Particle Trajectory-Dependent Ionic Current Blockade in Low-Aspect-Ratio Pores.

Resistive pulse sensing with nanopores having a low thickness-to-diameter aspect-ratio structure is expected to enable high-spatial-resolution analysis of nanoscale objects in a liquid. Here we investigated the sensing capability of low-aspect-ratio pore sensors by monitoring the ionic current blockades during translocation of polymeric nanobeads. We detected numerous small current spikes due to partial occlusion of the pore orifice by particles diffusing therein reflecting the expansive electrical sensing zone of the low-aspect-ratio pores. We also found wide variations in the ion current line-shapes in the particle capture stage suggesting random incident angle of the particles drawn into the pore. In sharp contrast, the ionic profiles were highly reproducible in the post-translocation regime by virtue of the spatial confinement in the pore that effectively constricts the stochastic capture dynamics into a well-defined ballistic motion. These results, together with multiphysics simulations, indicate that the resistive pulse height is highly dependent on the nanoscopic single-particle trajectories involved in ultrathin pore sensors. The present finding indicates the importance of regulating the translocation pathways of analytes in low-aspect-ratio pores for improving the discriminability toward single-bioparticle tomography in liquid.

Single-nanoparticle detection using a low-aspect-ratio pore.

We explored single-particle translocation through a low thickness-to-diameter aspect ratio Si(3)N(4) pore mimicking graphene nanopore structure by a resistive pulse method. Ionic conductance of 0.05 aspect ratio pores scales linearly with the diameter, indicating predominant contribution of the access resistance to the ion transport. We find that the access resistance changes little during particle translocation. Furthermore, we observe enhanced particle capture rates via the strong electric field extended outside the low-aspect-ratio pore mouth. We also demonstrate electrical discrimination of two different sized particles using the low-aspect-ratio pore sensor with the constant access resistance assumption. The present findings indicate the potential utility of nucleotide-sized graphene nanopores as an electrical sensing platform for single-base identification via transmembrane ionic current blockade detections.

Determination of single nucleotide polymorphisms in N-acetyltransferase2 gene using an electrochemical DNA chip and an automated DNA detection system.

An electrochemical DNA chip using an electrochemically active intercalator and DNA probe immobilized on a gold electrode has been developed for genetic analysis. In this study, N-acetyltransferase2 (NAT2) gene polymorphisms (C481T G590A G857A) were determined by the electrochemical DNA chip and the automated DNA detection system that performs hybridization reaction, washing, detection, and data analysis. Human genomic DNAs were extracted from blood and DNA fragments containing the three polymorphisms were amplified by the polymerase chain reaction (PCR) method. Double-stranded PCR products were treated with T7 exonuclease and single-stranded target DNAs were obtained. A sample containing the single-stranded target DNAs was injected into a cassette including the electrochemical DNA chip and set in an automated system. The turnaround time for genotyping with this system was 90 min. A total of 38 samples were automatically genotyped by an SNP determination algorithm. The results of genotype were completely consistent with those determined by the polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) analysis. Consequently, this method requires no labeling step and has the advantage of realizing a compact and automatic system, and so the system is expected to contribute to personalized medicine based on genotype.

Specific and quantitative detection of PCR products from Clostridium piliforme, Helicobacter bilis, H. hepaticus, and mouse hepatitis virus infected mouse samples using a newly developed electrochemical DNA chip.

We developed a microfabricated electrochemical DNA chip for detection of polymerase chain reaction (PCR) products from 16S rRNA sequences of Clostridium piliforme (Cp), Helicobacter bilis (Hb) and Helicobacter hepaticus (Hh), and the nucleocapsid protein gene of mouse hepatitis virus (MHV). This chip does not require DNA labeling, and the hybridization signal can be detected as an anodic current. The average anodic currents of 9 (Cp), 5 (Hb), 8 (Hh) and 7 (MHV) PCR positive samples derived from feces of spontaneously infected mice (Cp, Hb and Hh) and MHV-contaminated tumor cells were 27.9+/-7.2, 31.9+/-8.1, 29.3+/-10.1, and 27.6+/-3.0 nA, respectively. On the other hand, the average anodic currents of 19 (Cp), 27 (Hb), 18 (Hh), and 13 (MHV) PCR negative samples were 0.3+/-2.9, 3.7+/-2.4, -1.0+/-1.7, and -2.3+/-2.7 nA, respectively. The anodic current increased with increasing concentrations of pathogens. For experimentally infected samples, the results of PCR/electrophoresis were in complete accord with those of this system when anodic currents of 6.1 (Cp), 8.5 (Hb), 2.4 (Hh), and 3.1 nA (MHV) were taken as the cut-off value. The results suggested that the electrochemical DNA chip system is useful for specific and quantitative detection of PCR products.