Development of photocatalytic biosensor for the evaluation of biochemical oxygen demand

The photocatalytic biosensor of flow system using semiconductor TiO2 was developed to evaluate biochemical oxygen demand (BOD) levels in river water. Photocatalysis of sample was carried out in a photoreactor with TiO2 and a 6W black-light blue fluorescent tube as light source. Sample from a photoreactor outlet was measured by an oxygen electrode with a biofilm. The sensor response of photocatalytic biosensor was between 5 and 10 min depending on concentration of biochemical in the samples. At BOD of 1 mgl-1, the sensor response increased 1.33-fold in comparison with that without photocatalysis. The degradation of tannic acid and humic acid with photocatalysis were 51.8 and 38.4%, respectively. Gum arabic and linear alkylbenzene sulfonate (LAS) were degraded a little, but gave the responses of more than double to the sensor. Free radicals yielded by photocatalysis in a photoreactor did not affect the sensor response because their lifetime is extremely short. Fairly good correlation (r=0.983) between the sensor method and the conventional method was obtained for test samples. This biosensor using photocatalytic pretreatment improved the sensitivity.     

Detection of PCR products of Escherichia coli O157:H7 in human stool samples using surface plasmon resonance (SPR)

A method for the rapid detection of verotoxin-producing Escherichia coli O157:H7 in stools was evaluated. Strains possessing Shiga toxin-2 (stx-2) genes were isolated from stool samples and amplified using oligonucleotide primers. Stools spiked with cultured E. coli O157:H7 (strain 298 or strain 1646) were detected to be polymerase chain reaction (PCR) positive at 10(2) cfu per 0.1 g of stool. Stool samples from patients and healthy carriers showed a high correlation between positive results for a PCR and the presence of verotoxin-producing E. coli O157:H7, confirmed by isolation of serotype O157:H7 on sorbitol MacConkey medium (10 of 10 stool samples). These PCR products could be detected using a BIAcore 2000 surface plasmon resonance device using peptide nucleic acid as a sensor probe. In this report we use this method for the rapid detection of DNA from significant pathogenic organisms.     

Aptamer Selection Based on G4-Forming Promoter Region

We developed a method for aptamer identification without in vitro selection. We have previously obtained several aptamers, which may fold into the G-quadruplex (G4) structure, against target proteins; therefore, we hypothesized that the G4 structure would be an excellent scaffold for aptamers to recognize the target protein. Moreover, the G4-forming sequence contained in the promoter region of insulin can reportedly bind to insulin. We thus expected that G4 DNAs, which are contained in promoter regions, could act as DNA aptamers against their gene products. We designated this aptamer identification method as “G4 promoter-derived aptamer selection (G4PAS).” Using G4PAS, we identified vascular endothelial growth factor (VEGF)165, platelet-derived growth factor-AA (PDGF)-AA, and RB1 DNA aptamers. Surface plasmon resonance (SPR) analysis revealed that the dissociation constant (K _d) values of VEGF165, PDGF-AA, and RB1 DNA aptamers were 1.7 × 10⁻⁷ M, 6.3 × 10⁻⁹ M, and 4.4 × 10⁻⁷ M, respectively. G4PAS is a simple and rapid method of aptamer identification because it involves only binding analysis of G4 DNAs to the target protein. In the human genome, over 40% of promoters contain one or more potential G4 DNAs. G4PAS could therefore be applied to identify aptamers against target proteins that contain G4 DNAs on their promoters.     

A polymerase chain reaction-based ribosomal DNA detection technique using a surface plasmon resonance detector for a red tide causing microalga, Alexandrium affine

A detection technique with a DNA probe was developed for the bloom‐forming alga Alexandrium affine harvested in Japan. The design of this probe was based on the sequence polymorphism within the 28S ribosomal DNA (rDNA) of this strain using the BIAcore? 2000 biosensor, which determines surface plasmon resonance. The specific DNA sequence in 28S rDNA for A. affine was determined by sequence data analysis, and a probe was designed for the detection of A. affine. A fragment of the 28S rDNA from A. affine was amplified by polymerase chain reaction and applied to the BIAcore? sensor system, and the target DNA was selectively recognized by species‐specific hybridization using two DNA probes: a fluorescein isothiocyanate (FITC)‐labeled probe and a biotin‐labeled DNA probe. Using FITC‐labeled anti‐immunogloblin G antibody, enhancement of the response for the target DNA can be detected directly as a resonant unit change. In this detection method, a difference within only 20 base pairs of the target could be detected, and specific detection of A. affine was achieved intraspecifically.     

Application of peptide nucleic acid to the direct detection of deoxyribonucleic acid amplified by polymerase chain reaction.

Double-stranded DNA amplified by polymerase chain reaction (PCR) was detected by peptide nucleic acid (PNA) using a BIAcore 2000 biosensor based on surface plasmon resonance (SPR). PNA is an artificial oligo amide that is capable of forming highly stable complexes with complementary oligonucleotides. We succeeded in the direct detection of double-stranded DNA, amplified by PCR with high-sequence specificity. It was shown that the target DNA was available for detection over the range of 40-160 nM. Therefore, the detection limit was 7.5 pmol of the target DNA (143 bases, applied volume 30 microliters). Our DNA detection system, the combination of BIAcore and the probe PNA, could detect the target DNA with good reproducibility. In this report, we show that our system is a powerful tool for the diagnosis of pathologically significant DNA.