Directed Covalent Immobilization of Aminated DNA Probes on Aminated Plates

Abstract

A new protocol that enables the immobilization of DNA probes on aminated micro-titer plates activated with aldehyde-dextran via an amino group artificially introduced in the 3' end of the oligonucleotide probe is reported in this work. The method is based on the use of hetero-functional-dextran as a long and multifunctional spacer arm covalently attached to an aminated surface capable of immobilizing DNA oligonucleotides. The immobilization occurred only via the amino introduced in the 3' end of the probe, with no implication of the DNA bases in the immobilization, ensuring that the full length of the probe is available for hybridization. These plates having immobilized oligonucleotide probes are able to hybridize complementary DNA target molecules. The tailor-made hetero-functional aldehyde-aspartic-dextran together with the chemical blocking of the remaining primary amino groups on the support using acetic anhydride avoid the nonspecific adsorption of DNA on the surface of the plates. Using these activated plates, (studying the effect of the probe concentration, temperature, and time of the plate activation on the achieved signal), thus, the covalent immobilization of the aminated DNA probe was optimized, and the sensitivity obtained was similar to that achieved using commercial biotin-streptavidin systems. The new DNA plates are stable under very drastic experimental conditions (90% formamide, at 100 degrees C for 30 min or in 100 mM NaOH).

Synthesis and functionalization of biocompatible Tb:CePO4 nanophosphors with spindle-like shape

“…The resulting dispersions were sonicated in cold water during 1 h. After this treatment, the so functionalized nanoparticles were washed several times with MilliQ water by centrifugation and finally redispersed in MilliQ water. The aspartic-dextran polymers solutions were prepared following a procedure previously reported by Fuentes et al (2004) The crystalline structure of the prepared samples was assessed by X-ray diffraction (XRD, Panalytical, X´Pert Pro with an X-Celerator detector). Unit cell parameters were determined from the XRD data (collected at intervals of 0.03° (2) for an accumulation time for interval of 3000 s) by using the Rietveld method with the JANAsoftware (Petricek et al 2006).…”

Gold nanoparticle-assisted single base-pair mismatch discrimination on a microfluidic microarray device

“…The uncovered amine groups on the glass surface were capped by acetyl groups by filling through the microchannels with acetic anhydride. 29 After washing the microchannels with 95% ethanol followed with citrate buffer solutions, the microchip was dried out for subsequent probe immobilization.…”

Improved reactivation of immobilized-stabilized lipase from Thermomyces lanuginosus by its coating with highly hydrophilic polymers

“…Preparation of dextran-aldehyde-glycine: Dextran (MW = 70,000 Da) with a 50% oxidation degree was used (Fuentes et al, 2004a). The protocol for dextran modification was similar to that previously described to prepare dextran-aspartic (Fuentes et al, 2004b).…”