Development of a molecular diagnosis assay based on electrohybridization at plastic electrodes and subsequent PCR.
Technologies enabling specific recognition of medically relevant nucleic acid sequences will play a pivotal role in future medical diagnosis. Whereas many approaches to molecular diagnosis systems include DNA microarrays on chips and fluorometric detection, the basis of our approach is the use of inexpensive components like plastic or metal thin film electrodes with low multiplexing and an electrochemical detection unit. To increase the sensitivity, PCR can be used as an intermediate step. For selective enrichment, specific nucleic acid probes were covalently attached at their 5'-ends to conducting polycarbonate/carbon fiber electrodes. Complementary oligonucleotides were enriched at the electrodes by cyclic inversion of an electrochemical potential, transferred into a PCR vial and thermally or electrochemically desorbed. The analysis of the PCR product shows the efficiency and selectivity of the electrochemical enrichment. Hybridization of DNA was shown by electrochemical methods, in this work especially by differential pulse voltammetry (DPV) using the single strand specific hybridization redox indicator osmium(VIII)-tetroxide, and potentiometric stripping analysis (PSA). This combination of experimental methods is the basis for a molecular diagnosis system including a disposable nucleic acid modified working electrode for specific enrichment, detection and quantification, and an optional capillary PCR module for fast amplification.
Hassmann
J
J
november AG, Ulrich-Schalk-Strasse 3, 91056 Erlangen, Germany. hassmann@november.de
Misch
A
A
Schülein
J
J
Krause
J
J
Grassl
B
B
Müller
P
P
Bertling
W M
WM
D015374:Biosensing Techniques / Q000379:methods*; D004247:DNA / Q000235:genetics* / Q000302:isolation & purification*; D019937:Diagnostic Techniques and Procedures; D004563:Electrochemistry; D004566:Electrodes; D004582:Electronics, Medical; D006801:Humans; D008855:Microscopy, Electron, Scanning; D009693:Nucleic Acid Hybridization; D010969:Plastics; D016133:Polymerase Chain Reaction
D010969:Plastics; D004247:DNA