Genetically engineered bacteriophages as novel nanomaterials: applications beyond antimicrobial agents.

Bacteriophages, also known as phages, are viruses that replicate in bacteria and archaea. Phages were initially discovered as antimicrobial agents, and they have been used as therapeutic agents for bacterial infection in a process known as "phage therapy." Recently, phages have been investigated as functional nanomaterials in a variety of areas, as they can function not only as therapeutic agents but also as biosensors and tissue regenerative materials. Phages are nontoxic to humans, and they possess self-assembled nanostructures and functional properties. Additionally, phages can be easily genetically modified to display specific peptides or to screen for functional peptides via phage display. Here, we demonstrated the application of phage nanomaterials in the context of tissue engineering, sensing, and probing.

A Customizable Proteinic Bioadhesive Patch with Water-Switchable Underwater Adhesiveness, Adjustable Biodegradability, and Modifiable Stretchability for Healing Diverse Internal Wounds.

Customizable bioadhesives for individual organ requirements, including tissue type and motion, are essential, especially given the rise in implantable medical device applications demanding adequate underwater adhesion. While synthetic bioadhesives are widely used, their toxicity upon degradation shifts focus to biocompatible natural biomaterials. However, enhancing the adhesive strengths of these biomaterials presents ongoing challenges while accommodating the unique properties of specific organs. To address these issues, three types of customized underwater bioadhesive patches (CUBAPs) with strong, water-responsive adhesion and controllable biodegradability and stretchability based on bioengineered mussel adhesive proteins conjugated with acrylic acid and/or methacrylic acid are proposed. The CUBAP system, although initially nonadhesive, shows strong underwater adhesion upon hydration, adjustable biodegradation, and adequate physical properties by adjusting the ratio of poly(acrylic acid) and poly(methacrylic acid). Through ex vivo and in vivo evaluations using defective organs and the implantation of electronic devices, the suitability of using CUBAPs for effective wound healing in diverse internal organs is demonstrated. Thus, this innovative CUBAP system offers strong underwater adhesiveness with tailored biodegradation timing and physical properties, giving it great potential in various biomedical applications.

Multiplex 16S rRNA-derived geno-biochip for detection of 16 bacterial pathogens from contaminated foods.

Foodborne diseases caused by various pathogenic bacteria occur worldwide. To prevent foodborne diseases and minimize their impacts, it is important to inspect contaminated foods and specifically detect many types of pathogenic bacteria. Several DNA oligonucleotide biochips based on 16S rRNA have been investigated to detect bacteria; however, a mode of detection that can be used to detect diverse pathogenic strains and to examine the safety of food matrixes is still needed. In the present work, a 16S rRNA gene-derived geno-biochip detection system was developed after screening DNA oligonucleotide specific capture probes, and it was validated for multiple detection of 16 pathogenic strains that frequently occur with a signature pattern. rRNAs were also used as detection targets directly obtained from cell lysates without any purification and amplification steps in the bacterial cells separated from 8 food matrixes by simple pretreatments. Thus, the developed 16S rRNA-derived geno-biochip can be successfully used for the rapid and multiple detection of the 16 pathogenic bacteria frequently isolated from contaminated foods that are important for food safety.

Hybrid microarray based on double biomolecular markers of DNA and carbohydrate for simultaneous genotypic and phenotypic detection of cholera toxin-producing Vibrio cholerae.

Life-threatening diarrheal cholera is usually caused by water or food contaminated with cholera toxin-producing Vibrio cholerae. For the prevention and surveillance of cholera, it is crucial to rapidly and precisely detect and identify the etiological causes, such as V. cholerae and/or its toxin. In the present work, we propose the use of a hybrid double biomolecular marker (DBM) microarray containing 16S rRNA-based DNA capture probe to genotypically identify V. cholerae and GM1 pentasaccharide capture probe to phenotypically detect cholera toxin. We employed a simple sample preparation method to directly obtain genomic DNA and secreted cholera toxin as target materials from bacterial cells. By utilizing the constructed DBM microarray and prepared samples, V. cholerae and cholera toxin were detected successfully, selectively, and simultaneously; the DBM microarray was able to analyze the pathogenicity of the identified V. cholerae regardless of whether the bacteria produces toxin. Therefore, our proposed DBM microarray is a new effective platform for identifying bacteria and analyzing bacterial pathogenicity simultaneously.

Surface-independent antibacterial coating using silver nanoparticle-generating engineered mussel glue.

During implant surgeries, antibacterial agents are needed to prevent bacterial infections, which can cause the formation of biofilms between implanted materials and tissue. Mussel adhesive proteins (MAPs) derived from marine mussels are bioadhesives that show strong adhesion and coating ability on various surfaces even in wet environment. Here, we proposed a novel surface-independent antibacterial coating strategy based on the fusion of MAP to a silver-binding peptide, which can synthesize silver nanoparticles having broad antibacterial activity. This sticky recombinant fusion protein enabled the efficient coating on target surface and the easy generation of silver nanoparticles on the coated-surface under mild condition. The biosynthesized silver nanoparticles showed excellent antibacterial efficacy against both Gram-positive and Gram-negative bacteria and also revealed good cytocompatibility with mammalian cells. In this coating strategy, MAP-silver binding peptide fusion proteins provide hybrid environment incorporating inorganic silver nanoparticle and simultaneously mediate the interaction of silver nanoparticle with surroundings. Moreover, the silver nanoparticles were fully synthesized on various surfaces including metal, plastic, and glass by a simple, surface-independent coating manner, and they were also successfully synthesized on a nanofiber surface fabricated by electrospinning of the fusion protein. Thus, this facile surface-independent silver nanoparticle-generating antibacterial coating has great potential to be used for the prevention of bacterial infection in diverse biomedical fields.

Specific discrimination of three pathogenic Salmonella enterica subsp. enterica serotypes by carB-based oligonucleotide microarray.

It is important to rapidly and selectively detect and analyze pathogenic Salmonella enterica subsp. enterica in contaminated food to reduce the morbidity and mortality of Salmonella infection and to guarantee food safety. In the present work, we developed an oligonucleotide microarray containing duplicate specific capture probes based on the carB gene, which encodes the carbamoyl phosphate synthetase large subunit, as a competent biomarker evaluated by genetic analysis to selectively and efficiently detect and discriminate three S. enterica subsp. enterica serotypes: Choleraesuis, Enteritidis, and Typhimurium. Using the developed microarray system, three serotype targets were successfully analyzed in a range as low as 1.6 to 3.1 nM and were specifically discriminated from each other without nonspecific signals. In addition, the constructed microarray did not have cross-reactivity with other common pathogenic bacteria and even enabled the clear discrimination of the target Salmonella serotype from a bacterial mixture. Therefore, these results demonstrated that our novel carB-based oligonucleotide microarray can be used as an effective and specific detection system for S. enterica subsp. enterica serotypes.

A facile and sensitive detection of pathogenic bacteria using magnetic nanoparticles and optical nanocrystal probes.

We report a facile and sensitive analytical method for the detection of pathogenic bacteria. Salmonella bacteria in milk were captured by antibody-conjugated magnetic nanoparticles (MNPs) and separated from analyte samples by applying an external magnetic field. The MNP-Salmonella complexes were re-dispersed in a buffer solution then exposed to antibody-immobilized TiO(2) nanocrystals (TNs), which absorb UV light. After magnetically separating the MNP-Salmonella-TN complexes from solution, the UV-Vis absorption spectrum of the unbound TN solution was obtained. Because the light absorption intensity was reversely proportional to the Salmonella concentration, the assay exhibited high sensitivity toward low concentrations of Salmonella bacteria. The detection limit of Salmonella in milk was found to be more than 100 cfu mL(-1).

Specific multiplex analysis of pathogens using a direct 16S rRNA hybridization in microarray system.

For the rapid multiplex analysis of pathogens, 16S rRNAs from cell lysates were directly applied onto a DNA microarray at room temperature (RT) for RNA-DNA hybridization. To eliminate the labeling step, seven fluorescent-labeled detector probes were cohybridized with 16S rRNA targets and adjacent specific capture probes. We found that eight pathogens were successfully discriminated by the 16S rRNA-based direct method, which showed greater specificity than the polymerase chain reaction (PCR)-labeled method due to chaperone and distance effects. A new specificity criterion for a perfect match between RNA and DNA was suggested to be 21-41% dissimilarity using correlation analysis between the mismatch and the sequence according to the guanine-cytosine (GC) percentage or the distribution of mismatches. Six categories of food matrix (egg, meat, milk, rice, vegetable, and mixed) were also tested, and the target pathogen was successfully discriminated within statistically significant levels. Finally, we found that the intrinsic abundance of 16S rRNA molecules successfully substituted PCR-based amplification with a low limit of detection of 10-10(3) cells mL(-1) and a high quantitative linear correlation. Collectively, our suggested 16S rRNA-based direct method enables the highly sensitive, specific, and quantitative analysis of selected pathogens at RT within 2 h, even in food samples.