New diagnostic system for specific and fast detection of multiple nucleic acid mutations by PNA hybridization on an array sensor

Informazioni relative al progetto

ID dell’accordo di sovvenzione: BMH4961081

Progetto chiuso

Data di avvio 1 Luglio 1996

Data di completamento 31 Dicembre 2000

Finanziato da

• Specific research, technological development and demonstration programme in the field of biomedicine and health, 1994-1998

Costo totale € 0,00

Contributo UE Nessun dato

Coordinato da

Kobenhavns Universitet
Denmark

Risultati finali

PNA Surface plasmon fluorescence array

We have developed a parallel multiple read-out scheme for monitoring on-line hybridization reactions in real time between surface-attached DNA oligonucleotides and complementary strands (oligonucleotides or DNA) from solution by using our earlier introduced surface-plasmon field-enhanced fluorescence scheme in a microscopic mode. The substrate, a 50nm Au-coated glass chip, is functionalised by an array of individual spots exposing different oligonucleotide sequences as catcher probes using an ink-jet-(drop-on-demand) based principle for the preparation of the sensor matrix elements. This chip is then mounted into a surface plasmon microscope and coupled to a flow-through cell. Resonant excitation of surface plasmon modes at the Au/catcher/aqueous buffer phase-interface results in a strong fluorescence if the chromophore-labeled complements bind to their respective sensor spot. This fluorescence is monitored on-line with a suitable imaging optical and a very sensitive CCD camera. Hybridization kinetic constants (kon, koff), as well as affinity constants can be determined as a function of temperature, ionic strength, mismatch conditions, etc. It was shown that a single base mismatch can easily be detected this way. By the combination of surface plasmon microscopic and fluorescence detection schemes a novel, highly sensitive technique for the parallel monitoring of many simultaneous binding events (hybridization between a specific surface-attached oligonucleotide catcher probe sequence and a complementary strand or PCR product from solution) to an array of sensor spots has been developed: The resonant excitation of surface plasmon modes at the surface (in contact with the analyte solution) of the sensor array leads to a greatly enhanced optical field for the excitation of fluorescent probes monitoring the binding event. These probes can be covalently attached to the target strands, or the catcher sequences, or both or can be dyes intercalating into the double-stranded hybrids. The emitted fluorescence photons are detected in a spatially resolved manner by an imaging optics and a highly sensitive CCD camera as the detection element. Transfer of the images taken, e.g., in real time in order to evaluate reaction rate constants, to a computer and image analysis software routines, allow for the quantification of the hybridization events on the various sensor spots in a highly parallel fashion. In addition to the detection scheme we also developed the bio-active surface layers, based on thiol coupling chemistry and self-assembly strategies, optimised for the specific surface reaction, e.g., for maximum mismatch discrimination, minimizing non-specific binding and matrix effects leading to artefacts in the kinetic and affinity constants.