RESUMO
Catalase (CAT) is an important enzyme that protects biomolecules against oxidative damage by breaking down hydrogen peroxide (H2O2) into water and oxygen. CAT is present in all aerobic microbes, animals, and plants. It is, however, absent from normal human urine but can be detected in pathological urine. CAT testing can thus help to detect such urine. This study presents a novel spectrophotometric method for determining CAT activity characterized by its simplicity, sensitivity, specificity, and rapidity. The method involves incubating enzyme-containing samples with a carefully chosen concentration of H2O2 for a specified incubation period. Subsequently, a solution containing ferrous ammonium sulfate (FAS) and sulfosalicylic acid (SSA) is added to terminate the enzyme activity. A distinctive maroon-colored ferrisulfosalicylate complex is formed. The formation of this complex is a direct result of the reaction between FAS and any residual peroxide present. This leads to the generation of ferric ions when coordinated with SSA. The complex has a maximum absorbance of 490 nm. This advanced method eliminates the need for concentrated acids to stop CAT activity, making it safer and easier to handle. A comparative analysis against the standard ferrithiocyanate method showed a correlation coefficient of 0.99, demonstrating the new method's comparable effectiveness and reliability. In conclusion, a simple and reliable protocol for assessing CAT activity, which utilizes a cuvette or microplate, has been demonstrated in this study. This interference-free protocol can easily be used in research and clinical analysis with considerable accuracy and precision.
RESUMO
MicroRNA (miRNA) is a small non-coding RNA with the size of 18-22 nucleotide. MiRNAs play a major role in the gene expression and regulation. They influence more than 50% of protein coding genes in mammalian genome which regulate many cellular functions. Their dysregulation can lead to cancer and other diseases. MiRNAs have been shown to be associated with breast cancers. Previously q-PCR based assays have been used successfully for detection and quantification of miRNAs, however, these assays are expensive and cumbersome. Here, we report the detection of few breast cancer microRNA sequences using fluorescence displacement assay. The assay employed a fluorophore-quencher pair by hybridization of fluorescently labelled cDNA of miRNA and a quencher labelled short DNA. Upon hybridization, the fluorophore and the quencher become in close vicinity to each other leading to significant fluorescence quenching. In the presence of target miRNA, the fluorophore and quencher are separated due to the duplex dissociation and form stable cDNA-miRNA double strand which leads to increase in the fluorescence intensity. The assay has dynamic range from 0.2 to 250â¯nM miRNA concentration. We could detect miRNA as low as 1â¯pM level. The method was applied to detect the miRNA spiked to the total RNA extracted from whole blood. The fluorescence based method has been validated using standard Q-PCR method. This could prove a promising method for the detection of miRNAs.
