Real-time PCR for direct aptamer quantification on functionalized graphene surfaces

Viviane C F Dos Santos,Thiago A S L De Sousa,Flávio Plentz,Eduardo N D Araujo,Nathalie B F Almeida,Antero S R De Andrade

doi:10.1038/s41598-019-55892-3

Abstract

In this study, we develop a real-time PCR strategy to directly detect and quantify DNA aptamers on functionalized graphene surfaces using a Staphylococcus aureus aptamer (SA20) as demonstration case. We show that real-time PCR allowed aptamer quantification in the range of 0.05 fg to 2.5 ng. Using this quantitative technique, it was possible to determine that graphene functionalization with amino modified SA20 (preceded by a graphene surface modification with thionine) was much more efficient than the process using SA20 with a pyrene modification. We also demonstrated that the functionalization methods investigated were selective to graphene as compared to bare silicon dioxide surfaces. The precise quantification of aptamers immobilized on graphene surface was performed for the first time by molecular biology techniques, introducing a novel methodology of wide application.

Highlights

In this study, we develop a real-time PCR strategy to directly detect and quantify DNA aptamers on functionalized graphene surfaces using a Staphylococcus aureus aptamer (SA20) as demonstration case
Graphene-based biosensors functionalized with aptamers have already been demonstrated to detect a wide range of targets, such as cancer molecules[15], Staphylococcus aureus[16], DNA17, glucose[18], bacteria on tooth enamel[13] and immunoglobulin E19,20
The S. aureus binding aptamer SA2030 was modified in the 5′ end with a 3C spacer followed by a pyrene cap phosphoramidite (SA20-pyrene) and in the 5′ end with a 6C spacer followed by an amine moiety (SA20-amino)

Summary

Introduction

We develop a real-time PCR strategy to directly detect and quantify DNA aptamers on functionalized graphene surfaces using a Staphylococcus aureus aptamer (SA20) as demonstration case. In comparison with traditional ligands, such as antibodies, aptamers present some advantages They are chemically stable, cost-effective, more resistant to pH and temperature variations, and are more flexible in the design of their structures[2]. Good examples are the ability to even detect single molecules and single micro-organisms[12,13] This makes graphene an ideal candidate for sensing applications, for instance as a transducer in biosensors devices[14]. Graphene-based biosensors functionalized with aptamers have already been demonstrated to detect a wide range of targets, such as cancer molecules[15], Staphylococcus aureus[16], DNA17, glucose[18], bacteria on tooth enamel[13] and immunoglobulin E19,20. This can only be accomplished by means of highly efficient and reproducible functionalization strategies

Objectives

Methods

Results