Single-mismatch position-sensitive detection of DNA based on a bifunctional ruthenium complex.
A ruthenium complex, pentaamine ruthenium [3-(2-phenanthren-9-yl-vinyl)-pyridine] (which we refer to as RuL in the text) generated in situ has been used as a sensitive and selective electrochemical indicator in DNA sensing. The complex incorporates dual functionalities with the Ru center providing a redox probe and the ligand (L) providing a fluorescent tag. The presence of the aromatic groups in the ligand endows the complex with an intercalative character and makes it capable of binding to double-stranded DNA (dsDNA) more efficiently than to single-stranded DNA (ssDNA). Combining spectroscopic and electrochemical techniques, we have elucidated the nature of the interactions. From these data we conclude that the binding mode is fundamentally intercalative. The ligand-based fluorescence allows characterization of the complex formation as well as for melting experiments to be carried out. The metal-based redox center is employed as an electrochemical indicator to detect the hybridization event in a DNA biosensor. The biosensor has been developed by immobilization of a thiolated capture probe sequence from Helicobacter pylori onto gold electrodes. With the use of this approach, complementary target sequences of Helicobacter can be quantified over the range of 106 to 708 pmol with a detection limit of 92+/-0.4 pmol and a linear correlation coefficient of 0.995. In addition, this approach allows the detection, without the need for a hybridization suppressor in solution, such as formamide, of not only a single mismatch but also its position in a specific sequence of H. pylori, due to the selective interaction of this bifunctional ruthenium complex with dsDNA.
García
T
T
Departamento de Química Analítica y Análisis Instrumental, Facultad de Ciencias, Universidad Autónoma de Madrid, Madrid 28049, Spain.
Revenga-Parra
M
M
Abruña
H D
HD
Pariente
F
F
Lorenzo
E
E
D000818:Animals; D020137:Base Pair Mismatch; D001483:Base Sequence; D015374:Biosensing Techniques / Q000379:methods*; D002417:Cattle; D004247:DNA / Q000032:analysis* / Q000235:genetics*; D004563:Electrochemistry; D004566:Electrodes; D005453:Fluorescence; D006046:Gold / Q000737:chemistry; D016480:Helicobacter pylori; D007364:Intercalating Agents / Q000138:chemical synthesis / Q000737:chemistry; D008969:Molecular Sequence Data; D009691:Nucleic Acid Denaturation; D009942:Organometallic Compounds / Q000138:chemical synthesis / Q000737:chemistry*; D010084:Oxidation-Reduction; D017975:Ruthenium Compounds / Q000138:chemical synthesis / Q000737:chemistry*; D017422:Sequence Analysis, DNA; D013050:Spectrometry, Fluorescence
D007364:Intercalating Agents; D009942:Organometallic Compounds; D017975:Ruthenium Compounds; C527540:ruthenium (3-(2-phenanthren-9-yl-vinyl)-pyridine); D006046:Gold; D004247:DNA