Electrochemically active peptide nucleic acid probes

Abstract

DNA sensors are useful in various applications including medical diagnosis, environmental monitoring, food quality control, and forensic science. The sensor usually consists of a sensing element known as a “probe” that can bind specifically with the DNA target. The binding between the probe and the target creates a should allow rapid detection, provide high specificity and sensitivity, require small sample volumes and require low cost instrumentation. Electrochemical DN sensors are one type of DNA sensors that meet all the above requirements. Peptide nucleic acid or PNA is a unique DNA analogue that is often employed as the sensing element in DNA sensors due to its superior affinity and specificity in recognition of DNA than natural DNA or other DNA analogues. To combine the advantages of PNA and electrochemistry in DNA sensing, an electrochemical DNA sensor based on pyrrolidinyl peptide nucleic acid (acpcPNA) probe that does not require probe immobilization was developed. Various kinds of redox-active redox reporters including anthraquinone and methylene blue were synthesized and introduced onto acpcPNA via acylation chemistry. The redox-active acpcPNA were purified by reverse phase HPLC and characterized by MALDI – TOF MS. The labeled acpcPNA formed hybrids to DNA with high thermal stability and specificity according to UV melting experiments. The immobilization-free electrochemical DNA detection platform was next developed on an inexpensive screen-printed carbon electrode (SPCE) by using a square-wave voltammetric (SWV) technique. Electrostatic interactions between the acpcPNA probe, the acpcPNA hybrid and the electrode surface modulate the electron transfer between the labeled acpcPNA probe and the electrode, providing the basis of the signal transduction. As an example, a highly specific signal-on detection of DNA by SWV with LOD and LOQ in the low nanomolar range was achieved using anthraquinone-labeled acpcPNA probes on PQDMAEMA-modified SPCE electrodes. Applications of the technique for analysis of real DNA samples that had been amplified by LAMP or PCR were demonstrated. Furthermore, the effects of the charges on the surface of the electrode and on the acpcPNA probe were studied to obtain further insights into the mechanism of the signal transduction. Accordingly, a novel and highly promising immobilization-free electrochemical DNA sensor based on redox-active acpcPNA prode on SPCE was successfully developed.