Towards a Real-Time, Label-Free, Diamond-Based DNA Sensor

Abstract

Most challenging in the development of DNA sensors is the ability to distinguish between fully complementary target ssDNA (single-strand DNA) and 1-mismatch ssDNA. To deal with this problem, we performed impedance spectroscopy on DNA-functionalized nanocrystalline diamond (NCD) layers during hybridization and denaturation. In both reactions, a difference in behavior was observed for 1-mismatch target DNA and complementary target DNA in real-time. During real-time hybridization, a decrease of the impedance was observed at lower frequencies when the complementary target DNA was added, while the addition of 1-mismatch target ssDNA caused no significant change. Fitting these results to an electrical circuit demonstrates that this is correlated with a decrease of the depletion zone in the space charge region of the diamond. During real-time denaturation, differentiation between 1-mismatch and complementary target DNA was possible at higher frequencies. Denaturation of complementary DNA showed the longest exponential decay time of the impedance, while the decay time during 1-mismatch denaturation was the shortest. The real-time hybridization and denaturation experiments were carried out on different NCD samples in various buffer solutions at temperatures between 20 and 80 degrees C. It was revealed that the best results were obtained using a Microhyb hybridization buffer at 80 degrees C and 10x PCR buffer at 30 degrees C for hybridization and 0.1 M NaOH at temperatures above 40 degrees C for denaturation. We demonstrate that the combination of real-time hybridization spectra and real-time denaturation spectra yield important information on the type of target. This approach may allow a reliable identification of the mismatch sequence, which is the most biologically relevant.

Optical properties of heavily boron-doped nanocrystalline diamond films studied by spectroscopic ellipsometry

“…As such films can be deposited on a plethora of materials, 1 possible applications include NEMS and MEMS structures, 2 biosensors, 3 tribology, 4 optical coatings 5 and thermal management. 6 By adding boron, NCD films can be turned into a p-type semiconductor.…”

Synthetic diamond films as a platform material for label‐free protein sensors

“…The field-effect in NCD substrates, caused by negatively charged DNA at the surface, has been reported in our previous work on real-time impedimetric DNA detection [10] as well as in other publications [9,28] and will not be discussed here. A negligible field-effect in the NCD substrate was observed during CRP treatment, reflected by the lack of impedance variations in the semi-circle.…”

“…This molecule is frequently used in PCR-ELISA, and the FITC-label acts as an antigen. Moreover, the fact that DNA was attached to the label allowed us to thoroughly distinguish between the impedimetric effects caused by DNA [10] and the impedimetric effects caused by a protein. Finally, the reaction wells were rinsed and refilled with pure PBS buffer.…”

“…This was done using the ZSimpWin software from Princeton Applied Research (Massachusetts, USA). The equivalent circuit was the same as was used in previous work focusing on the construction of a NCD-based DNA-sensor [10]. The fits were performed over the total frequency range from 100 Hz to 1 MHz.…”

“…Recently, we used diamond for a label-free impedimetric DNA-sensor and demonstrated that real-time differentiation between fully complementary and 1-mismatch DNA was possible on lightly boron (B)-doped diamond substrates using electrochemical impedance spectroscopy (EIS) [10]. In this current study, the NCD-based impedimetric biosensor setup is transferred to an immunological setting.…”