Biomedical subjects

F Fixe

Publications and source records attributed to F Fixe.

5 recordsLinked to original sources

PubMed2005

Single base mismatch detection by microsecond voltage pulses.

A single square voltage pulse applied to metal electrodes underneath a silicon dioxide film upon which DNA probes are immobilized allows the discrimination of DNA targets with a single base mismatch during hybridization. Pulse duration, magnitude and slew rate of the voltage pulse are all key factors controlling the rates of electric field assisted hybridization. Although pulses with 1 V, lasting less than 1 ms and with a rise/fall times of 4.5 ns led to maximum hybridization of fully complementary strands, lack of stringency did not allow the discrimination of single base mismatches. However, by choosing pulse conditions that are slightly off the optimum, the selectivity for discriminating single base mismatches could be improved up to a factor approximately 5 when the mismatch was in the middle of the strand and up to approximately 1.5 when the mismatch was on the 5'-end and. These results demonstrate that hybridization with the appropriate electric field pulse provides a new, site-specific, approach to the discrimination of single nucleotide polymorphisms in the sub-millisecond time scale, for addressable DNA microarrays.

Base Pair Mismatch↗

PubMed2004

Immobilization and hybridization by single sub-millisecond electric field pulses, for pixel-addressed DNA microarrays.

Single square voltage pulses applied to buried electrodes result in dramatic rate increases for (1) selective covalent bonding (immobilization) of single-stranded DNA (ssDNA) probes to a functionalized thin film SiO(2) surface on a plastic substrate and (2) hybridization of ssDNA to the immobilized probe. DNA immobilization and hybridization times are 100 ns and 10 micros, respectively, about 10(9) times faster than the corresponding passive reactions without electric field. Surface coverage is comparable. Duration, magnitude and slew rate of the voltage pulse are all key factors controlling the rates of ssDNA immobilization and hybridization. With rise times of 4.5 ns, pulses shorter than 1 ms and voltages below 1V are effective. The ssDNA adsorbed on the surface is reoriented by the rapidly changing electric field. This reduces steric barriers and speeds the immobilization and hybridization reactions. These results open the way for pixel-addressed microarrays driven by silicon microelectronics circuits.

Adsorption↗

PubMed2004

An on-chip thin film photodetector for the quantification of DNA probes and targets in microarrays.

A flat microdevice which incorporates a thin-film amorphous silicon (a-Si:H) photodetector with an upper layer of functionalized SiO2 is used to quantify the density of both immobilized and hybridized DNA oligonucleotides labeled with a fluorophore. The device is based on the photoconductivity of hydrogenated amorphous silicon in a coplanar electrode configuration. Excitation, with near UV/blue light, of a single-stranded DNA molecule tagged with the fluorophore 1-(3-(succinimidyloxycarbonyl)benzyl)-4-(5-(4-methoxyphenyl)oxazol-2-yl) pyridinium bromide (PyMPO), results in the emission of visible light. The emitted light is then converted into an electrical signal in the photodetector, thus allowing the optoelectronic detection of the DNA molecules. The detection limit of the present device is of the order of 1 x 10(12) molecules/cm2 and is limited by the efficiency of the filtering of the excitation light. A surface density of 33.5 +/- 4.0 pmol/cm2 was measured for DNA covalently immobilized to the functionalized SiO2 thin film and a surface density of 3.7 +/- 1.5 pmol/cm2 was measured for the complementary DNA hybridized to the bound DNA. The detection concept explored can enable on-chip electronic data acquisition, improving both the speed and the reliability of DNA microarrays.

Base Pairing↗

PubMed2004

One-step immobilization of aminated and thiolated DNA onto poly(methylmethacrylate) (PMMA) substrates.

Direct immobilisation of modified DNA oligonucleotides (aminated or thiolated) onto a plastic substrate, poly(methylmethacrylate), (PMMA) is described. Using the methyl esters present on non-modified PMMA, it was possible to establish a covalent bond between the electron donor of a DNA probe and the C terminal ester of the PMMA substrate. Since the procedure consists of a single brief wash in isopropanol or ethanol, the procedure is simple and environmentally friendly. The new immobilization strategy was characterized by analysing DNA microarray performance. The new procedure resulted in probe- and hybridization densities that were greater or equivalent to those obtained with commercially available surfaces and other procedures to immobilize DNA onto PMMA. The described chemistry selectively immobilized the DNA via terminal thiol or amine groups indicating that probe orientation could be controlled. Furthermore, the chemical bond between the immobilized DNA and the PMMA could endure repeated heat cycling with only 50% probe loss after 20 cycles, indicating that the chemistry could be used in integrated PCR/microarray devices.

Amines↗

PubMed2004

Functionalization of poly(methyl methacrylate) (PMMA) as a substrate for DNA microarrays.

A chemical procedure was developed to functionalize poly(methyl methacrylate) (PMMA) substrates. PMMA is reacted with hexamethylene diamine to yield an aminated surface for immobilizing DNA in microarrays. The density of primary NH2 groups was 0.29 nmol/cm2. The availability of these primary amines was confirmed by the immobilization of DNA probes and hybridization with a complementary DNA strand. The hybridization signal and the hybridization efficiency of the chemically aminated PMMA slides were comparable to the hybridization signal and the hybridization efficiency obtained from differently chemically modified PMMA slides, silanized glass, commercial silylated glass and commercial plastic Euray trade mark slides. Immobilized and hybridized densities of 10 and 0.75 pmol/cm2, respectively, were observed for microarrays on chemically aminated PMMA. The immobilized probes were heat stable since the hybridization performance of microarrays subjected to 20 PCR heat cycles was only reduced by 4%. In conclusion, this new strategy to modify PMMA provides a robust procedure to immobilize DNA, which is a very useful substrate for fabricating single use diagnostics devices with integrated functions, like sample preparation, treatment and detection using microfabrication and microelectronic techniques.

Base Sequence↗