Development of a protein-free amplification assay for small-molecule target using aptamer, graphene oxide, and fenton reagents

Abstract

DNA-based aptamers are synthetic single-stranded oligonucleotides that can bind to various target analytes with high affinity and specificity. The use of aptamers as recognition probes has offered new avenues for the detection of small molecule biomarkers. Fluorophore-labelled aptamer coupled with graphene oxide provided a quantitative model for the development of a sensitive and high-throughput fluorescence-based assay. With new aptamers being reported, a simple, general, and cost-effective aptamer/GO fluorescence assay for small molecules is required. In this study, adenosine (a signalling metabolite) was used as a model biomarker. The key focus is to design an adenosine aptamer/GO fluorescence assay to detect adenosine in human serum. Without using any strategy, the specific signal from the serum containing adenosine could not be distinguished from the background serum signal. This was due to the severe nonspecific aptamer probe desorption from graphene oxide that induced by the interference molecules (such as proteins) in serum. A 3,000 Da ultrafiltration centrifugal device was then used to fractionate human serum specimens based on their molecular weights. By doing so, the issue of the undesirable false-positive signal was resolved and selective adenosine detection in ultrafiltered serum was achieved. Apart from the assay specificity improvement, the sensitivity enhancement is another important aspect of detection. Fenton reagents, comprising ferrous iron, EDTA, and hydrogen peroxide were added to the assay. A mixture of these chemicals generated hydroxyl radicals that triggered the target recycling signal amplification. The limit of detection in buffer and ultrafiltered serum was boosted by a factor of 100 and 9, respectively. In conclusion, the "protein-free" reagent assay (i.e., no antibodies and nucleases involved) herein developed can be an alternative to conventional protein-based biosensing methods.