Detection system based on the conformational change in an aptamer and its application to simple bound/free separation

Aptamers are good molecular recognition elements for biosensors. Especially, their conformational change, which is induced by the binding to the target molecule, enables the development of several types of useful detection systems. We applied this property to bound/free separation, which is a crucial process for highly sensitive detection. We designed aptamers which change their conformation upon binding to the target molecule and thereby expose a single-strand bearing the complementary sequence to the capture probe immobilized onto the support. We named the designed aptamers "capturable aptamers" and the capture probe "capture DNA". Three capturable aptamers were designed based on the PrP aptamer, which binds to prion protein. One of these capturable aptamers was demonstrated to recognize prion protein and change its conformation upon binding to it. A detection system using this designed capturable aptamer for prion protein was developed. Capturable aptamers and capture DNA allow us to perform simple bound/free separation with only one target ligand.     

Effect of incident angle of light on sensitivity and detection limit for layers of antibody with surface plasmon resonance spectroscopy

The effect of the incident angle of light on sensitivity and the detection limit for surface-plasmon resonance spectroscopy were examined. The sensitivities and the detection limit were experimentally measured using an antibody as a modeled analyte in the incident angles of a light region of 66-76 degrees. The results showed that the sensitivity of a smaller incident angle was higher than that of a larger one. For instance, the sensitivity of a 66 degree incident angle was three times higher than that of a 76 degree incident angle. The detection limit with a 66 degree incident angle was one-tenth of that with a 76 degree incident angle. These sensitivities and detection limits were compared with those of a commercially produced surface-plasmon resonance instrument. This comparison demonstrated that a wavelength resolution of the order of less than 10(-2) nm was necessary to obtain satisfactory sensitivities and detection limits. In addition, the refractive index and thickness of the antibody layer formed on a sensor surface was proposed by experimental results and theoretical calculation.     

Purification of single stranded DNA from asymmetric PCR product using the SMART system

A method has been developed using the SMART system for the purification of single stranded DNA from a mixture containing single- and double-stranded DNA amplified using asymmetric PCR. The asymmetric PCR product was separated into single- and double-stranded DNA using an anion exchange column which took 15 min. Compared to another method in which biotinylated symmetric PCR products were applied to an immobilized streptavidin column, this method was simple and could purify single- and double-stranded DNA. © Rapid Science Ltd. 1998     

Selection of DNA aptamers against VEGF<Subscript>165</Subscript> using a protein competitor and the aptamer blotting method

Two DNA aptamers against a tumor marker protein, human vascular endothelial growth factor (VEGF₁₆₅) were identified. In the screening process, another protein was used as the competitor to isolate those aptamers that have high specificity for the target. In addition, we evaluated the affinities of the enriched library by means of aptamer blotting. The isolated aptamers bound to VEGF₁₆₅ with a K _d value in the range of a few hundred nanomoles, and did not bind to the competitor. This selection method enabled us to efficiently select the specific aptamers against the target protein. These specific aptamers would be useful sensor elements for cancer diagnosis.     

PCR-Based Ribosomal DNA Detection Technique for Microalga (Heterosigma carterae) Causing Red Tide and Its Application to a Biosensor Using Labeled Probe

A technique for detecting Raphidophycean, a bloom-forming genus of algae, was developed using a specific DNA probe. The design of the probe was based on a sequence polymorphism within the small subunit (SSU) ribosomal RNA gene (rDNA) of this strain by using fluorescence polarization (FP) analysis and the BIAcore 2000 biosensor, which utilized surface plasmon resonance (SPR). The specific sequence in SSU rDNA for Heterosigma carterae was determined by sequence data analysis. One pair of polymerase chain reaction (PCR) probes was designed for use in making the identification. H. carterae SSU rDNA was amplified by PCR. Using a fluoroscein isothiocyanate–labeled or biotin-labeled oligonucleotide probe, the PCR-amplified rDNA was selectively detected as an FP-intensity change via FP analysis or as a resonance-unit change via SPR. Although total time for final detection after sampling was within 3 hours, specific rDNA could be detected within 10 minutes after PCR through these detection methods.     

A surface plasmon resonance probe with a novel integrated reference sensor surface

A surface plasmon resonance (SPR) sensor probe with integrated reference surface is described. In order to fabricate the integrated reference surface, two dielectric layers with different thickness were deposited on the single gold SPR sensor surface via plasma polymerization of hexamethyldisiloxane. The working sensor surface was a 34 nm dielectric layer with immobilized bovine serum albumin (BSA) antigen and an adjacent thin 1 nm dielectric layer without BSA provided reference surface. A specific immunoreaction of anti-BSA antibody was detected after immersion of the SPR probe into sample solution. Simultaneous observation of reference and working surface response enabled determination of the immunoreaction without the need for the baseline measurement. Moreover, compensation of nonspecific adsorption could be confirmed using anti-human serum albumin antibody.     

Interaction of three-way DNA junctions with steroids

DNA aptamers that bind to cholic acid were previously isolated by an in vitro selection method. Secondary structural prediction and deletion-mutant experiments suggested that the cholic-acid binding regions of 19 sequenced clones could form three-way-junction structures. In this article, the secondary structures of the sequenced clones and the structural requirements for binding to cholic acid were evaluated. A course of mutational-analysis and chemical-modification experiments provided strong support for the predicted secondary structure and also indicated that the binding site is located at the branching point of the three-way junction. Sequence analysis revealed that the sequences of the three base pairs flanking the junction of the three stems are highly conserved among selected clones. The evaluation of the relative binding of several bile acids and structurally related steroids with the aptamer was also carried out. The results revealed a broad range of selectivity and preference for hydrophobic steroids rather than for cholic acid upon binding, indicating that the binding is driven by a hydrophobic interaction. The experimental results reported here allowed us to propose a structural model of a binding site formed by three Watson–Crick base pairs.