AVS 49th International Symposium
    Biomaterials Thursday Sessions
       Session BI+HS+SS-ThM

Paper BI+HS+SS-ThM8
ToF-SIMS Analysis of PNA/DNA Hybridization on Thiolated Biosensor Chips

Thursday, November 7, 2002, 10:40 am, Room C-201

Session: Biosensors and Biodiagnostics
Presenter: M. Schröder, Westfälische Wilhelms-Universität Münster; Germany
Authors: M. Schröder, Westfälische Wilhelms-Universität Münster; Germany
J.C. Feldner, Westfälische Wilhelms-Universität, Germany
S. Sohn, Westfälische Wilhelms-Universität, Germany
H.F. Arlinghaus, Westfälische Wilhelms-Universität, Germany
Correspondent: Click to Email
We have investigated a diagnostic method that uses peptide nucleic acid (PNA) biosensor chips to detect hybridization of unlabeled DNA. Using two different approaches, different PNAs were immobilized onto Au-coated spots with an approximate diameter of 100µm. One method was to immobilize thiolated PNA in a single-step reaction to the Au-surface via an Au-S-bond. The other method was to crosslink the N-terminal end of the PNA to a preformed layer of 11-mercaptoundecanoic acid (MUA) in a reaction consisting of two steps forming an amide bond. These layers were hybridized with complementary and non-complementary unlabeled single-stranded DNAs (ssDNA). Since the backbone of DNA, in contrast to PNA, contains phosphorous, it is possible to identify DNA-PNA-hybrids with time-of-flight mass spectrometry (ToF-SIMS) via DNA-specific phosphate-related ions at the masses 63 amu (PO@sub 2@@super -@) and 79 amu (PO@sub 3@@super -@). In addition to these signals, the deprotonated bases M-H@super -@ were detected in both immobilization approaches. In the case of the two-step-immobilization, it was possible to independently control the different steps by measuring characteristic peaks of MUA-fragments. Due to the manifold control-possibilities, especially variation of surface-density of the immobilized PNA and saturation of the remaining active Au-binding-sites with different thioles, it is possible to optimize hybridization conditions and suppression of uncharacteristic bonding of the ssDNA to the Au-surface. From the obtained data it can be concluded that both PNA immobilization approaches are very promising for designing PNA biosensors and that ToF-SIMS is a useful tool for identifying DNA-PNA-hybrids on these biosensor chips with good discrimination.