Different Strategies of Covalent Attachment of Oligonucleotide Probe onto Glass Beads and the Hybridization Properties |
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Authors: | Han Sheng Bang-Ce Ye |
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Institution: | (1) Laboratory of Bioanalysis and Biosystems, State Key Laboratory of Bioreactor Engineering, East China University of Science & Technology, Shanghai, 200237, China |
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Abstract: | The glass bead is a new biochip support material for immobilization biomolecules, due to its independence and convenient rearrangement.
In order to optimize the immobilization efficiency of oligonucleotides onto glass beads and obtain the highest hybridization
efficiency, three commonly used coupling strategies have been studied for covalently attaching oligonucleotides onto large
glass beads. Glass beads with 250 μm diameter were amino-silaned with 2% 3-aminopropyltrimethoxysilane (APTMS) and then reacted
separately with glutaraldehyde, succinic anhydride and 1,4-phenylene diisothiocyanate (PDITC) to derive CHO beads, COOH beads
and isothiocyanate-modified beads (NCS-Beads) accordingly. Afterwards, amino-terminal oligonucleotides were covalently attached
onto the surface of beads achieved by three strategies mentioned above. The immobilization efficiency were studied to compare
the three strategies, which turned out 2.55 × 1013 probes/cm2 for CHO-Beads, 3.21 × 1013 probes/cm2 for COOH beads and 6.68 × 1013 probes/cm2 for NCS beads. It meant that the immobilization efficiency based on NCS beads was most acceptable. And the method, developed
by attaching amino-terminal oligonucleotides onto these cyanate active beads, could be regarded as an efficient one for immobilizing
oligonucleotides onto a solid surface. Moreover, in this paper, the hybridization properties of NCS bead-based oligonucleotides
have been studied by employing Cy5-tagged complementary oligonucleotides. It was found that the high probe density NCS beads
led to low hybridization efficiency possibly due to the existence of steric crowding. In addition, the equilibrium binding
constant K
A was determined by employing Langmuir isotherm model, which was 7.0 × 106 M−1 for NCS beads with the density of 6.7 × 1013 probes/cm2. Furthermore, it only took 60 min to reach hybridization equilibrium. These large microspheres (>100 μm) can be employed
in the mesofluidic systems for automated heterogeneous assays. |
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Keywords: | Covalent attachment Immobilization Oligonucleotides Beads Hybridization |
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