Detection of Mismatched DNA on Partially Negatively Charged Diamond Surfaces by Optical and Potentiometric Methods

Abstract

The effects of surface charge density on DNA hybridization have been investigated on a mixture of hydrogen-, oxygen-, and amine-terminated diamond surfaces. A difference in the hybridization efficiencies of complementary and mismatched DNA was clearly observed by fluorescence and potentiometric observations at a particular coverage of oxygen. In the fluorescence observation, singly mismatched DNA was detected with high contrast after appropriate hybridization on the surface with 10-20% oxygen coverage. The amount of oxygen in the form of C-O(-) (deprotonated C-OH) producing the surface negative-charge density was estimated by X-ray photoelectron spectroscopy. Electrolyte solution gate field-effect transistors (SGFETs) were used for potentiometric observations. The signal difference (change in gate potential) on the SGFET, which was as large as approximately 20 mV, was caused by the difference in the hybridization efficiencies of complementary target DNA (cDNA) and singly mismatched (1MM) target DNA with a common probe DNA immobilized on the same SGFET. The reversible change in gate potential caused by the hybridization and denaturation cycles and discriminating between the complementary and 1MM DNA targets was very stable throughout the cyclical detections. Moreover, the ratio of signals caused by hybridization of the cDNA and 1MM DNA targets with the probe DNA immobilized on the SGFET was determined to be 3:1 when hybridization had occurred (after 15 min on SGFET), as determined by real-time measurements. From the viewpoint of hybridization kinetics, the rate constant for hybridization of singly mismatched DNA was a factor of approximately 3 smaller than that of cDNA on this functionalized (oxidized and aminated) diamond surface.

Diamond/β-SiC Composite Thin Films: Preparation, Properties and Applications

“…However, while fabricating thin film based biosensors, the surfaces are more than often chemically or physically treated to make them clean and highly active [36][37][38], aiming at enhancing the adhesion of the chemical or biochemical species on the solid surface while minimizing the nonspecific adsorption of those species which disturbs the sensing process [39,40]. In this context, the wettability of the thin film surface is important to explain its interactions with chemicals or biological species.…”

“…Because of the OH-terminated β-SiC phase, the surface of the chemically treated composite films is charged in solutions with different pH values [40,42]. While considering a modified thin film surface for the biosensoric applications, this implication is very important, since the biomolecules are also charged in the solutions.…”

“…If the charge of the surface is of the opposite sign with respect to that of the biomolecules to be sensed, non-specific adsorption of the biomolecules onto the substrate surface will take place. This will drastically reduce the specificity and sensitivity of the sensor [40]. On the OH-terminated β-SiC phase in the composite films, the ionized bonds are present as…”

“…This is not surprising because only the OH-terminated β-SiC phase will be influenced by the pH change, and the PZC of the composite films are determined by the PZC of β-SiC. At pH = 7.5-8.5, which is most suitable pH for DNA hybridization activities [52], the composite surfaces will be negatively charged enhancing the sensitivity of the DNA sensor [40]. The advantages mentioned above motivated the fabrication of the composite films as DNA biosensors.…”

Nucleotides and Nucleic Acids; Oligo- and Polynucleotides

“…708 Oligonucleotides have been attached to carbon electrodes for the electrochemical detection of SNPs 709 and to detect cytosine methylation, 710 and to diamond gate field effect transistors to detect mismatched DNA. 711 Oligonucleotides (amino-modified) have also been attached to the polycarbonate surface of a compact disc as an analytical platform for the detection of SNPs, 712 and to TiO 2 for the detection of duplex mismatches. 713 Biotinylated shine-Delgarno sequences have been bound to a quart-crystal microbalance via neutravidin to measure the association/dissociation constants of binding by the 70S ribosome.…”