A robust technique for assembly of nucleic acid hybridization chips based on electrochemically templated chitosan.
A nucleic acid hybridization assay was assembled onto a robust and readily addressable silicon-based chip using polysaccharide chitosan as a scaffold for the covalent coupling of probe DNA to the chip's surface. Chitosan is a unique polymer, ideally suited for this application because its net charge and solubility are pH dependent. Specifically in this work, gold-patterned electrodes were created using standard photolithographic techniques, chitosan was electrodeposited in a spatially resolved manner onto the polarized electrodes, probe DNA was covalently assembled onto the chitosan, and both DNA:DNA and DNA:mRNA hybridization detection schemes were evaluated. Hybridization of target nucleic acid was quantifiable, reproducible, and robust; the surface was regenerated and rehybridized up to eight times without loss of signal. Finally, transcriptional upregulation of the Escherichia coli chaperone, DnaK, which is an indicator of cellular stress, was observed using the hybridization chip sandwich assay. Thus, this method enables rapid and facile monitoring of gene expression in a format that is reusable and requires minimal reagent quantities.
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
Ghodssi
Reza
R
Rubloff
Gary W
GW
Payne
Gregory F
GF
Bentley
William E
WE
D002686:Chitin / Q000031:analogs & derivatives* / Q000737:chemistry*; D048271:Chitosan; D004277:DNA, Single-Stranded / Q000737:chemistry; D004563:Electrochemistry; D004926:Escherichia coli / Q000737:chemistry; D005976:Glutaral / Q000737:chemistry; D006046:Gold / Q000737:chemistry; D008856:Microscopy, Fluorescence; D009691:Nucleic Acid Denaturation; D009693:Nucleic Acid Hybridization / Q000379:methods; D020411:Oligonucleotide Array Sequence Analysis / Q000295:instrumentation* / Q000379:methods; D012313:RNA / Q000032:analysis / Q000235:genetics / Q000302:isolation & purification; D012825:Silicon / Q000737:chemistry
D004277:DNA, Single-Stranded; D002686:Chitin; D012313:RNA; D006046:Gold; D048271:Chitosan; D005976:Glutaral; D012825:Silicon