Comparative Transcriptomic Analysis of Flagellar-Associated Genes in Salmonella Typhimurium and Its rnc Mutant.

Salmonella enterica serovar Typhimurium (S. Typhimurium) is a globally recognized foodborne pathogen that affects both animals and humans. Endoribonucleases mediate RNA processing and degradation in the adaptation of bacteria to environmental changes and have been linked to the pathogenicity of S. Typhimurium. Not much is known about the specific regulatory mechanisms of these enzymes in S. Typhimurium, particularly in the context of environmental adaptation. Thus, this study carried out a comparative transcriptomic analysis of wild-type S. Typhimurium SL1344 and its mutant (∆rnc), which lacks the rnc gene encoding RNase III, thereby elucidating the detailed regulatory characteristics that can be attributed to the rnc gene. Global gene expression analysis revealed that the ∆rnc strain exhibited 410 upregulated and 301 downregulated genes (fold-change > 1.5 and p < 0.05), as compared to the wild-type strain. Subsequent bioinformatics analysis indicated that these differentially expressed genes are involved in various physiological functions, in both the wild-type and ∆rnc strains. This study provides evidence for the critical role of RNase III as a general positive regulator of flagellar-associated genes and its involvement in the pathogenicity of S. Typhimurium.

MESIA: Magnetic force-assisted electrochemical sandwich immunoassays for quantification of prostate-specific antigen in human serum.

We propose a new immunoassay technique, called magnetic-force assisted electrochemical sandwich immunoassay (MESIA), where serum biomarkers can be determined by magnetic actuation and electrochemical detection of gold-coated iron oxide nanoparticles as probes for immunocomplex formation. In MESIA, neither washing buffer nor fluidic parts are necessary, because the formation of immunocomplexes and the removal of unbound probes are controlled by magnetic forces. Electrochemical pretreatment and measurement of the gold-coated magnetic probes allows highly sensitive, precise, and robust system for quantification of target analytes. Using MESIA, the concentration of prostate-specific antigen (PSA) in 10 µl of human serum is determined within 5 min. The limit of detection is 0.085 ng/mL, and the average coefficient of variance is 8.85% for five different PSA concentrations ranging from 0 to 25 ng/mL. This method shows good precision and reproducibility (<10%) and high correlation with cobas e 801 (r = 0.997) for clinical patient samples. We believe this technique to be useful in the development of a point-of-care testing platform for diagnosis and prognosis of various diseases, such as cancer, based on quantification of biomarkers in a drop of blood.

Plastic enzyme-linked immunosorbent assays (ELISA)-on-a-chip biosensor for botulinum neurotoxin A.

A plastic ELISA-on-a-chip (EOC) employing the concept of cross-flow immuno-chromatographic analysis was applied to the measurement of botulinum neurotoxin A (BoNT/A) as agent for bio-terrorism. Two monoclonal antibodies specific to the heavy chain of the toxin were raised and identified to form sandwich binding complexes as the pair with the analyte. For the construction of an immuno-strip, one was utilized as the capture antibody immobilized onto nitrocellulose membrane and the other as the detection coupled to an enzyme, horseradish peroxidase. The two plates of EOC used in this study were fabricated by injection molding of polycarbonate to improve the reproducibility of manufacture and, after inclusion of the immuno-strip, bonded using a UV-sensitive adhesive. Under optimal conditions of analysis, the chip produced a color signal in proportion to the analyte dose and the signal was quantified using a detector equipped with a digital camera. From the dose-response curve, the detection limit of BoNT/A was 2.0 ng mL(-1), approximately five times more sensitive than a commercial-version detection kit employing colloidal gold tracer.

Plastic ELISA-on-a-chip based on sequential cross-flow chromatography.

A plastic chip that can perform immunoassays using an enzyme as signal generator, i.e., ELISA-on-a-chip, was developed by incorporating an immunostrip into channels etched on the surfaces of the chip. To utilize an analytical concept of cross-flow chromatography, the chip consisted of two cross-flow channels in the horizontal and vertical directions. In the vertical channel, we placed a 2-mm-wide immunostrip for cardiac troponin I (cTnI), which was identical to a conventional rapid test kit except for the utilization of an enzyme, horseradish peroxidase (HRP), as tracer. An enzyme substrate supply channel and a horizontal flow absorption pad compartment were transversely arranged on each lateral side of the signal generation pad of the strip, respectively. Upon application of a sample containing cTnI, it migrated vertically through the membrane strip by capillary action, and antigen-antibody binding occurred. After 15 min, the horizontal flow was initiated by the addition of a chromogenic substrate solution for HRP into the supply channel and by partial superimposition of the horizontal flow absorption pad onto the signal generation pad. A color signal proportional to the analyte concentration was produced on this pad, measured after 5 min as optical densities using a digital camera-based detector, and quantified by integration of the densities under the peak after normalization. Its calibration curve indicated that the detection limit of the chip was approximately 0.1 ng/mL and its quantification limit was 0.25 ng/mL. In measuring blindly prepared samples, the chip performance correlated with that of a reference system, Beckman Coulter Access, within 2.5-fold discrepancy at the detection limit.