DNA conjugation and reversibility on chitosan surfaces Rubloff Research Group Accomplishments

DNA conjugation and reversibility on chitosan surfaces Accomplishment Single string DNA (ssDNA, probe) was conjugated to chitosan surface via glutaraldehyde activation Target ssDNA was hybridized to matching probe ssDNA via self-assembly Reversible DNA hybridization was achieved under urea denaturation and hybridization Significance The chitosan-based DNA hybridization is simple to achieve spatially selective assembly The probe is sensitive and is applied to sandwich assay of total RNA sample The probe is robust and provides reproducible measurements with a high signal-to-noise ratioseven after repeated hybridization and denaturation cycles People involved Hyunmin Yi, Greg Payne, Bill Bentley, Gary Rubloff Collaborations with Li-Qun Wu, Reza Ghodssi,, Links Hyunmin Yi et al, Anal. Chem. 76 (2), 365-372 (2004)

Chitosan Electrodeposition Unusual polysaccharide Low pH, soluble High pH, insoluble O H N 3 + * n 2 2n Chitosan electrodeposition High pH region at negative electrode due to hydrogen evolution Chitosan molecules deprotonated, immobilized at electrode surface Cathode - pH gradient NH3+ pH>6.3 2H+ + 2e-  H2 H2 Chitosan biopolymer is emerging candidate for our application due to its intrinsic properties. This material has pH dependant solubility since the the primary amine group is protonated at low pH and deprotonated in high pH. (Nitrogen in amine group has a lone electron pair available which can accept a H+ ion.) (Polarity of amine of chitosan molecule contribute to the solubility due to hydrogen bonding between water molecules ) This property enables electrodeposition of chitosan film on the negative electrode. When we applied potential, Proton consumption occurs at the cathode and instantly water molecules dissociate to compensate proton and OH accumulate. Therefore pH gradient is generated near the surface of negative electrode. Chitosan molecule which is electrostatically transported to the cathode and in the high pH region near cathode, are deprotonated and immobilized. (Or it could be reduction of O2 and the reduction (addition of electron, hydrogen removal of oxygen) of H2O,

DNA Hybridization on Chitosan Probe ssDNA Target ssDNA Activated Surface

Reversible DNA Hybridization on Chitosan

Reversible DNA Hybridization Yi et al, Anal. Chem. 76 (2), 365-372 (2004)

Analysis of Purified Total RNA from Escherichia coli culture by Sandwich Assay Yi et al, Anal. Chem. 76 (2), 365-372 (2004) Hybridization ssDNA (sandwich probe) mRNA (analyte) Hybridization Electrode  Chitosan  Glutaraldehyde  ssDNA (probe)

Virus Assembly on Chitosan 300 nm Yi et al, Nano Lett 5 (10) 1931-1936 (2005)