Chitosan scaffolds for biomolecular assembly: coupling nucleic acid probes for detecting hybridization.
Chitosan, a naturally occurring biopolymer, was used as a scaffold for the covalent binding of single-stranded DNA oligonucleotide probes in a fluorescence-based nucleic acid hybridization assay. Chitosan's pH dependent chemical and electrostatic properties enable its deposition on electrodes and metal surfaces, as well as on the bottom of microtiter plates. A combinatorial 96-well microtiter plate format was used to optimize chemistries and reaction conditions leading to hybridization experiments. We found the coupling of oligonucleotides using relatively common glutaraldehyde chemistry was quite robust. Our hybridization results for complementary ssDNA oligonucleotides (E. coli dnaK sequences) demonstrated linear fluorescence intensity with concentration of E. coli dnaK-specific oligonucleotide from 0.73 microM to 6.6 microM. Moreover, hybridization assays were specific as there was minimal fluorescence associated with noncomplementary groEL oligonucleotide. Finally, these results demonstrate the portability of a DNA hybridization assay based on covalent coupling to chitosan, which, in turn, can be deposited onto various surfaces. More arduous surface preparation techniques involving silanizing agents and hazardous washing reagents are eliminated using this technique.
Yi
Hyunmin
H
Center for Biosystems Research, University of Maryland Biotechnology Institute, 5115 Plant Sciences Building, College Park, Maryland 20742, USA.
Wu
Li-Qun
LQ
Sumner
James J
JJ
Gillespie
James B
JB
Payne
Gregory F
GF
Bentley
William E
WE
D002686:Chitin / Q000031:analogs & derivatives* / Q000737:chemistry*; D048271:Chitosan; D004277:DNA, Single-Stranded; D005976:Glutaral / Q000737:chemistry; D009693:Nucleic Acid Hybridization; D015341:Nucleic Acid Probes; D015345:Oligonucleotide Probes; D012680:Sensitivity and Specificity
D004277:DNA, Single-Stranded; D015341:Nucleic Acid Probes; D015345:Oligonucleotide Probes; D002686:Chitin; D048271:Chitosan; D005976:Glutaral