A new approach for immobilization of oligonucleotides onto piezoelectric quartz crystal for preparation of a nucleic acid sensor for following hybridization.
The aim of this study is to develop a nucleic acid sensor based on piezoelectric crystal microbalance system (QCM) for following hybridization. Piezoelectric quartz crystal surfaces were first treated in a glow-discharge apparatus with ethylene diamine (EDA) plasma at 15 W (discharge power), 2.5 min (incubation time) and 35 ml/min (monomer flow rate) to create amino groups on the crystal surfaces. The thickness of the EDA-plasma film formed was about 43+/-24 A. Then, the amino groups on the crystal surfaces were converted to aldehyde groups by reacting the amino groups with glutaraldehyde (GA) at different conditions. A GA concentration of 2.5% and an incubation time of 2 h were selected as optimal values at this step, corresponding to a GA surface concentration of about 270 ng/cm2. A double strand Oligonucleotides, having one extra base on 5'-end of one of the complementary strands, were immobilized through the amino groups of this base onto the GA-modified crystals. Optimal immobilization conditions were as follows: oligonucleotide concentration: 1 microg/ml; time: 3 h; pH: 9.2 carbonate buffer; ionic strength: 0.1; and temperature: 20 degrees C. The QCM sensor carrying the covalently bound strand was used in the hybridization experiments, which showed that equilibrium is achieved in about 5 min, and the frequency shift measured is related to the concentration of the target strand to be measured within the medium.
Duman
Memed
M
Department of Chemical Engineering and Bioengineering Division, Hacettepe University, 06532 Beytepe, Ankara, Turkey.
Saber
Reza
R
Pişkin
Erhan
E
D000327:Adsorption; D015374:Biosensing Techniques / Q000295:instrumentation / Q000379:methods*; D004563:Electrochemistry / Q000295:instrumentation / Q000379:methods*; D009696:Nucleic Acids / Q000032:analysis / Q000737:chemistry / Q000648:ultrastructure; D020411:Oligonucleotide Array Sequence Analysis / Q000295:instrumentation / Q000379:methods*; D015345:Oligonucleotide Probes / Q000138:chemical synthesis* / Q000737:chemistry; D009841:Oligonucleotides / Q000032:analysis* / Q000737:chemistry*; D011791:Quartz / Q000737:chemistry*; D014159:Transducers
D009696:Nucleic Acids; D015345:Oligonucleotide Probes; D009841:Oligonucleotides; D011791:Quartz