A subfemtomolar electrochemical DNA biosensor realized by in-situ grafting of gold nanoparticle/neutral red on the terminal of hairpin probe as the signal tag

Abstract

Hairpin-structured oligonucleotide (HSO) probe presents higher analytical specificity than the traditional linear analog since the analytical signal driven by conformation change of HSO strictly follows the base-pairing principle. However, the homogeneous liquid-based preparation of electroactive HSO is complicated and time/reagent-consuming, and the signal output is limited. Herein, a novel strategy for electroactive HSO synthesis is proposed by direct labeling of gold nanoparticle (GNP) and neutral red (NR) on the terminal of the HSO probe that pre-confined on a gold electrode, which easily realizes the synthesis of the electroactive HSO and fabrication of electrochemical DNA biosensor synchronously. Electrochemical results show that the assembled electroactive tag (NR-GNP) presents excellent electroactivity and stronger electrochemical signal intensitiy than the single-point labeling HSO. The HSO probe after in-situ labeling of electroactive NR-GNP tag remains the intrinsic outstanding hybridization specificity of the pristine one. A good linear relationship between the signal attenuation of the biosensor and the logarithm of target DNA concentration is achieved in seven orders (1.0 fM ~ 1.0 nM). The detection limit is down to subfemtomolar level. When the developed biosensor is applied for target DNA analysis in a 10% (V/V) diluted serum sample, satisfactory recoveries from 93.6 to 101.6%, showing promising values of the strategy in practical clinic diagnosis.