The Design And Application Of Label-free Electrochemical Biosensor Based On DNA Super-sandwich Structure

Three electrochemical biosensors have been designed based on DNA super-sandwich structure. In the experiment, we have studied the manufacture method, the optimal test conditions, the self-assembly and hybridization conditions of DNA. At last, the performance of these sensors has been determined by electrochemical impedance (EIS) and square wave voltammetry (SWV) techniques. The main contents of this paper are presented as follows:1. A Simple and Label-Free Electrochemical Biosensor for DNA Detection Based on the Super-Sandwich AssayHerein. we report on a new label-free DNA biosensor for oligonucleotides detection based on super-sandwich assay by electrochemical impedance spectroscopy (EIS) technique. We designed a signal-up configuration assay. In this configuration. linker probes hybridize with two regions of the target DNA. thus creating long concatemers containing multiple target molecules and linker probes and leading to improved signal and detection limit. To further confirm this phenomenon, we adopted the gel electrophoresis to investigate this super-sandwich assay and the results were in good agreement with our speculation. The oligonucleotide with negative charge was self-assembled on the gold electrode, which would block the electron transfer. The more the oligonucleotide sequences were self-assembled on the gold electrode, the harder the electrons would approach upon the electrode surface. So. we can conclude that the "super-sandwich" structure could be more effective to block the electron transfer than the traditional-sandwich structure. Based on these results, a simple and label-free electrochemical biosensor for oligonucleotide sequence was constructed, and the detected concentration of target can be as low as1.7nM.2. A Super-Sandwich Assay for DNA Detection based on Label-Free Signal Decreased Electrochemical BiosensorHerein, we report a signal decreased label-free DNA biosensor for oligonucleotides detection based on the super-sandwich assay by square wave voltammetry (SWV) technique. In this type of super-sandwich assay, there are four linker probes, which were recorded as L-1, L-2. L-3, and L-4. On the5end of the DNA sequence, the L-1was labeled with sulfydryl (-SH). So that L-1could be fixed on the surface of the electrode by self-assembly. The linker probe L-2hybridizes with two regions of linker probe L-3, thus creating long concatemers contain multiple L-2and L-3and leading to improved signal and detection limit. The linker probe L-4could hybridize with L-1and L-2which is at the end of the super-sandwich assay. Therefore, a long concatemers were fixed on the surface of the electrode. When the target is present, because the L-4is the complementary strand of the target, and the melting temperature (Tm) of double stand DNA formed by L-4and target is higher than the Tm of double stand DNA formed by L-4, L-1, and L-2, the long concatemers of super-sandwich was far away and unfixed on the surface of electrode. The signal molecules RuHex would approach upon the electrode surface by the DNA phosphoric acid skeleton with negative charge. We can conclude that the super-sandwich structure could adsorb a lot of RuHex when the target was absent, and a large signal of RuHex could be observed. However, because the target hybridizes with L-4, the long concatemers of super-sandwich were unfixed on the surface of electrode. Consequently, a substantial decrease in the signal of RuHex is monitored by SWV. On the basis of these results, a signal decreased and label-free electrochemical biosensor based on the super-sandwich assay for oligonucleotide sequence was constructed. The detected concentration of target can be as low as2.4nM.3. A New Type of Super-Sandwich Assay for DNA Detection Based on Label-Free Electrochemical BiosensorHerein, we report a label-free DNA biosensor for oligonucleotides detection based on a new type of super-sandwich assay by square wave voltammetry (SWV). In this type of super-sandwich assay, there are two capture probes, which were recorded as C-1and C-2. The C-1was labeled with sulfydryl (-SH) on the5end of the DNA sequence. The configuration of C-2is two molecular beacons in one DNA sequence when the target is absent, so that C-1and C-2could not hybridize with each other, although they have complementary base sequence. That is because the hybrid toehold in C-2is closed by the configuration of molecular beacon. The C-2also could not hybridize with itself in this way. The target named C-T could hybridize with C-2to open the configuration of molecular beacon. This ds-DNA could both hybridize with itself and C-1to form a new type of super-sandwich assay. To further confirm this phenomenon, we adopted the gel electrophoresis to investigate this super-sandwich assay and the results were in good agreement with our speculation. We employed RuHex as a signaling molecule in this DNA sensor. Square wave voltammetry was employed to characterize the electrochemistry of RuHex on the gold electrode surface with DNA/MCH monolayers. The oligonucleotide with negative charge was self-assembled on the gold electrode, which would adsorb RuHex with positive charge. The more the oligonucleotide sequences were self-assembled on the gold electrode, the more the RuHex would approach upon the electrode surface. Consequently, a substantial change ratio in the signal of RuHex is monitored by SWV. On the basis of these results, a simple and label-free electrochemical biosensor based on the new type of super-sandwich assay for oligonucleotide sequence was constructed. The detected concentration of target can be as low as17pM.