PNA Probes and Novel Intercalators for Impedance and Field-Effect based DNA Detection

Electrochemical DNA sensors based on the detection of a target DNA by sensing its intrinsic negative charge, can be strongly improved by the use of Peptide Nucleic Acid (PNA) probes. The hybridization of the uncharged PNA probe with the negatively charged DNA target results in a dramatic increase in the charge of the biolayer. The use of PNA has the added advantage of increased specificity and stability. The increase in surface charge can be easily measured with electrochemical impedance spectroscopy (EIS), where variations of charge transfer resistance in the order of 400% can be observed [1]. Non-Faradaic EIS measurements (i.e. without redox markers in solution) are useful for measuring any capacitance changes that occur on the biolayer. On the other hand, both charge variations and capacitance variations can be detected using biologically-sensitive field-effect transistors (BioFETs), where the biolayer is attached to the metal gate of the transistor [2]. In this study we use PNA-DNA redox intercalators to change the surface potential of the electrodes. We have developed and fully characterized an electroactive ferrocenyl PNA-DNA intercalator [3]. The presence of the intercalator allows the direct amperometric detection of the DNA target as well as enhancing the electrochemical impedance of the system. Of significant interest for BioFETs is that the presence of the intercalator induces a change in the formal potential of the system; this in turn modulates the threshold voltage of the transistor providing a sensitive and robust sensing method. An added advantage of the BioFETs is that they can be easily implemented as a monolithically integrated array of sensors providing high-throughput screening of DNA sequences. We have demonstrated the use of the BioFETs for the detection of Clostridium difficile. The bacterium is the primary cause of antibiotic associated diarrhea in humans and is a significant cause of morbidity and mortality. Thus the rapid and accurate identification of this pathogen in clinical samples (such as feces) is a key step in reducing the devastating impact of this disease. We have achieved the BioFET and EIS detection of DNA of Clostridium difficile specific toxins extracted via a microwave-based technique [4].