Study Of Aptasensors Based On DNA Molecular Switches

DNA-based molecular switches are DNA assemblies that can flip reversiblybetween two or more states in a controllable manner. External stimuli that trigger thechange of the state concludes photons, temperature, pressure, magnetic or electricfields, and change of chemical environments. The state of DNA assemblies could bechanged in response to temperature, photoisomerization, presence or depletion ofvarious ions, and protein binding. Aptamer, a single-stranded nucleic acid moleculewith high selective property and affinity, is a reasonable component of moleculerecognition and has been extensively used in the preparation of biosensors. Thebiosensors based on the aptamer can be used in the diagnostics, therapy andproteomics research.In this paper, we prepared three types of DNA-switch-based aptasensors forproteins and small molecules, and investigated the properties of these three sensors byelectrochemical methods and surface-enhanced Raman spectrometry (SERS). Detailsare presented in this work as follows:1. A three-way junction aptasensor for lysozyme detectionA well-designed three-way junction (TWJ) aptasensor for lysozyme detectionwas developed based on target-binding-induced conformational change ofaptamer-complementary DNA (cDNA) as probe. A ferrocene (Fc)-tagged cDNA ispartially hybridized with an anti-lysozyme aptamer to form a folded structure wherethere is a coaxial stacking of two helices and the third one at an acute angle. Inaddition, the fabrication of the sensor was achieved via the single-step method, whichoffered a good condition for sensing. In the absence of lysozyme, electron transfer(eT), through the coaxial two helices called ‘‘conductive path’’, is allowed betweenFc-labeled moiety and the electrode. The binding of lysozyme to the aptamer blockseT, leading to diminished redox signal. This aptasensor with an instinct signalattenuation factor shows a high sensitivity to lysozyme, and the response data is fittedby nonlinear least-squares to Hill equation. Detection limit is0.2nM with a dynamicrange extending to100nM. Compared with existing electrochemical impedance spectroscopy (EIS)-based approaches, TWJ-DNA aptasensor was demonstrated to bemore specific for detection and simpler for regeneration procedure.2. Aptamer-based assay for ATP on an electrode-supported lipid bilayerA sensitive approach for SERS detection of cytochrome c using targetbinding-induced conformational change of signal transduction probe (STP) wasestablished. STP labeled with a SERS-active molecule, carboxy-X-rhodamine (ROX),is immobilized on the substrate where the formation of a rigid triplex switchingstructure with aptamer does not allow SERS amplification to take place. The targetbinding event leads to enhancement in SERS intensity of ROX adsorbed on the goldsurface. Meanwhile, we found that an appropriate STP surface density could shieldSERS signal produced by proteins adsorption which would foul the sensing surface.In addition, cytochrome c aptamers used were not original sequence but reorganizedin the nonspecific binding site to adapt to our design. This method provides a lowdetection limit of2nM (10fmol within5μL sample solution), and shows a goodselectivity to cytochrome c from interfering proteins such as hemoglobin andimmunoglobulin G. The general strategy of the method can also be extended toaptamer or DNA based sensors.3. Aptasensor based on triplex switch for SERS detection of cytochrome cAn aptamer-based bioassay for adenosine triphosphate (ATP) on anelectrode-supported lipid bilayer was developed. The electrochemical bioassay isbased on the mechanism that aptamer binding-induced conformational change canmodulate the electron transfer in the conductive path, and the electrochemical signals,as a consequence, can be easily detected. The results showed high sensitivity, stabilityand repeatability of this method. The detection limit is estimated to be50.1nM with adynamic range extending to3.2μM, which covers the range of concentration of ATPin the lysis solution (0.1~1μM). With the astonishing progress in nanotechnology, wehold a promise to transplant this method on a submicro-or nano-level electrode so asto monitor the intracellular surroundings in a single cell.