SERS imaging analyses of bacteria cells among plant tissues.

A chemical imaging method to mass surveil bacteria cells among plant tissues in situ is reported. Bacteria cells were pre-labeled with 3-mercaptophenylboronic acid for complexation with gold nanoparticles. Surface-enhanced Raman spectra were collated en masse to generate panoramic chemical images of bacteria populations. The approach was successfully employed to study the distribution of mass bacteria populations directly on and in selected plant tissues. This study demonstrates the great potential with which SERS imaging can be utilized for the study of bacterial cells among complex matrices, in some ways that are superior to electron and fluorescent microscopies.

Understanding and Advancing the 3-mercaptophenylboronic Acid Chemical Label for Optimal Surface-enhanced Raman Spectroscopic Analysis of Bacteria Populations.

3-mercaptophenylboronic acid (3-MPBA) was applied as a capturer and label for bacteria detection using surface-enhanced Raman spectroscopy (SERS). The objective of this study was to further understand and advance 3-MPBA as a SERS labeling chemical to study bacteria populations using SERS. We report that the coating of bacteria cells with 3-MPBA was very strong, with bacteria producing 3-MPBA SERS signals after five thorough rinse water applications. The procedure was also found to implement harm to bacterial ecology, and the trend was quantitatively different based on the initial cell population being labeled. SERS imaging by this approach measured all labeled bacteria cells, regardless of viability. Nonculturable cells are therefore detectable by this SERS approach. Nanoparticle administrations were optimal when bacteria cells were suspended in a liquid or applied to substrates which already possessed nanostructures. Circumstances in which dried bacteria cells were present upon a substrate prior to nanoparticle administrations warranted lower SERS signals in comparison. Bacteria were analyzed at the single-cell scale using this approach, and the data revealed that microscopic objective lenses and overall bacteria population influenced the SERS limit-of-detection in this respect. The information obtained from this study provides useful guidelines for advancing 3-MPBA labeling for the SERS analysis of bacteria.

Comparison of label-free and label-based approaches for surface-enhanced Raman microscopic imaging of bacteria cells.

Surface-enhanced Raman spectroscopic (SERS) approaches are emerging for bacteria analysis whereby bacteria cells can be measured based on their biochemical composition (label-free) or with the aid of a chemical label to enhance the SERS signal. Combining a microscope, SERS microscopy is capable of imaging bacteria populations en masse based on specific spectrophotometric peaks. Here, we compared the label-free and label-based approaches to study Escherichia coli O157:H7 that was utilized as a model bacterium for SERS imaging analyses. Gold (Au) nanoparticles were utilized to enhance Raman scattering during this study and 3-mercaptophenylboronic acid was utilized as a model chemical label for comparison against label-free conditions. The result shows that SERS images of bacteria cells yielded measurable differences in precision, depending on the application of chemical labels. Chemical labels enabled SERS imaging of whole bacteria populations with single-cell precision. Bacteria coated with labels were also easier to bring into focus using high-magnification optical microscopy, without the need for immersion oil. Label-free analyses of single-cells were lower in geographic precision but provided opportunities to study the natural biochemistry of bacteria cells with strong accuracy. SERS analyses of label-free bacteria cell components were conclusively improved in vitro on a time-dependent basis. This concept can serve as an important benchmark when biochemically profiling or characterizing bacteria cells based on SERS. Electron micrographs proved that chemical labels can be utilized to increase nanoparticle contact with bacteria cells and reduce free nanoparticles that contribute to background noise in SERS spectra. We also demonstrate the use of both 3-mercaptophenylboronic acid and propidium iodide to discriminate live and dead bacteria through the simultaneous collection of data from these two chemical labels. Label-free approaches to SERS bacteria analyses are better suited for biochemical characterization and label-based approaches are better suited when accounting for individual cells among a population.

In situ assembly of well-dispersed Ag nanoparticles on the surface of polylactic acid-Au@polydopamine nanofibers for antimicrobial applications.

Nanofibrous membranes which exhibit bacteriostatic functions are a good strategy to prevent microorganisms from adhering to the surface of biomaterials. Here, we report the synthesis of such a nanofibrous membrane which can be applied to biological coatings to reduce bacteriostatic functionality. Ascorbic acid was utilized to reduced chloroauric acid to gold nanoparticles (AuNPs). Dopamine was then polymerized upon AuNP surfaces by ultrasound-assistance, to synthesize core-shell structured polydopamine-coated AuNPs (Au@PDA NPs). The Au@PDA NPs were then mixed with polylactic acid (PLA) for electrospinning into cylindrical nanofibers (136.6 nm diameter). PLA-Au@PDA nanofibrous membranes were finally immersed in silver nitrate for in situ reduction into a silver nanoparticle (AgNP) coating to yield PLA-Au@PDA@Ag nanofibers. The PLA-Au@PDA@Ag nanofibers were characterized based on field emission scanning electron microscopy, Fourier transform infrared spectroscopy, X-ray photoelectron spectroscopy, and contact angle analyses. The antibacterial properties of the PLA-Au@PDA@Ag nanofibers were examined based on the optical density absorbance of bacterial cell suspensions, traditional colony plate counts, zone inhibition analyses, and field-emission scanning electron microscopy. Escherichia coli and Staphylococcus aureaus respectively served as Gram negative and positive bacterial models of industrial relevance. The data conclusively illustrates the antimicrobial and biomedical applications of PLA-Au@PDA@Ag nanofibers.

Purification and characterization of the thermostable protease produced by Serratia grimesii isolated from channel catfish.

BACKGROUND: Microbial spoilage of fishery products accounts for significant financial losses, yearly on a global scale. Psychrotrophic spoilage bacteria often secrete extracellular enzymes to break down surrounding fish tissue, rendering the product unsuitable for human consumption. For a better understanding of bacterial spoilage due to enzymatic digestion of fish products, proteases in Serratia grimesii isolated from North American catfish fillets (Ictalurus punctatus) were investigated. RESULTS: Mass spectrometric evidence demonstrated that S. grimesii secretes two distinct extracellular proteases and one lipase. Protease secretion displayed broad thermostability in the 30-90 °C range. The major protease-secretion (O-1) was most active under alkaline conditions and utilized manganese as a co-factor. Organic solvents significantly disrupted the efficacy of S. grimesii extracellular enzymes and, in a series of bactericidal detergents, protease activity was highest when treated with Triton X-100. Ethylenediaminetetraacetic acid (EDTA) and phenylmethylsulfonyl fluoride (PMSF) significantly inhibited the enzyme activity, while protease was moderately stable under freeze-thaw and refrigerated storage. CONCLUSION: The influence of fish spoilage-related enzymes, depending on various factors, is discussed in this paper. This study will provide new insight into enzymatic spoilage and its control, which can be exploited to enhance food safety and the shelf-life of fishery products worldwide. © 2018 Society of Chemical Industry.

Lecithin doped electrospun poly(lactic acid)-thermoplastic polyurethane fibers for hepatocyte viability improvement.

The development of hepatocyte cultures in vitro holds great significance in the study of bioartificial liver support systems. Electrospun fiber cultures have received widespread attention as an effective method to culture hepatocytes in vitro. Polylactic acid (PLA) -a synthetic polymer with high biocompatibility and biodegradability- is widely used to fabricate electrospun fibers in the biomedical field. However, the use of PLA is limited in cell cultures due to its brittleness, strong hydrophobicity, and lack of biologically active functional groups. In this study, thermoplastic polyurethane (TPU) and lecithin (Lec) were used to modify PLA by spiking them into the PLA electrospun solution in attempt to establish a suitable fiber scaffold for hepatocyte culture in bioreactors. TPU and lecithin incorporation into PLA increases the flexibility, hydrophilicity, and biologically active groups of the fibers which further promotes the growth, proliferation, and viability of hepatocytes. The morphology, wettability, and biocompatibility of the as-prepared PLA-TPU-Lec fibers were carefully characterized. The results showed that the PLA-TPU-Lec fibers possessed favorable morphology and hydrophilicity, as well as high biocompatibility ability. HepG2 cells on the PLA-TPU-Lec fibers and tissue culture plates (TCP) were exposed to hepatotoxins for 24 h and we found that HepG2 cells on the PLA-TPU-Lec fibers had higher viability than cells on TCP. The PLA-TPU-Lec fibers are therefore expected to be used in vitro for hepatocyte culture to improve cellular activity in artificial liver bioreactors.

Preparation and study of the antibacterial ability of graphene oxide-catechol hybrid polylactic acid nanofiber mats.

The functionalization of electrospun mats with antimicrobial nanomaterials is an attractive strategy when developing functional graphene oxide coating materials to prevent bacterial colonization on surfaces. In this study, we demonstrated a simple approach to produce antimicrobial electrospun mats by dip-coating a polylactic acid (PLA) nanofiber into a graphene oxide-catechol derivative. PLA was first electrospun to yield narrow-diameter polymeric nanofibers. We then modified the graphene oxide (GO) with a catechol derivative - dopamine methacrylamide monomer (DMA) - to synthesize a GO-DMA nanocomposite material which exhibited robust antimicrobial properties. The catechol groups promote the immobilization of graphene oxide onto the PLA nanofibers and possess strong antimicrobial properties. We therefore selected this functional group to modify GO. We dipped the GO-DMA onto the PLA nanofiber to produce the final functionalized electrospun mats. The PLA mats which were functionalized using the GO-DMA nanocomposite (PLA-GO-DMA) displayed antibacterial activity against both Gram-negative (Escherichia coli) and Gram-positive (Staphylococcus aureus) bacteria. Furthermore, we studied the biocompatibility of the mats by culturing the cell lines (HepG2, A549, and HUVEC-C) of PLA-GO-DMA among the nanofibers which exhibited excellent biocompatibility. These results collectively demonstrate the potential of PLA-GO-DMA nanofiber mats as antimicrobial biomaterials and provide fundamental information toward the establishment of future biomedical applications.

A Triple Functional Approach To Simultaneously Determine the Type, Concentration, and Size of Titanium Dioxide Particles.

The large-scale manufacturing and use of titanium dioxide (TiO2) particles in food and consumer products significantly increase the likelihood of human exposure and release into the environment. We present a simple and innovative approach to rapidly identify the type (anatase or rutile), as well as to estimate, the size and concentration of TiO2 particles using Raman spectroscopy and surface-enhanced Raman spectroscopy (SERS). The identification and discrimination of rutile and anatase were based on their intrinsic Raman signatures. The concentration of the TiO2 particles was determined based on Raman peak intensity. Particle sizes were estimated based on the ratio between the Raman intensity of TiO2 and the SERS intensity of myricetin bound to the nanoparticles (NPs), which was proven to be independent of TiO2 nanoparticle concentrations. The ratio that was calculated from the 100 nm particles was used as a cutoff value when estimating the presence of nanosized particles within a mixture. We also demonstrated the practical use of this approach when determining the type, concentration, and size of E171: a mixture that contains TiO2 particles of various sizes which are commonly used in many food products as food additives. The presence of TiO2 anatase NPs in E171 was confirmed using the developed approach and was validated by transmission electron micrographs. TiO2 presence in pond water was also demonstrated to be an analytical capability of this method. Our approach shows great promise for the rapid screening of nanosized rutile and anatase TiO2 particles in complex matrixes. This approach will strongly improve the measurement of TiO2 quality during production, as well as the survey capacity and risk assessment of TiO2 NPs in food, consumer goods, and environmental samples.

Comparison of extracellular DNase- and protease-producing spoilage bacteria isolated from Delaware pond-sourced and retail channel catfish (Ictalurus punctatus).

BACKGROUND: Spoilage of fishery products begins immediately following filleting due to microbial growth that degrades fish tissue quality prior to consumption. Extensive research has been conducted to identify such bacterial populations. A better understanding of the mechanisms involved in fish spoilage is necessary as a novel remedy for microbial spoilage inhibition has yet to be established for fish tissue. The present study identified, for the first time, bacterial populations that produce extracellular DNase and protease from Delaware and local retail distributed channel catfish (Ictalurus punctatus) fillets. RESULTS: A clear trend was identified between bacteria derived from catfish filleted under aseptic conditions where Pseudomonas was the dominant genus. Bacteria isolated from retail catfish contained high quantities of DNase-producing isolates, in contrast to aseptic-filleted catfish tissue which had none. Both types of catfish sample maintained high populations of protease-producing bacterial colonies throughout the duration of the study. Most bacteria isolated from catfish intestines exhibited DNase production with no protease production. CONCLUSION: Specific spoilage organism populations were significantly higher on retail-derived catfish in comparison to lab-filleted Delaware cultured catfish tissue. It is suggested that DNase production and protease production contribute to the spoilage of fish tissue as a result of mishandling and septic filleting being the major cause of rapid catfish tissue spoilage.