Generation of epitope-specific hCG aptamers through a novel targeted selection approach.

Human chorionic gonadotropin (hCG) is a glycoprotein hormone used as a biomarker for several medical conditions, including pregnancy, trophoblastic and nontrophoblastic cancers. Most commercial hCG tests rely on a combination of antibodies, one of which is usually specific to the C-terminal peptide of the ß-subunit. However, cleavage of this region in many hCG degradation variants prevents rapid diagnostic tests from quantifying all hCG variants in serum and urine samples. An epitope contained within the core fragment, ß1, represents an under-researched opportunity for developing immunoassays specific to most variants of hCG. In the study described here, we report on a SELEX procedure tailored towards the identification of two pools of aptamers, one specific to the ß-subunit of hCG and another to the ß1 epitope within it. The described SELEX procedure utilized antibody-blocked targets, which is an underutilized strategy to exert negative selection pressure and in turn direct aptamer enrichment to a specific epitope. We report on the first aptamers, designated as R4_64 and R6_5, each capable of recognising two distinct sites of the hCG molecule-the ß-subunit and the (presumably) ß1-epitope, respectively. This study therefore presents a new SELEX approach and the generation of novel aptamer sequences that display potential hCG-specific biorecognition.

Generation and screening of histamine-specific aptamers for application in a novel impedimetric aptamer-based sensor.

Histamine is an important biomarker in both biomedical and food quality assurance sectors. Current methods of monitoring this compound via fluorescent, electrochemical, and enzymatic means have several drawbacks, preventing routine detection. This work reports on the isolation of single-stranded DNA-based, histamine-targeting aptamers generated by the Systematic Evolution of Ligands by Exponential Enrichment (SELEX) and the characterisation of these candidates via bioinformatics analysis. Aptamer binding affinity was determined by magnetic bead-based enzyme linked oligonucleotide assays, followed by the detection of unmodified histamine at a physiological pH via electrochemical impedance spectroscopy (EIS). Aptamer H47 demonstrated the lowest apparent binding affinity (72.8 ±â€¯13.9 nmol L-1) towards bead immobilised histamine. When immobilised to a gold surface, H47 demonstrated the largest biosensor response (ΔRct = 6.83 ±â€¯2.00) compared to other single-stranded DNA sequences in the presence of dissolved histamine. The H47 EIS aptasensor also displayed a highly selective, concentration-dependent response towards histamine (linear range = 1 µmol L-1 - 5 mmol L-1), compared to other similar small molecules. Possessing an apparent binding affinity, limit of detection and limit of quantification of 7.80 ±â€¯1.70 mmol L-1, 4.83 mmol L-1 and 16.08 mmol L-1, respectively, the H47 EIS aptasensor demonstrates promise towards the development of aptasensors in applications which require the rapid detection of histamine in solution.

Quantitative methylene blue decolourisation assays as rapid screening tools for assessing the efficiency of catalytic reactions.

Identifying the most efficient oxidation process to achieve maximum removal of a target pollutant compound forms the subject of much research. There exists a need to develop rapid screening tools to support research in this area. In this work we report on the development of a quantitative assay as a means for identifying catalysts capable of decolourising methylene blue through the generation of oxidising species from hydrogen peroxide. Here, a previously described methylene blue test strip method was repurposed as a quantitative, aqueous-based spectrophotometric assay. From amongst a selection of metal salts and metallophthalocyanine complexes, monitoring of the decolourisation of the cationic dye methylene blue (via Fenton-like and non-Fenton oxidation reactions) by the assay identified the following to be suitable oxidation catalysts: CuSO4 (a Fenton-like catalyst), iron(II)phthalocyanine (a non-Fenton oxidation catalyst), as well as manganese(II) phthalocyanine. The applicability of the method was examined for the removal of bisphenol A (BPA), as measured by HPLC, during parallel oxidation experiments. The order of catalytic activity was identified as FePc > MnPc > CuSO4 for both BPA and MB. The quantitative MB decolourisation assay may offer a rapid method for screening a wide range of potential catalysts for oxidation processes.

Novel detection of Escherichia coli beta-D-glucuronidase activity using a microbially-modified glassy carbon electrode and its potential for faecal pollution monitoring.

The electrochemical detection of Escherichia coli beta-D-glucuronidase activity as a means of monitoring water pollution by faecal material was investigated using separate Moraxella- and Pseudomonas putida-modified glassy carbon electrodes. The former was more sensitive and selective. The Moraxella-modified biosensor was 100 times more rapid and sensitive than the spectrophotometric detection of beta-D-glucuronidase activity. The experimental limit of detection of the biosensor was two c.f.u. per 100 ml polluted water sample within 20 min. The biosensor gave a linear response to commercial beta-D-glucuronidase concentration between 0.2 ng and 2 microg ml(-1). The biosensor detected activity of beta-D-glucuronidase from viable but non-culturable (VBNC) cells and can therefore serve as a presence or absence device for rapid water quality monitoring.