Hybridization-Modulated Ion Fluxes through Peptide-Nucleic-Acid- Functionalized Gold Nanotubes. A New Approach to Quantitative Label-Free DNA Analysis

Abstract

The inner walls of gold nanotubules, prepared by template synthesis in the nanopores of polycarbonate track etch membranes, have been chemically modified with peptide nucleic acid (PNA) and used for label-free quantification of complementary DNA sequences. Selective binding of DNA to the PNA modified nanotubules are shown to decrease the flux of optically detected anionic markers through the nanotubules in a concentration-dependent manner. The strong dependence of the biorecognition-modulated ion transport through the nanopores on the ionic strength suggests a dominantly electrostatic exclusion mechanism of the ion flux decrease as a result of DNA binding to the PNA-modified nanopores.The extremely low detection limits required for bioanalysis are usually achieved with some chemical or physical amplification mechanism. 1,2 In ion channel-mimetic sensors, the target species functions as a stimulus, actuating (opening or closing) ion channels and, thus, modulating the flux of the marker ions to be detected. 3 Amplification is achieved because the concentration of markers exceeds the concentration of the target species by many orders of magnitude. In one approach, electrodes are modified with artificial receptors having intramolecular channels that can be blocked by the formation of inclusion complexes with the analyte and, thus, control the heterogeneous redox reactions of electroactive species. 4 Another method in these lines makes use of genetically and/or chemically engineered receptors built into cell membranes. 5-7 In a third approach based on gold nanopores, pioneered by the group of Martin, 8-10 limitations due to the fragility of lipid bilayer membranes, in which biological nanopores are suspended, are eliminated. Modulations of the ionic permeability due to polarity changes (hydrophobic to hydrophilic) 11 or steric effects (high molecular weight analytes binding to small molecular weight receptors in the nanopores) have already been explored. 12,13 In a previous study, we have demonstrated the feasibility of the ion channel-mimetic biosensing with gold nanoporous membranes modified by biotin. The selective recognition of avidin modulates the Ca 2+ ion transport, which is detected with ion-selective potentiometry. 13 Later, the protein detection system has been extended by using single conically shaped gold nanopores. 12In this paper, for the first time, gold nanopores are functionalized, with a peptide nucleic acid (PNA) 14 in view of a label-free detection of complementary DNA strands. The PNAs are shown to bind their complementary nucleic acid sequence 15 with higher affinity and specificity NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript compared with DNA probes. 16 The nanopores were prepared by electroless Au plating of track etch polycarbonate membranes. 10 In order to obtain regular pore diameters and better control of the plating process, the original recipe was slightly modified in that the membranes were dried in N 2 atmosphere before placing them in the Au pla...

Conference of MSc students – Abstracts of the best contributions November 2010

“…We aimed to simulate the reaction mechanism of both the native and the evolved enzyme and compare the major catalytic factors. The activation barriers obtained by the Empirical Valence Bond (EVB) method [3] were in accord with the experimental data. We found that in course of the laboratory evolution the reorganization energy was optimized significantly, meanwhile the activation barrier only modestly decreased.…”

“…Sensing with nanopores/nanotubes for molecular detection emerged recently in the forefront of nanostructure-based chemical sensors [1] due to its potential for: (a) ultrafast DNA sequencing; [2] (b) biosensing with selective receptor modified nanopores; [3] (c) macromolecule and nanoparticle counting [4]. Sensing with nanopores most often implies monitoring changes in the applied voltage driven ion flow across a nanopore that separates two electrolyte chambers as particles are passing through or residing within the nanopore sensing zone.…”

Immunoassays Using Artificial Nanopores

“…As with redox indicators, the diffusion of an optical indicator can be measured through a nanoporous membrane (Jágerszki et al, 2007). Such sensing schemes can potentially take advantage of distinctive recognition/transport phenomena that occur in nanostructures.…”

Electrochemical Detection of miRNAs

“…Additionally, PNA probes have excellent chemical, biochemical and thermal stability. Their electroneutrality makes them ideal probes for electrochemical transducers based on detection of surface charge variations upon oligonucleotide hybridization, e.g., ion channel sensors [31], field effect transistors (FET) [31], and nanopore sensors [32,33]. As a low cost alternative of PNA, antisense morpholino oligos (MCPs), which have higher affinity to complementary RNA than natural nucleic acid probes, were also reported [34].…”