Sensitive Electrochemiluminescence Immunosensor for Detection of N-Acetyl-β-d-glucosaminidase Based on a "Light-Switch" Molecule Combined with DNA Dendrimer.

Abstract

Here, a novel "light-switch" molecule of Ru (II) complex ([Ru(dcbpy)2dppz](2+)-DPEA) with self-enhanced electrochemiluminescence (ECL) property is proposed, which is almost nonemissive in aqueous solution but is brightly luminescent when it intercalates into DNA duplex. Owing to less energy loss and shorter electron-transfer distance, the intramolecular ECL reaction between the luminescent [Ru(dcbpy)2dppz](2+) and coreactive tertiary amine group in N,N-diisopropylethylenediamine (DPEA) makes the obtained "light-switch" molecule possess much higher light-switch efficiency compared with the traditional "light-switch" molecule. For increasing the loading amount and further enhancing the luminous efficiency of the "light-switch" molecule, biotin labeled DNA dendrimer (the fourth generation, G4) is prepared from Y-shape DNA by a step-by-step assembly strategy, which provides abundant intercalated sites for [Ru(dcbpy)2dppz](2+)-DPEA. Meanwhile, the obtained nanocomposite (G4-[Ru(dcbpy)2dppz](2+)-DPEA) could well bind with streptavidin labeled detection antibody (SA-Ab2) due to the existence of abundant biotin. Through sandwiched immunoreaction, an ECL immunosensor was fabricated for sensitive determination of N-acetyl-β-d-glucosaminidase (NAG), a typical biomarker for diabetic nephropathy (DN). The detemination linear range was 0.1 pg mL(-1) to 1 ng mL(-1), and the detection limit was 0.028 pg mL(-1). The developed strategy combining the ECL self-enhanced "light-switch" molecular and DNA nanotechnology offers an effective signal amplification mean and provides ample potential for further bioanalysis and clinical study.