DNA detection using a triple readout optical/AFM/MALDI planar microwell plastic chip.
A ready-to-spot disposable DNA chip for specific and sensitive detection of DNA was developed. Plastic copolymeric substrate chemistry was optimized to selectively couple the target DNA with the active chip surface. At the same time, the developed substrate limits the unspecific adsorption of probe DNA molecules or additional polar contaminants in the test samples to the chip surface. The combination of glycidyl and n-butyl methacrylates was found to best fit the requirements of the assay. The fabricated DNA microarrays have mechanical properties similar to those of the glass or silicon substrates and, at the same time, provide chemically reactive surfaces that do not require lengthy chemical modification. An additional advantage of the plastic microchip is its compatibility with different analytical readout techniques, such as mass spectrometry (MALDI-TOF/MS), optical detection (fluorescence and enzyme-induced metal deposition), and imaging techniques (atomic force microscopy). These multiple readout techniques have given us the ability to compare the sensitivity, selectivity, and robustness of current state-of-the-art bioanalytical methods on the same platform exemplified by successful DNA-based detection of human cytomegalovirus. The obtained sensitivity for enzymatically enhanced silver deposition (10(-15) M) surpasses that of conventional fluorescence readouts. In addition, the assay's dynamic range (10(-6)-10(-15) M), reproducibility, and reliability of the DNA probe detection speaks for the silver deposition method. At compromised sensitivity (10(-9) M), the length of the DNA probes could be checked and, alternatively, DNA single point polymorphisms could be analyzed.
Mass Spectrometry Research Group, Max Planck Institute for Chemical Ecology, Hans-Knoell-Strasse 8, 07745, Jena, Germany.
Alfredo J
AJ
Ibáñez
Thomas
T
Schüler
Robert
R
Möller
Wolfgang
W
Fritzsche
Hans-Peter
HP
Saluz
Ales
A
Svatos
D003587:Cytomegalovirus / Q000302:isolation & purification; D004247:DNA / Q000032:analysis* / Q000235:genetics; D015342:DNA Probes / Q000032:analysis; D006801:Humans; D046228:Microarray Analysis / Q000295:instrumentation*; D018625:Microscopy, Atomic Force / Q000592:standards*; D020411:Oligonucleotide Array Sequence Analysis; D020641:Polymorphism, Single Nucleotide; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000592:standards*
D015342:DNA Probes; D004247:DNA