Novel DNA Biosensor Based On Electrically Neutral Metal Complexe As Hybridization Indicator

As one of the key components of an electrochemical biosensor, designing anddeveloping of the novel hybridization indicator has been the research hotspot in the field oflife analysis. However, currently widely used indicator usually presents positive attributes inthe test condition, which largely extent restricted the sensitive of the biosensor due to thenonspecific electrostatic interaction between single-or double-strand DNA. In the work, weemploy the negative charge molecule that containing hydroxyl or carboxyl groups （such aspyridine carboxylic acid） as a charge control ligand to synthesis a lot of electrically neutralmetal complexes to reduce the non-specific binding between the complexes and DNA,selecting excellent hybridization indicator from them and used in DNA electrochemicalsensor to construct new method for DNAbiosensor detection. The main research contents areas follows:（1） A novel electrically neutral osmium complex [Os（DPPZ）（PC）（H₂O）]（DPPZ=dipyrido（3,2-a:2’,3’-c）phenazine, PC=2,6-pyridine dicarboxylic acid） wassynthesized by using DPPZ as intercalate ligand and PC as charge control ligand. Theinteraction of [Os（DPPZ）（PC）（H₂O）] with double-strand DNA （dsDNA）was investigated byUV absorption spectroscopy, cyclic voltammetry, normal pulse voltammetry andchronocoulometry. UV of the complex was changed when dsDNA was added, suggesting theinteraction of the two species. Cyclic voltammetric results of the complex on a glassy carbonelectrode showed a pair of quasi-reversible redox peaks corresponding to the electron transferof Os（III）/Os（II） couple. When a certain amount of dsDNAwas added, the peak current of thecomplex decreased, with a positive shift of peak potential, indicating the intercalative modebetween [Os（DPPZ）（PC）（H₂O）] and dsDNA. The normal pulse voltammetry-based titrationexperiments showed that the peak currents of the complex decreased with the increase ofdsDNA concentrations, and a binding constants of2.7（±0.3）×10⁵L/mol were obtained bynon-linear fitting. When the electrically neutral osmium complex was utilized as anelectrochemical hybridization indicator for the detection of oligonucleotides related toCaMV35S promoter gene, there was not any electrochemical response on probe DNA modified electrode, but a sensitive electrochemical signal was obtained on the hybridelectrodes. The complementary target sequence could be quantified over the range from8.0×10-10mol/L to2.8×10^-9mol/L. The hybridization specificity experiments furtherindicated that the biosensor could well discriminate the complementary sequence from thebase-mismatched and the non-complementary sequences.（2） An electrically neutral copper complex, Cu（PC）₂·H₂O （PC=pyridine2carboxylate）was synthesized and its interaction with double-stranded DNA （dsDNA） wascomprehensively studied by electrochemical methods on a glassy carbon electrode （GCE）.The experimental results revealed that the copper complex could interact with dsDNA via aspecific intercalate-binding mode with an affinity constant of K=3.1（±0.9）×10⁵L/mol. Thesurface-based studies showed that Cu（PC）₂·H₂O could electrochemically accumulate withinthe immobilized dsDNA layer rather than single-stranded DNA （ssDNA） layer. Based on thisfact, the copper complex was utilized as an electrochemical hybridization indicator for thedetection of oligonucleotides related to CaMV35S promoter gene. The results showed that thedeveloped biosensor presented very low background interference due to the Cu（PC）₂·H₂Ocomplex can not interact with ssDNA. The hybridization specificity experiments furtherindicated that the biosensor could well discriminate the complementary sequence from thebase-mismatched and the non-complementary sequences. The complementary targetsequence could be quantified over the range from1.0×10^-14mol/L to1.0×10^-6mol/L with adetection limit of1.0×10^-15mol/L.（3） A dumbbell-shaped bi-acetylferrocene ethylenediamine complex,（AFc）₂-en （AFc=acetylferrocene, en=ethylenediamine） was synthesized and its interaction withdouble-stranded DNA（dsDNA） was comprehensively studied by electrochemical methods ona gold electrode. The experimental results reveal that the highly electroactive （AFc）₂-encomplex can interact with dsDNA via a specific bis-groove binding mode. The bindingconstant and binding site size between the two species were determined to be3.7×10⁵L/moland4.2, respectively. The surface-based studies further show that （AFc）₂-en presents thestronger electrochemical response on dsDNA modified electrode than on the single-stranded DNA（ssDNA） modified electrode, suggesting that the complex can well discriminate dsDNAfrom ssDNA. Based on this characteristic, the bi-acetylferrocene complex was utilized as anelectrochemical hybridization indicator in a DNA biosensor for the detection ofoligonucleotides from CaMV35S promoter gene. The specificity experiments indicate that thebiosensor can well discriminate the complementary sequence from the base-mismatched andthe non-complementary sequences. The complementary target sequence can be quantifiedover a wide range from1.0×10^-14mol/L to1.0×10^-6mol/L with a detection limit of2.0×10^-15mol/L.