Microplate differential calorimetric biosensor for ascorbic acid analysis in food and pharmaceuticals.

In this paper we report on the development of a label-free low-volume (12.5 microL), high-throughput microplate calorimetric biosensor for fast ascorbic acid quantification in food and pharmaceutical products. The sensor is based on microplate differential calorimetry (MiDiCal) technology in which the heat generation, due to the exothermic reaction between ascorbic acid and ascorbate oxidase, is differentially monitored between two neighboring wells of an IC-built wafer. A severe discrepancy is found between expected and observed sensor readings. To investigate the underlying mechanisms of these findings a mathematical model, taking into account the biochemical reactions and diffusion properties of oxygen, ascorbic acid, and ascorbate oxidase, is developed. This model shows that oxygen depletion in the microliter reaction volumes, immediately after injection of sample (ascorbic acid) into the well, causes the enzymatic reaction to slow down. Calibration experiments show that the sensor's signal is linearly correlated to the area under the output versus time profile for the ascorbic acid concentration range from 2.4 to 350 mM with a limit of detection of 0.8 mM. Validation experiments on fruit juice samples, food supplements, and a pain reliever supplemented with ascorbic acid reveal that the designed method correlates well with HPLC reference measurements. The main advantages of the presented biosensor are the low analysis cost due to the low amounts of enzyme and reagents required and the possibility to integrate the device in fully automated laboratory analysis systems for high-throughput screening and analysis.

Actinomyces naeslundii fimbrial protein Orf977 shows similarity to a streptococcal adhesin.

The nucleotide sequence of the chromosomal DNA, upstream of Actinomyces naeslundii T14V fimbrial gene fimA, was determined. One open reading frame (orf977) encoding 977 amino acids was found, preceded by a gene homologous to elongation factor TU. Database searches revealed that Orf977 was homologous to CshA, a Streptococcus gordonii protein involved in cell adhesion. Previous studies had already determined two genes in the type 2 fimbrial gene cluster of A. naeslundii T14V: the structural subunit fimA, and orf365 with unknown function, followed by ribosomal genes. This study completes the type 2 fimbrial gene cluster sequence.

Nucleotide sequence and characterization of the cryptic Bacillus thuringiensis plasmid pGI3 reveal a new family of rolling circle replicons.

The complete nucleotide sequence of plasmid pGI3 from Bacillus thuringiensis subsp. thuringiensis H1.1. was obtained. Although this 11,365-bp molecule contained at least 11 putative open reading frames (ORFs), extensive database searches did not reveal any homologous sequences with the exception of ORF6, which displayed similarity to the largest ORF of pSTK1, a 1,883-bp cryptic plasmid isolated from Bacillus stearothermophilus. Deletion analysis to determine the pGI3 minimal replicon revealed that ORF6 is the rep gene. Replication occurred via a single-stranded DNA (ssDNA) intermediate, as demonstrated by S1 treatment and Southern hybridization in nondenaturating conditions. Interestingly, however, no homology was found between the pGI3 (ORF6) and pSTK1 (ORF3) rep genes and those from other single-stranded DNA plasmids, nor was there any DNA similarity to the double-strand origins of replication characterized so far, indicating that pGI3 and pSTK1 form another, new family of ssDNA plasmids. PCR analysis revealed that the pGI3 rep gene is largely distributed among B. thuringiensis strains but can also be found in B. cereus and B. mycoides strains, albeit at a lower frequency. Finally, segregation experiments performed with B. subtilis and B. thuringiensis showed that the pGI3 derivatives, including the minimal replicon, were segregationally stable at temperatures suitable for B. thuringiensis growth (<43 degrees C).