Selection process generating peptide aptamers and analysis of their binding to the TiO2 surface of a surface acoustic wave sensor.
The set-up presented in this article is intended for the selection of peptides which serve as specific binders to suitable materials. Additionally, the interaction of such binders with material surfaces can be characterized. Using this approach, a subset of peptides which adhere to the mineral TiO(2) was generated by means of a cell surface display library. The peptides are constrained by a thioredoxin scaffold. Selection of proteins was carried out on a silicium wafer sputtered with TiO(2) in anatase conformation. To verify binders and to analyze the binding kinetics of the diluted suspension of the purified proteins, the chip-based S-sens K5 surface acoustic wave sensor system was used. The surface of the sensor chips was also TiO(2), resembling the material of the Si wafer selection target retaining the peptides. Several peptides were identified. The respective binding behavior differed. The data derived from real-time interaction analysis were evaluated to select for strong and specific binders. For one of these peptides, the binding kinetics was analyzed. On- and off-rate binding constants were extracted from the fitted curves. With the resulting association rate constant k(on) and the dissociation constant k(off), the affinity of the peptide for the TiO(2) surface was calculated, represented by the equilibrium dissociation constant K(D)=81 nM.
Gronewold
Thomas M A
TM
Biosensor GmbH, Ludwig-Erhard-Allee 2, D-53175 Bonn, c/o caesar, Germany.
Baumgartner
Antje
A
Weckmann
Andreas
A
Knekties
Jaqueline
J
Egler
Christian
C
D000162:Acoustics; D000595:Amino Acid Sequence; D052158:Aptamers, Peptide / Q000737:chemistry* / Q000235:genetics; D001483:Base Sequence; D015374:Biosensing Techniques; D003001:Cloning, Molecular; D017931:DNA Primers; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D013499:Surface Properties; D013879:Thioredoxins / Q000737:chemistry / Q000235:genetics; D014025:Titanium / Q000737:chemistry*
D052158:Aptamers, Peptide; D017931:DNA Primers; C009495:titanium dioxide; D013879:Thioredoxins; D014025:Titanium