The impact of pH and temperature on the green gold nanoparticles preparation using Jeju Hallabong peel extract for biomedical applications.

The utilization of gold nanoparticles (AuNPs) has garnered significant attention in recent times, particularly in the field of biomedical research. The utilization of AuNPs in chemical synthesis procedures raises apprehensions regarding their potential toxicity in living organisms, which is inconsistent with their purported eco-friendly and cost-effective aspects. In this investigation, AuNPs were synthesized via the green synthesis approach utilizing Jeju Hallabong peel extract (HPE), a typical fruit variety indigenous to South Korea. The visible-range absorption spectrum of gold nanoparticles from green synthesis (HAuNPs) that are red wine in color occurs at a wavelength of λ = 517 nm. The morphology and particle size distribution were analysed using transmission electron microscopy (TEM) and ImageJ software. The TEM images reveal that the HAuNPs exhibit a high degree of dispersion and uniformity in their spherical shape, with an average size of approximately 7 nm. Moreover, elevating the initial pH level of the mixed solution has an impact on the decrease in particle dimensions, as evidenced by the blue shift observed in the UV-visible spectroscopy absorbance peak. Elevating the reaction temperature may accelerate the synthesis duration. However, it does not exert a substantial impact on the particle dimensions. The outcomes of an avidin-biocytin colorimetric assay provide preliminary analyses of possible sensor tunability using HAuNPs. The cytotoxicity of HAuNPs was evaluated through in vitro studies using the MTT assay on RAW 264.7 cell lines. The results indicated that the HAuNPs exhibited lower cytotoxicity compared to both chemically reduced gold nanoparticles (CAuNPs).

A Poisson-Independent Approach to Precision Nucleic Acid Quantification in Microdroplets.

Digital PCR (dPCR) has become indispensable in nucleic acid (NA) detection across various fields, including viral diagnostics and mutant detection. However, misclassification of partitions in dPCR can significantly impact accuracy. Despite existing methods to minimize misclassification bias, accurate classification remains elusive, especially for nonamplified target partitions. To address these challenges, this study introduces an innovative microdroplet-based competitive PCR platform for nucleic acid quantification in microfluidic devices independent of Poisson statistics. In this approach, the target concentration (T) is determined from the concentration of competitor DNA (C) at the equivalence point (E.P.), where C/T is 1. Competitive PCR ensures that the ratio of target to competitor DNA remains constant during amplification, reflected in the resultant fluorescence intensity, allowing the quantification of target DNA concentration at the equivalence point. The unique amplification technique eliminates Poisson distribution, addressing misclassification challenges. Additionally, our approach reduces the need for post-PCR procedures and shortens analytical time. We envision this platform as versatile, reproducible, and easily adaptable for driving significant progress in molecular biology and diagnostics.

Optimization of photocatalytic ceramic membrane filtration by response surface methodology: Effects of hydrodynamic conditions on organic fouling and removal efficiency.

The effects of the operating conditions, including the applied pressure, feed organic concentration, and recirculation flowrate along the TiO2-coated ceramic membrane, on the normalized membrane permeability and organic removal efficiency were systematically investigated by operating a photocatalytic membrane reactor (PMR). Response surface methodology (RSM) was conducted to better understand the interactive effect of operational conditions as well as their individual and combined effects to control membrane performance. Our results showed that the applied pressure and feed organic concentration, as single parameter, affected the normalized membrane permeability and organic removal efficiency more dominantly than the recirculation flowrate. The polynomial performance equations generated by RSM successfully predicted the membrane performance of the PMR. The responses to the normalized membrane permeability and organic removal efficiency with respect to the operational conditions were less sensitive to any combination of operational conditions than to their individual impacts. The combined effects of the operating conditions were less pronounced in promoting the catalytic performance of organic contaminants on the TiO2 surface. Our RSM analysis based on experimental observations designed by Box-Behnken Design (BBD) suggested that 1.3 bar of applied pressure, 44 mg/L of feed organic dye concentration and 0.8 L/min as recirculation flowrate as optimum conditions achieved more than 98% of organic removal efficiency and less than 5% of decline in normalized membrane permeability. This research shows that the RSM provides effective tool to optimize operational conditions to determine fouling rate and organic removal in PMR.

Solvent-Free Fabrication of Anisotropic Microparticles with Precise 3D Shape Control Using Dipping-Based Micromolding.

We present an innovative solvent-free micromolding technique for rapidly fabricating complex polymer microparticles with three-dimensional (3D) shapes utilizing a surface tension-induced dipping process. Our fabrication process involves loading a photocurable solution into micromolds through mold dipping. The loaded solution, induced by surface tension, undergoes spatial deformation upon mold removal caused by surface forces, ultimately acquiring an anisotropic shape before photopolymerization. Results show that the amount of photocurable solution loaded depends on the degree of capillary penetration, which can be adjusted by varying the dipping time and mold height. It enables the production of polymer particles with precisely controlled 3D shapes without diluting them with volatile organic solvents. Sequential micromolding enables the spatial stacking of the polymer domain through a bottom-up approach, facilitating the creation of complex multicompartmental microparticles with independently controlled compartments. Finally, we demonstrated the successful simultaneous conjugation of multiple model-fluorescent proteins through the biofunctionalization of microparticles, indicating functional stability and effective conjugation of hydrophilic molecules such as proteins. We also extend our capacity to create bicompartmental microparticles with distinct functionalities in each compartment, revealing spatially controlled functional structures. In summary, these findings demonstrate a straightforward, rapid, and reliable method for producing highly uniform complex particles with precise control over the 3D shape and compartmentalization, all accomplished without the use of organic solvents.

Hysteresis-Free, Elastic, and Tough Hydrogel with Stretch-Rate Independence and High Stability in Physiological Conditions.

Many existing synthetic hydrogels are inappropriate for repetitive motions because of large hysteresis, and their mechanical properties in warm and saline physiological conditions remain understudied. In this study, a stretch-rate-independent, hysteresis-free, elastic, and tough nanocomposite hydrogel that can maintain its mechanical properties in phosphate-buffered saline of 37 °C similar to warm and saline conditions of the human body is developed. The strength, stiffness, and toughness of the hydrogel are simultaneously reinforced by biomimetic silica nanoparticles with a surface of embedded circular polyamine chains. Such distinctive surfaces form robust interfacial interactions by local topological folding/entanglement with the polymer chains of the matrix. Load transfer from the soft polymer matrix to stiff nanoparticles, along with the elastic sliding/unfolding/disentanglement of polymer chains, overcomes the traditional trade-off between strength/stiffness and toughness and allows for hysteresis-free, strain-rate-independent, and elastic behavior. This robust reinforcement is sustained in warm phosphate-buffered saline. These properties demonstrate the application potential of the developed hydrogel as a soft, elastic, and tough bio-strain sensor that can detect dynamic motions across various deformation speeds and ranges. The findings provide a simple yet effective approach to developing practical hydrogels with a desirable combination of strength/stiffness and toughness, in a fully swollen and equilibrated state.

Development of fluorescence-linked immunosorbent assay for rapid detection of Staphylococcus aureus.

Staphylococcus aureus is a major pathogen that causes infections and life-threatening diseases. Although antibiotics, such as methicillin, have been used, methicillin-resistant S. aureus (MRSA) causes high morbidity and mortality rates, and conventional detection methods are difficult to be used because of time-consuming process. To control the spread of S. aureus, a development of a rapid and simple detection method is required. In this study, we generated a fluorescent anti-S. aureus antibody, and established a novel fluorescence-linked immunosorbent assay (FLISA)-based S. aureus detection method. The method showed high sensitivity and low limit of detection toward MRSA detection. The assay time for FLISA was 5 h, which was faster than that of conventional enzyme-linked immunosorbent assay (ELISA) or rapid ELISA. Moreover, the FLISA-based detection method was applied to diagnose clinically isolated MRSA samples that required only 5.3 h of preincubation. The FLISA method developed in this study can be widely applied as a useful tool for convenient S. aureus detection. KEY POINTS: • A fluorescence-linked immunosorbent assay-based S. aureus detection method • Simultaneous quantification of a maximum of 96 samples within 5 h • Application of the novel system to diagnosis clinical isolates.

Fabrication and Morphological Control of Nonspherical Alginate Hydrogel Particles.

We report a simple platform for the fabrication of nonspherical alginate hydrogel particles using a dripping method. Hydrogel particles with novel morphologies, such as vortex ring, teardrop, disk, sphere, and mushroom, are fabricated by controlling various parameters. We monitored the deformation process of the hydrogel particles after they penetrated the crosslinking solution using a high-speed camera. Then, we proposed a mechanism showing a unique morphological transformation from a spherical to a disk shape. We demonstrated how controlling the collecting height that causes the drop impact force against the crosslinking solution surface was critical to producing hydrogel particles with these intriguing shapes. In particular, disk-shaped alginate particles show their ability as potential platforms for culturing mouse adrenocortical tumor cells (Y1) and a hippocampal neuronal cell (HT-22). To modify alginate particles, cell-adhesive gelatin is incorporated into the alginate matrix and then alginate particles are coated with poly(allylamine hydrochloride). Two modified alginate particles show good adhesion and proliferation rates on their surfaces. In particular, the hybrid hydrogel particles provide great potential to be developed into promising materials for cell culture, drug delivery, and tissue engineering.

Structure-Activity Relationship of Truncated 2,8-Disubstituted-Adenosine Derivatives as Dual A2A/A3 Adenosine Receptor Antagonists and Their Cancer Immunotherapeutic Activity.

Based on hA2AAR structures, a hydrophobic C8-heteroaromatic ring in 5'-truncated adenosine analogues occupies the subpocket tightly, converting hA2AAR agonists into antagonists while maintaining affinity toward hA3AR. The final compounds of 2,8-disubstituted-N6-substituted 4'-thionucleosides, or 4'-oxo, were synthesized from d-mannose and d-erythrono-1,4-lactone, respectively, using a Pd-catalyst-controlled regioselective cross-coupling reaction. All tested compounds completely antagonized hA2AAR, including 5d with the highest affinity (Ki,A2A = 7.7 ± 0.5 nM). The hA2AAR-5d X-ray structure revealed that C8-heteroaromatic rings prevented receptor activation-associated conformational changes. However, the C8-substituted compounds still antagonized hA3AR. Structural SAR features and docking studies supported different binding modes at A2AAR and A3AR, elucidating pharmacophores for receptor activation and selectivity. Favorable pharmacokinetics were demonstrated, in which 5d displayed high oral absorption, moderate half-life, and bioavailability. Also, 5d significantly improved the antitumor effect of anti-PD-L1 in vivo. Overall, this study suggests that the novel dual A2AAR/A3AR nucleoside antagonists would be promising drug candidates for immune-oncology.

Ferritin-nanocaged copper arsenite minerals with oxidative stress-amplifying activity for targeted cancer therapy.

We report copper(II) arsenite-encapsulated ferritin nanoparticles (CuAS-FNs) as oxidative stress-amplifying anticancer agents. The CuAS-FNs were fabricated through CuAS mineralization in the cavity of the FNs. The formation of crystalline CuAS complex minerals in the FNs was systematically identified using various analytical tools, including X-ray photoelectron spectroscopy (XPS) and transmission electron microscopy (TEM)-associated energy-dispersive X-ray spectroscopy (TEM-EDS). The CuAS-FNs showed pH-dependent release behavior, in which the CuAS mineral was effectively retained at physiological pH, in contrast, at lysosomal pH, the CuAS complex was dissociated to release arsenite and Cu2+ ions. At lysosomal pH, the release rate of arsenite (HAsO32-) and Cu2+ ions from the CuAS-FNs more accelerated than at physiological pH. Upon transferrin receptor-1-mediated endocytosis, the CuAS-FNs simultaneously released arsenite and Cu2+ ions in cells. The released arsenite ions can increase the intracellular concentration of hydrogen peroxide (H2O2), with which the Cu2+ ions can elevate the level of hydroxyl radicals (·OH) via Fenton-like reaction. Thus, the CuAS-FNs could target cancer cell through the recognizing ability of FNs and kill cancer cells by amplifying the ·OH level through the synergistic activity of Cu2+ and arsenic ions. Importantly, MCF-7 tumors were effectively suppressed by CuAS-FNs without systemic in vivo toxicity. Therefore, the CuAS-FNs is a promising class of Fenton-like catalytic nanosystem for cancer treatment.

Hemicyanine-Based Near-Infrared Fluorescence Off-On Probes for Imaging Intracellular and In Vivo Nitroreductase Activity.

Nitroreductase (NTR) has the ability to activate nitro group-containing prodrugs and decompose explosives; thus, the evaluation of NTR activity is specifically important in pharmaceutical and environmental areas. Numerous studies have verified effective fluorescent methods to detect and image NTR activity; however, near-infrared (NIR) fluorescence probes for biological applications are lacking. Thus, in this study, we synthesized novel NIR probes (NIR-HCy-NO2 1-3) by introducing a nitro group to the hemicyanine skeleton to obtain fluorescence images of NTR activity. Additionally, this study was also designed to propose a different water solubility and investigate the catalytic efficiency of NTR. NIR-HCy-NO2 inherently exhibited a low fluorescence background due to the interference of intramolecular charge transfer (ICT) by the nitro group. The conversion from the nitro to amine group by NTR induced a change in the absorbance spectra and lead to the intense enhancement of the fluorescence spectra. When assessing the catalytic efficiency and the limit of detection (LOD), including NTR activity imaging, it was demonstrated that NIR-HCy-NO2 1 was superior to the other two probes. Moreover, we found that NIR-HCy-NO2 1 reacted with type I mitochondrial NTR in live cell imaging. Conclusively, NIR-HCy-NO2 demonstrated a great potential for application in various NTR-related fields, including NTR activity for cell imaging in vivo.

Generation of a novel monoclonal antibody against inflammatory biomarker S100A8 using hybridoma technology.

OBJECTIVES: S100A8 is highly expressed in several inflammatory and oncological conditions. To address the current lack of a reliable and sensitive detection method for S100A8, we generated a monoclonal antibody with a high binding affinity to human S100A8 to enable early disease diagnosis. RESULTS: A soluble recombinant S100A8 protein with a high yield and purity was produced using Escherichia coli. Next, mice were immunized with recombinant S100A8 to obtain anti-human S100A8 monoclonal antibodies using hybridoma technology. Lastly, the high binding activity of the antibody was confirmed and its sequence was identified. CONCLUSIONS: This method, including the production of antigens and antibodies, will be useful for the generation of hybridoma cell lines that produce anti-S100A8 monoclonal antibodies. Moreover, the sequence information of the antibody can be used to develop a recombinant antibody for use in various research and clinical applications.

Multifunctional bone substitute using carbon dot and 3D printed calcium-deficient hydroxyapatite scaffolds for osteoclast inhibition and fluorescence imaging.

Multifunctional bone substitute materials (BSM) have gained considerable attention with the exponential increase in aging populations. The development of hybrid materials for diagnosis and therapy of bone-related diseases and dysfunctions, especially, has been a significant challenge in the biological and the biomedical field, due to the shortage of agents with specificity and selectivity toward bone. In this study, a hybrid material, referred as Alen-CDs@CDHA, fabricated from alendronate-conjugated carbon dots (Alen-CDs) and calcium-deficient hydroxyapatite (CDHA, the mineral component of bones) scaffolds is offered as a novel multifunctional BSM for in vivo osteoclasts deactivation and fluorescence imaging. The fluorescent Alen-CDs were hydrothermally prepared using phytic acid as carbon source, followed by conjugating alendronate, for controlled alendronate release and fluorescent imaging under acidic conditions. As-prepared fluorescent Alen-CDs were consecutively immobilized on surfaces of CDHA scaffolds, exhibiting high affinity by bisphosphonate group, easily fabricated from α-tricalcium phosphate (α-TCP) paste using three-dimensional (3D) printing system. The resultant Alen-CDs@CDHA caused a significant decrease (> 50%) in viability of osteoclasts at 7 days after in vitro treatment. Furthermore, when Alen-CDs@CDHA was implanted in balb/c nude mice for in vivo evaluation, we found Alen-CDs@CDHA to be suitable for bone imaging through fluorescence signals, without necrosis or inflammatory symptoms in the epidermal tissues. Thus, these observations offer new opportunities for a novel and revolutionary use of Alen-CDs@CDHA as highly specific multifunctional BSM for bone diagnosis and imaging, and as bone-specific drug delivery materials, eventually providing anti-osteoclastogenic treatments solution for degenerative bone disorders. STATEMENT OF SIGNIFICANCE: Alen-CDs@CDHA significantly reduced the viability of osteoclasts and fluorescently imaged in vivo after transplantation, releasing drug via pH modulation. The development of fluorescence materials for bone imaging remains still a major challenge in the biomedical field owing to the shortage of selectivity and specificity. The results could lead to improvements in bone treatment strategies, as it could reduce the invasiveness of procedures and the associated negative outcomes, and increase the precision of strategies. Further, we believe that this study will be of interest to the readership of your journal as clearly focuses on the advancement of a biomaterial, where we have engineered a substance to substitute bone and integrate with a living system.

Flow cytometry-based rapid detection of Staphylococcus aureus and Pseudomonas aeruginosa using fluorescent antibodies.

Staphylococcus aureus (S. aureus) and Pseudomonas aeruginosa (P. aeruginosa) are major pathogens frequently detected in food and beverage poisoning, and persistent infections. Therefore, the development of a rapid method that can detect these pathogens before serious multiplication is required. In this study, we established a flow cytometry (FCM)-based detection method that allows rapid acquisition of cell populations in fluid samples by using a fluorescent antibody against S. aureus or P. aeruginosa. Using this method, we detected these pathogens with a 103 to 105 CFU order of limit of detection value within 1 hour. The FCM-based method for the detection of S. aureus and P. aeruginosa offers the possibility of high-throughput analysis of pathogens in food, environmental, and clinical sources.

Comparison of Bacterial Assemblages Associated with Harmful Cyanobacteria under Different Light Conditions.

Harmful cyanobacterial blooms in freshwater ecosystems are closely associated with changes in the composition of symbiotic microbiomes, water quality, and environmental factors. In this work, the relationship between two representative harmful cyanobacterial species (Anabaena sp. and Microcystis sp.) and their associated bacterial assemblages were investigated using a 16S rRNA-based meta-amplicon sequencing analysis during a large-scale cultivation of cyanobacteria under different light conditions with limited wavelength ranges (natural light, blue-filtered light, green-filtered light, and dark conditions). During the cultivation periods, the growth pattern of cyanobacteria and bacterial composition of the phycosphere considerably varied in relation to light restrictions. Unlike other conditions, the cyanobacterial species exhibited significant growth during the cultivation period under both the natural and the blue light conditions. Analyses of the nitrogenous substances revealed that nitrogen assimilation by nitrate reductase for the growth of cyanobacteria occurred primarily under natural light conditions, whereas nitrogenase in symbiotic bacteria could also be activated under blue light conditions. Sphingobium sp., associated with nitrogen assimilation via nitrogenase, was particularly dominant when the cell density of Microcystis sp. increased under the blue light conditions. Thus, cyanobacteria could have symbiotic relationships with ammonium-assimilating bacteria under light-limited conditions, which aids the growth of cyanobacteria.

MnCO3-mineralized polydopamine nanoparticles as an activatable theranostic agent for dual-modality imaging-guided photothermal therapy of cancers.

Background: Single imaging modality is still insufficient to evaluate the biological and anatomical structures of tumors with high accuracy and reliability. Generation of non-specific contrast, leading to a low target-to-background signal ratio, results in low imaging resolution and accuracy. Tumor environment-specific activatable multifunctional contrast agents need to maximize the contrast signals, representing a dual imaging-guided photothermal therapy (PTT) at target tumor sites. Methods: Cellular uptake, cytotoxicity assay, and in vitro photothermal conversion efficiency of MnCO3-mineralized fluorescent polydopamine nanoparticles (MnCO3-FPNPs) were evaluated using 4T1 breast cancer cells. In vivo dual-modality imaging was performed using IVIS imaging and a 4.7 T animal MRI systems after injection into 4T1 tumor-bearing nude mice. The effects of photothermal therapeutic through PTT were measured after irradiation with an 808 nm laser (1.5 W/cm2) for 10 min, measuring the size of the tumors every 2 days. Results: At physiological pH (7.4), MnCO3-FPNP is efficiently quenched. Conversely, at acidic pH (5.4), the strong fluorescence (FL) is recovered due to the dissociation of Mn2+ from the FPNPs. At pH 7.4, MnCO3-FPNP activity is silenced to enhance water proton relaxation due to unionized MnCO3 maintenance; conversely, at acidic pH (5.4), MnCO3-FPNPs efficiently release Mn2+ ions, thereby resulting in T 1-weighted magnetic resonance (MR) contrast enhancement. MnCO3-FPNPs display a promising diagnostic ability for 4T1 breast cancer xenograft models, as well as exhibit a high photothermal conversion efficiency. A successful tumor treatment via their photothermal activity is accomplished within 14 days. Conclusions: Our studies exhibited unique "OFF-ON" activation abilities in FL/MR dual imaging and PTT functions. This approach suggests that the MnCO3-FPNPs may serve as a useful platform for various mineralization-based multimodal imaging-guided PTT models for many cancer theranostic applications.

Recent progress in the synthesis of all-aqueous two-phase droplets using microfluidic approaches.

An aqueous two-phase system (ATPS) is a system with liquid-liquid phase separation and shows great potential for the extraction, separation, purification, and enrichment of proteins, membranes, viruses, enzymes, nucleic acids, and other biomolecules because of its simplicity, biocompatibility, and wide applicability [1-4]. The clear aqueous-aqueous interface of ATPSs is highly advantageous for their implementation, therefore making ATPSs a green alternative approach to replace conventional emulsion systems, such as water-in-oil droplets. All aqueous emulsions (water-in-water, w-in-w) hold great promise in the biomedical field as glucose sensors [5] and promising carriers for the encapsulation and release of various biomolecules and nonbiomolecules [6-10]. However, the ultralow interfacial tension between the two phases is a hurdle in generating w-in-w emulsion droplets. In the past, bulk emulsification and electrospray techniques were employed for the generation of w-in-w emulsion droplets and the fabrication of microparticles and microcapsules in the later stage. Bulk emulsification is a simple and low-cost technique; however, it generates polydisperse w-in-w emulsion droplets. Another technique, electrospray, involves easy experimental setups that can generate monodisperse but nonspherical w-in-w emulsion droplets. In comparison, microfluidic platforms provide monodisperse w-in-w emulsion droplets with spherical shapes, deal with the small volumes of solutions and short reaction times and achieve portability and versatility in their design through rapid prototyping. Owing to several advantages, microfluidic approaches have recently been introduced. To date, several different strategies have been explored to generate w-in-w emulsions and multiple w-in-w emulsions and to fabricate microparticles and microcapsules using conventional microfluidic devices. Although a few review articles on ATPSs emulsions have been published in the past, to date, few reviews have exclusively focused on the evolution of microfluidic-based ATPS droplets. The present review begins with a brief discussion of the history of ATPSs and their fundamentals, which is followed by an account chronicling the integration of microfluidic devices with ATPSs to generate w-in-w emulsion droplets. Furthermore, the stabilization strategies of w-in-w emulsion droplets and microfluidic fabrication of microparticles and microcapsules for modern applications, such as biomolecule encapsulation and spheroid construction, are discussed in detail in this review. We believe that the present review will provide useful information to not only new entrants in the microfluidic community wanting to appreciate the findings of the field but also existing researchers wanting to keep themselves updated on progress in the field.

Artificial olfactory sensor technology that mimics the olfactory mechanism: a comprehensive review.

Artificial olfactory sensors that recognize patterns transmitted by olfactory receptors are emerging as a technology for monitoring volatile organic compounds. Advances in statistical processing methods and data processing technology have made it possible to classify patterns in sensor arrays. Moreover, biomimetic olfactory recognition sensors in the form of pattern recognition have been developed. Deep learning and artificial intelligence technologies have enabled the classification of pattern data from more sensor arrays, and improved artificial olfactory sensor technology is being developed with the introduction of artificial neural networks. An example of an artificial olfactory sensor is the electronic nose. It is an array of various types of sensors, such as metal oxides, electrochemical sensors, surface acoustic waves, quartz crystal microbalances, organic dyes, colorimetric sensors, conductive polymers, and mass spectrometers. It can be tailored depending on the operating environment and the performance requirements of the artificial olfactory sensor. This review compiles artificial olfactory sensor technology based on olfactory mechanisms. We introduce the mechanisms of artificial olfactory sensors and examples used in food quality and stability assessment, environmental monitoring, and diagnostics. Although current artificial olfactory sensor technology has several limitations and there is limited commercialization owing to reliability and standardization issues, there is considerable potential for developing this technology. Artificial olfactory sensors are expected to be widely used in advanced pattern recognition and learning technologies, along with advanced sensor technology in the future.

Generation of a recombinant antibody for sensitive detection of Pseudomonas aeruginosa.

Pseudomonas aeruginosa (P. aeruginosa) is a major pathogen that causes nosocomial infections and often exhibits antibiotic resistance. Therefore, the development of an accurate method for detecting P. aeruginosa is required to control P. aeruginosa-related outbreaks. In this study, we established an enzyme-linked immunosorbent assay method for the sensitive detection of three P. aeruginosa strains, UCBPP PA14, ATCC 27853, and multidrug-resistant ATCC BAA-2108. We produced a recombinant antibody (rAb) against P. aeruginosa V-antigen (PcrV), which is a needle tip protein of the type III secretion system of P. aeruginosa using mammalian cells with high yield and purity, and confirmed its P. aeruginosa binding efficiency. The rAb was paired with commercial anti-P. aeruginosa Ab for a sandwich ELISA, resulting in an antigen-concentration-dependent response with a limit of detection value of 230 CFU/mL. These results suggest that the rAb produced herein can be used for the sensitive detection of P. aeruginosa with a wide range of applications in clinical diagnosis and point-of-care testing.

Generation of Recombinant Antibodies in HEK293F Cells for the Detection of Staphylococcus aureus.

Staphylococcus aureus is a major resistant pathogen in clinical practice. Due to the increasing number of infections, rapid and sensitive detection of antibiotic-resistant S. aureus as well as antibiotic-sensitive S. aureus is important for the prevention and control of infectious diseases. In this study, we produced recombinant antibodies against S. aureus from mammalian human embryonic kidney 293 Freestyle cells with high yield and purity. These recombinant antibodies showed high binding affinity and low detection limit in both indirect and sandwich enzyme-linked immunosorbent assays for the detection of methicillin-resistant S. aureus and methicillin-sensitive S. aureus. These results suggest that the recombinant antibodies produced herein can be used for the accurate detection of S. aureus with a wild range of applications in medical diagnosis, food safety, and drug discovery.