RESUMEN
A well-designed three-way junction (TWJ) aptasensor for lysozyme detection was developed based on target-binding-induced conformational change of aptamer-complementary DNA (cDNA) as probe. A ferrocene (Fc)-tagged cDNA is partially hybridized with an anti-lysozyme aptamer to form a folded structure where there is a coaxial stacking of two helices and the third one at an acute angle. In addition, the fabrication of the sensor was achieved via the single-step method, which offered a good condition for sensing. In the absence of lysozyme, electron transfer (eT), through the coaxial two helices called "conductive path", is allowed between Fc-labeled moiety and the electrode. The binding of lysozyme to the aptamer blocks eT, leading to diminished redox signal. This aptasensor with an instinct signal attenuation factor shows a high sensitivity to lysozyme, and the response data is fitted by nonlinear least-squares to Hill equation. Detection limit is 0.2nM with a dynamic range extending to 100nM. Compared with existing electrochemical impedance spectroscopy (EIS)-based approaches, TWJ-DNA aptasensor was demonstrated to be more specific for detection and simpler for regeneration procedure.
Asunto(s)
Aptámeros de Nucleótidos/química , Técnicas Biosensibles/métodos , Espectroscopía Dieléctrica/métodos , Muramidasa/análisis , Animales , Pollos , ADN Complementario/química , Compuestos Ferrosos/química , Límite de Detección , Metalocenos , Modelos Moleculares , Conformación de Ácido NucleicoRESUMEN
We report a sensitive approach for SERS detection of cytochrome c using target binding-induced conformational changes of signal transduction probe (STP). STP labeled with a SERS-active molecule, carboxy-X-rhodamine (ROX), is immobilized on the substrate where the formation of a rigid triplex switching structure with aptamers does not allow SERS amplification to take place. The target binding event leads to an enhancement in SERS intensity of ROX adsorbed on the gold surface. Meanwhile, we found that an appropriate STP surface density could shield the SERS signal produced by protein adsorption which would foul the sensing surface. In addition, cytochrome c aptamers used were not the original sequence but reorganized in the nonspecific binding site to adapt to our design. This method provides a low detection limit of 2 nM (10 fmol within 5 µL sample solution), and shows good selectivity toward cytochrome c compared to interfering proteins such as hemoglobin and immunoglobulin G. The general strategy of the method can also be extended to aptamer or DNA based sensors.