title: Immobilization-free electrochemical DNA detection with anthraquinone-labeled pyrrolidinyl peptide nucleic acid probe.
abstract: Electrochemical detection provides a simple, rapid, sensitive and inexpensive method for DNA detection. In traditional electrochemical DNA biosensors, the probe is immobilized onto the electrode. Hybridization with the DNA target causes a change in electrochemical signal, either from the intrinsic signal of the probe/target or through a label or a redox indicator. The major drawback of this approach is the requirement for probe immobilization in a controlled fashion. In this research, we take the advantage of different electrostatic properties between PNA and DNA to develop an immobilization-free approach for highly sequence-specific electrochemical DNA sensing on a screen-printed carbon electrode (SPCE) using a square-wave voltammetric (SWV) technique. Anthraquinone-labeled pyrrolidinyl peptide nucleic acid (AQ-PNA) was employed as a probe together with an SPCE that was modified with a positively-charged polymer (poly quaternized-(dimethylamino-ethyl)methacrylate, PQDMAEMA). The electrostatic attraction between the negatively-charged PNA-DNA duplex and the positively-charged modified SPCE attributes to the higher signal of PNA-DNA duplex than that of the electrostatically neutral PNA probe, resulting in a signal change. The calibration curve of this proposed method exhibited a linear range between 0.35 and 50 nM of DNA target with a limit of detection of 0.13 nM (3SD(blank)/Slope). The sub-nanomolar detection limit together with a small sample volume required (20 μL) allowed detection of <10 fmol (<1 ng) of DNA. With the high specificity of the pyrrolidinyl PNA probe used, excellent discrimination between complementary and various single-mismatched DNA targets was obtained. An application of this new platform for a sensitive and specific detection of isothermally-amplified shrimp's white spot syndrome virus (WSSV) DNA was successfully demonstrated.
authors.affiliation: Organic Synthesis Research Unit, Department of Chemistry, Faculty of Science, Chulalongkorn University, Phayathai Road, Patumwan, Bangkok 10330, Thailand.
authors.forename: Jutatip
authors.initials: J
authors.lastname: Kongpeth
authors.affiliation: Program in Petrochemistry, Faculty of Science, Chulalongkorn University, Phayathai Road, Patumwan, Bangkok 10330, Thailand.
authors.forename: Sakda
authors.initials: S
authors.lastname: Jampasa
authors.affiliation: Laboratory of Plant Transgenic Technology and Biosensor, Department of Botany, Faculty of Science, Chulalongkorn University, Phayathai Road, Patumwan, Bangkok 10330, Thailand.
authors.forename: Piyasak
authors.initials: P
authors.lastname: Chaumpluk
authors.affiliation: Electrochemistry and Optical Spectroscopy Research Unit, Department of Chemistry, Faculty of Science, Chulalongkorn University, Phayathai Road, Patumwan, Bangkok 10330, Thailand.
authors.forename: Orawon
authors.initials: O
authors.lastname: Chailapakul
authors.affiliation: Organic Synthesis Research Unit, Department of Chemistry, Faculty of Science, Chulalongkorn University, Phayathai Road, Patumwan, Bangkok 10330, Thailand. Electronic address: vtirayut@chula.ac.th.
authors.forename: Tirayut
authors.initials: T
authors.lastname: Vilaivan
mesh_terms: D000880:Anthraquinones / Q000737:chemistry*; D001483:Base Sequence; D015374:Biosensing Techniques / Q000379:methods*; D002138:Calibration; D002244:Carbon / Q000737:chemistry; D004247:DNA / Q000032:analysis* / Q000737:chemistry / Q000235:genetics; D004279:DNA, Viral / Q000032:analysis; D004563:Electrochemistry; D015345:Oligonucleotide Probes / Q000737:chemistry* / Q000235:genetics; D020135:Peptide Nucleic Acids / Q000737:chemistry* / Q000235:genetics; D011759:Pyrrolidines / Q000737:chemistry*; D055672:Static Electricity
chemical_list: D000880:Anthraquinones; D004279:DNA, Viral; D015345:Oligonucleotide Probes; D020135:Peptide Nucleic Acids; D011759:Pyrrolidines; D002244:Carbon; D004247:DNA; C032519:pyrrolidine
keywords: DNA biosensor; Electrochemical detection; Peptide nucleic acid; Redox active label; Screen-printed carbon electrode