Rapid single-cell detection of pathogenic bacteria for in situ determination of food safety.

A highly sensitive in situ method to detect bacterial pathogens is of utmost importance in preventing the outbreak of foodborne diseases. In this study, a simple method enabling the detection of a single bacterial cell in a sample was developed based on magnetic capture particles (CPs), and europium-fluorescent labeling particles (LPs) functionalized with antibodies. After mixing the sample with the particles in a sample tube, the sample tube was connected to an assay chip, where the CP-bacteria-LP complex was transported from the sample chamber to a detection chamber using a simple assay device. The number of bacteria was quantitatively determined by measuring the fluorescence emitted from the detection chamber. This assay method enabled the detection of a single cell of Vibrio parahaemolyticus from 0.1 mL pure broth culture samples within 30 min. A simple enrichment method that can be performed using only the vibrating action of the assay device without any additional instruments was also developed for the analysis of food samples. By analyzing the enriched sample using the assay method, we could detect V. parahaemolyticus quantitatively with a detection limit of 1 colony forming unit from oyster samples within 130 min. Due to simplicity of this methodology and the instrumentation involved, and its capability of rapid single-cell detection, it may be considered as an in situ method for the determination of food safety.

Sensitive immunoassay-based detection of Vibrio parahaemolyticus using capture and labeling particles in a stationary liquid phase lab-on-a-chip.

In the present study, a method was developed for detection of Vibrio parahaemolyticus based on a stationary liquid phase lab-on-a-chip (SLP LOC). The present SLP LOC comprises a sample chamber, washing chamber, and detection chamber connected by two channels. The method utilizes two types of particles: capture particles (CPs), which are magnetic nanoparticles functionalized with antibody; and labeling particles (LPs), which are silica nanoparticles functionalized with horseradish peroxidase and antibody. Samples were added to the sample chamber with CPs and LPs, forming a CP-bacteria-LP complex, and the complex was transported to the detection chamber containing chromogenic substrate solution. The method allowed the detection of V. parahaemolyticus in the range of 101-105cfu within 45min. Additionally, contamination of oyster samples with V. parahaemolyticus was detected within 2.5h, including 2h of culturing. The present method has the advantage of being highly rapid and facile, and enabling the detection of bacteria with high sensitivity. Moreover, the LOC and LOC processing device used in this method possess simple structures, making the detection process economical and allowing miniaturization. Therefore, the present SLP LOC detection method is potentially useful for in situ determination of food safety.

Beneficial effects of fermented black ginseng and its ginsenoside 20(S)-Rg3 against cisplatin-induced nephrotoxicity in LLC-PK1 cells.

BACKGROUND: Nephrotoxicity is a common side effect of medications. Panax ginseng is one of the best-known herbal medicines, and its individual constituents enhance renal function. Identification of its efficacy and mechanisms of action against drug-induced nephrotoxicity, as well as the specific constituents mediating this effect, have recently emerged as an interesting research area focusing on the kidney protective efficacy of P. ginseng. METHODS: The present study investigated the kidney protective effect of fermented black ginseng (FBG) and its active component ginsenoside 20(S)-Rg3 against cisplatin (chemotherapy drug)-induced damage in pig kidney (LLC-PK1) cells. It focused on assessing the role of mitogen-activated protein kinases as important mechanistic elements in kidney protection. RESULTS: The reduced cell viability induced by cisplatin was significantly recovered with FBG extract and ginsenoside 20(S)-Rg3 dose-dependently. The cisplatin-induced elevated protein levels of phosphorylated c-Jun N-terminal kinase (JNK), p53, and cleaved caspase-3 were decreased after cotreatment with FBG extract or ginsenoside 20(S)-Rg3. The elevated percentage of apoptotic LLC-PK1 cells induced by cisplatin treatment was significantly abrogated by cotreatment with FBG and the ginsenoside 20(S)-Rg3. CONCLUSION: FBG and its major ginsenoside 20(S)-Rg3, ameliorated cisplatin-induced nephrotoxicity in LLC-PK1 cells by blocking the JNK-p53-caspase-3 signaling cascade.

Immunoassay of paralytic shellfish toxins by moving magnetic particles in a stationary liquid-phase lab-on-a-chip.

In this study, we devised a stationary liquid-phase lab-on-a-chip (SLP LOC), which was operated by moving solid-phase magnetic particles in the stationary liquid phase. The SLP LOC consisted of a sample chamber to which a sample and reactants were added, a detection chamber containing enzyme substrate solution, and a narrow channel connecting the two chambers and filled with buffer. As a model system, competitive immunoassays of saxitoxin (STX), a paralytic shellfish toxin, were conducted in the SLP LOC using protein G-coupled magnetic particles (G-MPs) as the solid phase. Anti-STX antibodies, STX-horseradish peroxidase conjugate, G-MPs, and a STX sample were added to the sample chamber and reacted by shaking. While liquids were in the stationary state, G-MPs were transported from the sample chamber to the detection chamber by moving a magnet below the LOC. After incubation to allow the enzymatic reaction to occur, the absorbance of the detection chamber solution was found to be reciprocally related to the STX concentration of the sample. Thus, the SLP LOC may represent a novel, simple format for point-of-care testing applications of enzyme-linked immunosorbent assays by eliminating complicated liquid handling steps.

A lipid-based method for the preparation of a piezoelectric DNA biosensor.

A piezoelectric DNA biosensor was prepared by immobilizing DNA probes on a quartz crystal microbalance (QCM) using a lipid-based method. A QCM electrode was coated with a hybrid bilayer membrane composed of an octadecanethiol monolayer and a lipid monolayer containing biotinylated lipids to establish biotin groups on the electrode surface. A DNA biosensor was prepared by sequentially immobilizing avidin and the biotinylated probe. The DNA biosensor was stable throughout repeated surface regeneration and showed higher sensitivity than that prepared by the conventional chemical method using diimide. We also optimized the surface regeneration conditions and flow rate for flow injection analysis.

Preparation of stabilized magnetic nanoparticles with polymerizable lipid and anchor compounds.

Although the lipid-based method for coating of magnetic nanoparticles (MNPs) is rapid and simple, the unstable state of the lipid layer is a major limitation for the practical application of this method. We devised a method to prepare stabilized MNPs by covalent modifications such as lipid polymerization and anchoring of the lipid layer. The stability of the modified lipid layer was demonstrated by the stable status of enzymes immobilized on the MNPs and the resistance of the MNPs to aggregation. We also determined the maximum ratio of nonpolymerizable lipophilic compounds that can be included in the layer without significantly reducing stability.

Functionalization of quartz crystal microbalances with liposomes containing the N-hydroxysuccinimide ester of palmitic acid.

We developed a fast and simple method to functionalize a quartz crystal microbalance (QCM) with liposomes composed of phosphatidylcholine lipid and the N-hydroxysuccinimide (NHS) ester of palmitic acid. The liposome was applied directly to a bare gold surface of a QCM to prepare the lipid bilayer presenting NHS groups on the surface. The whole functionalization process was completed within 1 h using stored lipid films. Streptavidin immobilization efficiency of the method was comparable to the 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC)/NHS chemical method, and the activity of the biotinylated antibody immobilized through the streptavidin was stably retained in repeated surface regeneration with the dissociation buffer.

Detection of Edwardsiella tarda by fluorometric or biosensor methods using a peptide ligand.

In this study, we identified a peptide ligand for Edwardsiella tarda from a phage peptide library and tested two approaches for sensitive detection of the bacteria with the peptide labeled with fluorescein or biotin. At first, the fluorescent peptide was proved to be advantageous in the fluorescence polarization (FP) assay because sensitivity of the assay is maximized when a fluorophore is linked to a small molecule. The FP assay using the fluorescent peptide enabled detection of E. tarda in a range from 5.2×10(3) to 2.1×10(5) cells. Second, we devised a new assay method using a quartz crystal microbalance (QCM) biosensor connected to a filter module. When a mixture of E. tarda and the biotinylated peptide was injected into the filter module, the E. tarda-peptide complex was separated from the unbound peptide by a filter and detected with a streptavidin-coated QCM sensor chip. On injection of samples containing the biotinylated peptide and E. tarda, concentration-dependent frequency change was observed in a range from 8×10(2) to 8×10(6) cells. The two approaches are expected to facilitate development of assay methods using other bacteria-binding peptides.

Identification of a polymeric ß-cyclodextrin-binding peptide from a phage-displayed peptide library.

In this work, a phage-displayed peptide library was applied to identification of ß-cyclodextrin (CD)-binding peptide tag, capable of being combined to target peptides or proteins in a homogeneous way by established methods such as peptide synthesis and the recombinant DNA technique. Four enriched sequences were obtained after five rounds of biopanning against polymeric ß-CD beads. One of the sequences showed high binding affinity to ß-CD beads with a dissociation constant of approximately 7×10(-6)M. The ß-CD-binding sequence was used for immobilization of a hepatitis C virus (HCV) antigenic peptide on ß-CD beads. The functionalized ß-CD beads were successfully used for immunoassay of anti-HCV antibody with a detection limit of 1 ng. These results demonstrate that the identified peptide sequence has the potential of being used as an affinity tag to ß-CD-containing surfaces.

Direct introduction of a hydrazide group into a quartz crystal microbalance surface with dodecanoic hydrazide embedded in a hybrid bilayer membrane.

Hydrazide group has a potential of immobilizing an antibody on a sensor surface in a way that ensures an optimal orientation and efficiency of the antibody. However, a multi-step chemical process, required for the preparation of a hydrazide group, is a barrier to its extensive application. This paper describes a new method to introduce a hydrazide group to a sensor surface by a one-step process using dodecanoic hydrazide. The method is based on an ability of the dodecanoic hydrazide to be incorporated into a hybrid bilayer membrane (HBM) layer, thereby presenting its hydrazide group to the surface. Liposome containing dodecanoic hydrazide was added to a hydrophobic self-assembled monolayer surface of a quartz crystal microbalance for the formation of a HBM. Then, the hydrazide group, presented in the surface of the HBM layer, was utilized for the oriented immobilization of an antibody via its carbohydrate moiety which was partially oxidized prior to the conjugation reaction. Activity and stable status of the incorporated dodecanoic hydrazide was revealed by the efficiency and reproducibility of the resulting immunosensor chip.

Optimization of the hybrid bilayer membrane method for immobilization of avidin on quartz crystal microbalance.

Hybrid bilayer membrane (HBM), comprising a lipid monolayer fused to a hydrophobic self-assembled monolayer (SAM), has a potential capability to provide a convenient tool for the preparation of functionalized sensor surfaces. In this work, the HBM approach was optimized for the preparation of avidin-containing quartz crystal microbalance (QCM) sensor chip which would be available for immobilization of biotinylated molecules. Lipid layer of HBM was composed of background lipid such as egg phosphatidyl choline and biotinylated lipid to which avidin was attached. Highest performance was obtained at 1:1 ratio of the biotinylated lipid and the background lipid, and sensitivity and stability of the resulting sensor chip was comparable to a sensor chip prepared by the conventional carbodiimide reaction. By utilizing the HBM method, construction of a stable avidin sensor chip was achieved within 40 min without any chemical steps. Thus the HBM method was proven to be a convenient and efficient way to immobilize avidin on sensor surfaces.

Development of QCM biosensor to detect a marine derived pathogenic bacteria Edwardsiella tarda using a novel immobilisation method.

QCM technology offers a real time output, simplicity of use and cost effectiveness in addition to high sensitivity. Sensitivity of QCM immunosensor can be enhanced by improving the immobilisation procedure on the quartz surface. The immobilisation strategy should be able to control both the amount and the orientation of the antibody (immunoglobulin; IgG) on the transducer for high affinity to antigens. This study introduced a new methodology recruiting oxidised IgG to expose aldehyde group in Fc region to cross-link to hydrazide conformed on self assembled monolayer (SAM) and compared with three conventional methods. Consequently, it was proved that considerable amount of antibody was immobilised and the sensitivity of new methodology was higher than other methods while ability of new methodology to immobilise IgG was lower than the conventional methods. The frequency shifts following bacterial cell injection were positively related to the frequency shifts after the injection of IgG and the amounts of bacterial cells, revealing that the frequency shifts after bacterial cell injection fully represented the weight change by specific attachments of bacterial cells to the IgG cross-linked on the gold surface. Specificity was tested on different bacteria including E. coli, V. vulnificus and A. hydrophila and showed no significant non-specific affinity on the tested bacteria. It was also demonstrated that the prepared sensor chip was stable enough to withstand repeated surface regeneration. Indeed, polyclonal antibody was more effective to detect antigen than monoclonal antibody which binds to only one epitope of antigen. Conclusively, the new methodology is appeared to be more sensitive than conventional methods tested and reusable for 10 times.

Purification and characterization of hepatic lipase from Todarodes pacificus.

Lipase was purified from squid (Todarodes pacificus) liver in an attempt to investigate the possibility of applying the enzyme for biotechnological applications. Crude extract of squid liver was initially fractionated by the batch type ion exchange chromatography. The fraction containing lipase activity was further purified with an octyl-Sepharose column. Finally, lipase was purified by eluting active protein from a non-dissociating polyacrylamide gel after zymographic analysis. Molecular weight of the purified enzyme was determined to be 27 kDa by SDS-polyacrylamide gel electrophoresis. The enzyme showed the highest activity at a temperature range of 35-40 degrees C and at pH 8.0. The activity was almost completely inhibited at 1 mM concentration of Hg(2+) or Cu(2+) ion. Partial amino acid sequence of the enzyme was also determined.

Identification of a deoxyribonuclease I inhibitor from a phage-peptide library.

Deoxyribonuclease I (DNase I) is a divalent cation dependent endonuclease and thought to be a significant barrier to effective gene delivery. The only known DNase I-specific inhibitor is monomeric actin which acts by forming a 1:1 complex with DNase I. Its use, however, is restricted because of tendency to polymerize under certain conditions. We screened two random phage peptide libraries of complexity 10(8) and 10(9) for DNase I binders as candidates for DNase I inhibitors. A number of DNase I-binding peptide sequences were identified. When these peptides were expressed as fusion proteins with Escherichia coli maltose binding protein, they inhibited the actin-DNase I interaction (IC50 = 0.1-0.7 microM) and DNA degradation by DNase I (IC50 = 0.8-8 microM). Plasmid protection activity in the presence of DNase I was also observed with the fusion proteins. These peptides have the potential to be a useful adjuvant for gene therapy using naked DNA.

A colorimetric microplate assay method for high throughput analysis of lipase activity.

The present work describes a colorimetric microplate assay for lipase activity based on the reaction between 5,5'-dithiobis(2-nitro benzoic acid) (DTNB) and the hydrolysis product of 2,3-dimercapto-1-propanol tributyrate (DMPTB). Reaction mixtures containing DTNB, DMPTB, and lipase were prepared in microplate wells, and the absorbance at 405nm was recorded after incubation at 37 degrees C for 30 min. A linear relationship was obtained in the range of 0.1-1 U of lipase activity by this method. The reaction conditions were also optimized for the range of 0.01-0.1 U or 1-10 U. When assaying crude tissue extracts, the reaction of DTNB with non-specific reducing agents created a major source of error. However, this error was corrected by the use of blank samples that did not contain DMPTB.

Microplate assay measurement of cytochrome p450-carbon monoxide complexes.

Cytochrome P450 in microsomes can be quantitated using the characteristic 450 nm absorption peak of the CO adduct of reduced cytochrome P450. We developed a simple microplate assay method that is superior to previous methods. Our method is less laborious, suitable for analyzing many samples, and less sensitive to sample aggregation. Microsome samples in microplate wells were incubated in a CO chamber rather than bubbled with CO gas, and then reduced with sodium hydrosulfite solution. This modification allowed a reliable and reproducible assay by effectively eliminating variations between estimations.