Authors: V. Vermeeren, N. Bijnens, L. Grieten, S. Wenmackers, N. Vanden Bon, K. Haenen, P. Wagner, L. Michiels
Affilation: Hasselt University, Belgium
Pages: 23 - 26
Keywords: bio sensors
Healthcare and diagnostics are moving towards molecular medicine. Hence, a lot of research is being performed on biosensors. However, rarely one has reached the commercialization phase because of the expensive infrastructure required for signal read-out. Electronic read-out methods, such as Electrochemical Impedance Spectroscopy (EIS), are preferred since they allow real-time and label-free signal generation and cheap implementation, because of the well-understood silicon (Si) microprocessing techniques. However, it is known that the covalent bond between Si and biomolecules is fairly weak, causing the gradual loss of bioreceptors from the surface, and hence a drift in signal and decrease in sensitivity and reliability1. Diamond is an attractive alternative, because of its semiconductive nature by doping, its chemical inertness, and its ability to be biofunctionalized. Furthermore, diamond, being a carbon (C) lattice, can form stable C-C bonds with biomolecules, enabling reuse of the surface without loss of receptor molecules1,2. For these reasons, our goal was to develop prototypes of impedimetric nanocrystalline diamond (NCD)-based DNA- and immunosensors, allowing real-time mutation and protein detection, respectively. We designed a simple, efficient two-step protocol for the covalent attachment of ssDNA onto NCD. ω-unsaturated fatty acids are photochemically bound to H-terminated NCD, yielding a COOH-terminated NCD surface. NH2-modified ssDNA is covalently linked to these COOH-groups, using 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC)3,4. The next step towards label-free DNA sensing involved the study of the electronic properties of functionalized diamond layers using EIS. The interface properties are expected to change differently upon hybridization with complementary versus non-complementary DNA. The impedance spectra were obtained in a frequency range between 100 Hz and 1 MHz. Complementary and 1-mismatch ssDNA can be impedimetrically differentiated in real-time during hybridization at mediate frequencies (~1 kHz), and during denaturation at high frequency (1 MHz). The response time of the latter was only 5 minutes. This is the first time that real-time denaturation with an electronic detection platform has been used for mutation detection, and the principle was based on the difference in melting temperatures of the perfect DNA duplex and the DNA duplex with a mismatch. This same discriminative parameter could possibly even allow for mutation identification5. We also developed a prototype of a label-free NCD-based impedimetric immunosensor by adsorption of anti-C-Reactive Protein (anti-CRP) onto H-terminated NCD. EIS was also used to detect CRP recognition in real-time, which is a risk marker for cardiovascular disease. Selective discrimination between the specific CRP antigen and non-specific plasminogen was reproducibly obtained in real-time at low frequencies (~100 Hz). We also obtained a clinically relevant sensitivity of 10 nM.