels.

In order to offer an easier way to study interactions between multiple cellular populations, we have developed a novel method to precisely place cells in a variety of nonoverlapping patterns using surface tension in laterally open microchannels. Our design is fundamentally different from previous strategies such as compartmentalization, stamping, stenciling, or mechanical approaches. It relies on capillary action or the propensity for liquid to move more readily through narrow spaces as a result of surface tension. Until now, capillary based patterning has been limited to coating chemically isolated areas. Here, we demonstrate, through use of surface tension and controlled flooding, that it is possible to pattern multiple cells and proteins using laterally open channels in a variety of designs. We demonstrate the relevance of the concept by coculturing different mammalian cell types and evaluating the behavior of engineered quorum sensing circuits in E. coli. In the future, we believe the laterally open channel designs shown here can be useful for rapidly creating and studying cellular ecologies using simple pipetting.

Isolation and characterization of bluensomycin biosynthetic genes from Streptomyces bluensis.

The biosynthetic gene cluster for bluensomycin, a member of the aminoglycoside family of antibiotics, was isolated and characterized from the bluensomycin producing strain, Streptomyces bluensis ATCC27420. PCR primers were designed specifically to amplify a segment of the dTDP-glucose synthase gene based on its conserved sequences among several actinomycete strains. By screening a cosmid library using amplified PCR fragments, a 30-kb DNA fragment was isolated. Sequence analysis identified 15 open reading frames (ORFs), eight of which had previously been identified by Piepersberg et al. But seven are novel to this study. We demonstrated that one of these ORFs, blmA, confers resistance against the antibiotic dihydrostreptomycin, and another, blmD, encodes a dTDP-glucose synthase. These findings suggest that the isolated gene cluster is very likely to be responsible for the biosynthesis of bluensomycin.