Gene Assays Based on Bio(Chemi)luminescence

Abstract

One of the first reports describing the use of recombinant aequorin as a reporter molecule in a bioluminometric nucleic acid hybridization assay was in 1996. 1 Microtiter wells were coated with anti-digoxigenin antibody. The target DNA was hybridized simultaneously with an immobilized digoxigenin-labeled capture probe and a biotinylated detection probe. The hybrids were determined using an aequorin-streptavidin conjugate followed by the measurement of luminescence in the presence of excess Ca 21 (Figure 9.1). The linearity of the assay was in the range 0.1-200 pM (5 amol well À1 to 10 fmol well À1) and the S/B ratio at 0.1 pM (5 amol well À1) was 5.3. A configuration in which the aequorinstreptavidin conjugate was replaced by a preformed complex of biotinylated aequorin to streptavidin resulted in equivalent signals and detectability. An advantage of using the preformed complex is that it was conveniently prepared by simply mixing the two components (streptavidin and biotinylated aequorin), thus providing a practical alternative to the use of aequorin-streptavidin conjugates prepared by covalent crosslinking techniques. The aequorin-biotin and the aequorin-streptavidin conjugates in this study were obtained commercially (the same group later reported the construction of a plasmid suitable for bacterial expression of in vivo-biotinylated aequorin, facilitating further the development of highly sensitive hybridization assays, 2 as well as a method for

Highly Sensitive Detection ofCampylobacter spp.in Chicken Meat using a Silica Nanoparticle Enhanced Dot Blot DNA Biosensor

“…Detection of 1 ng/µL CP3 under the optimized conditions using various concentrations of streptavidin-HRP and biotin-Si-NPs allowed to select 25 ng/µL of streptavidin-HRP and 10 6 nanoparticles/mL for the chemiluminometric reaction. It is worth to note that chemiluminometric reaction itself does not significantly contribute to the background staining as the emission of light arises from the enzymatic reaction without any photonic excitation (Laios et al 2010).…”

Absolute Quantification of the Alleles in Somatic Point Mutations by Bioluminometric Methods based on Competitive Polymerase Chain Reaction in the Presence of a Locked Nucleic Acid Blocker or an Allele-Specific Primer

“…The technology is based on a combination of microemulsion PCR on magnetic beads followed by allele-specific oligonucleotide hybridization and laser-induced fluorescence detection on a flow cytometer . On the other hand, end-point bio(chemi)luminometric assays have found wide applications in DNA/RNA assays since they provide higher detectability, wider linear range, high throughput, and simpler instrumentation (no need for excitation light). − Enzymes, along with chemiluminogenic substrates, or photoproteins have been employed as reporters in hybridization assays for the determination of PCR-amplified DNA, − determination of microRNA, and quantitative competitive PCR. , Bio(chemi)luminometric assays for genotyping of point mutations (e.g., single-nucleotide polymorphisms) comprise three steps: (i) exponential amplification, usually by PCR, (ii) an allele-discrimination reaction, e.g., primer extension reaction , or oligonucleotide ligation reaction, and (iii) detection of the reaction product by exploiting a bio(chemi)luminescent reporter. However, the aforementioned assays simply discriminate the three genotypes (normal, homozygote, and heterozygote) in inherited mutations without quantifying the amount of the mutant allele in the sample.…”