Electrochemical Biosensors Based On Functional Oligonucleotide Sensing Interface

Functional DNA referred to a class of short DNA with enzymatic function,biorecognition function selected by Systematic Evolution of Ligands by Exponential Enrichment?SELEX?or selected in vitro.Functional oligonucleotide was one kind of functional DNA.Electrochemical biosensor was more and more important in environmental monitoring,biological analysis,forensic identification,food safety and so on,because of its advantages of portability,simple operation,high sensitivity and small consumption.In this paper,we combined functional oligonucleotides and electrochemical sensing technology to build three new type of label-free impedance sensors,which were used for the detection of melamine and murcury ion(Hg²⁺).They had the advantages of simple preparation,high specificity and high sensitivity.The research contents were as followed:?1?Based on T-Mel-T structure formation of thymine?T?and melamine?Mel?by the hydrogen bond,a rapid and sensitive electrochemical aptasensor for Mel was fabricated for the detection of melamine in milk samples.First,a T-rich oligonucleotide?TRO?with a thiol terminal was immobilized on the surface of a bare gold electrode by S-Au bond.Then the TRO modified interface was obtained by blocking the exposed gold site with mercapto hexanol?MCH?.When the thymine?T?and melamine?Mel?reacted to form T-Mel-T structure,the conformational change of TRO would be induced by T-Mel-T structure,which would change the electrochemical behavior of the electrode.Using[Fe?CN?₆]^3-/4-as electrochemical probe,the performance of sensor for melamine was investigated by electrochemical impedance spectroscopy?EIS?.The results showed that there was a linear correlation between the impedance variation value(?R_ct)of charge transfer and the logarithm of the concentration of Mel(lgC_Mel)under the optimal conditions.Then,the sensor exhibited a wide dynamic range from 10.0 pM to 1.0?M with a detection limit of 1.2 pM.?2?Based on the formation of T-Hg²⁺-T structure by rich thymine?T?oligonucleotide and mercury ion(Hg²⁺),a new mercuric ion electrochemical sensor with multi sandwich oligonucleotides chain has been fabricated.First,the DNA?IS?were fixed on gold electrode by S-Au bond,and the electrode was sealed the exposed gold site with MCH.Then,the modified electrode hybridized alternately with DNA?MSO?and DNA?BS?that connected two MSO over and over again to form a hybridization chain linked by MSO-BS,forming multi"sandwich"oligonucleotides hybridization chain by MSO-BS connected alternatively.The hybridization chain could generate high impedance by multi oligonucleotide sequences.When the modified electrode was applied to Hg²⁺,the MSO of the sensor interface and Hg²⁺form U-shaped T-Hg²⁺-T structure,which makes the multi sandwich structure collapse rapidly,resulting in impedance reduction,thus achieving high sensitive detection for Hg²⁺.The performance of sensor for Hg²⁺was investigated by electrochemical impedance spectroscopy?EIS?.The results showed that there was a linear correlation between the impedance variation value of charge transfer(?R_ct)and the logarithm of the concentration of Hg²⁺(lgC_Hg2+)under the optimal conditions.Then,the sensor exhibited a wide dynamic range from 1.0 fM to 10.0pM with a detection limit of 0.16 fM.?3?Based on the repeated hybridization between ring-shaped DNA?RS?and amplificated auxiliary DNA?AAS?to form a DNA 3D hybrid network on the electrode surface,a new high-sensitivity electrochemical sensor for Hg²⁺was fabricated.First,the DNA?IS?strand was immobilized on the surface of the gold electrode by the S-Au bond,and the electrode was sealed the exposed gold site with MCH.Then IS was hybridized with DNA?MSO?and DNA?BS?in turn.Finally,the modified electrode hybridized alternately with RS and AAS over and over again to form DNA network,thus obtaining high impedance surface.The network structure impeded the transfer of electrons,so the impedance would increase dramatically.When Hg²⁺appeared in the analytical solution,MSO formed the T-Hg²⁺-T structure with Hg²⁺,causing the network structure to break,thus reducing EIS signal response of the sensor.The construction of the sensor was characterized by CV,EIS and AFM.And EIS was used to optimize the preparation of the sensor and the analysis of Hg²⁺.The result indicated that the electrochemical sensor exhibited excellent specific recognition for Hg²⁺under the optimal condition.The resistance variation value of charge transfer??Rct?versus the logarithm of the concentration of Hg²⁺(lgC_Hg2+)showed a wide linear range from 10.0aM to 1.0 pM with a detection limit of 7.2 aM.