Biology Reference
In-Depth Information
capacitance or electron transfer resistance of the film as a function
of hybridization. The latter method is highly versatile and highly
sensitive to the presence of mismatches as will be shown in this
chapter. Synthetic oligonucleotides are often used as the capture
probe. They are readily obtained in high purity and at low cost and
thebasesequencecanbeadjustedtosuitaparticulartarget.Peptide
nucleic acids (PNA) can also be used as a capture probe. PNA has
a higher stability and improved binding a
nity in comparison to
nucleic acids, but iscostly.
Often, as in the case of redox-labeled oligonucleotides, the
covalent attachment of a redox label such as a ferrocene (Fc) group
is achieved by imine and amide formation using Fc-carboxaldehyde
orFc-carboxylicacid,respectively,andalsobySonogashiracoupling
with the corresponding Fc-alkyne derivative [1, 2]. Redox labels
can be introduced either on the monomer stage, by a metal-
catalyzed reaction, or after assembly of the oligomer sequence
[3]. For example, Fc-conjugated nucleotides can be conveniently
used as building blocks in automated oligonucleotide synthesis [4].
Similarly, the Fc group can be introduced after ODN synthesis by
amide coupling of Fc-COOH to a 5'-amino group of a synthetic
ODN, as was reported by Ihara and co-workers [5]. However, the
introduction of a Fc-label into a DNA oligomer can decrease the
stability of the duplex. And the position of the Fc group, the nature
of its linkage to the ODN, and the nucleobase will all influence the
“melting point” of the duplex. The interested reader is referred to a
review [6], where different Fc oligomers are discussed.
In order to be useful for the detection of nucleotide basepair
mismatches, the electrochemical signature of the mismatched ds-
ODN must be significantly different from that of the fully hybridized
ds-ODN. In this chapter, we summarize the state of the art in this
field and provide an overview over capture strand immobilization
strategies andvarious mismatchdetections schemes.
7.2 Surface Immobilization
To design a functional DNA biosensor DNA, capture strands have to
beimmobilizedonanelectrodesurface.Thinfilmformationisoften
accomplishedbycovalentattachment,adsorptionora
nitybinding
