Immunological assay using a solid-state pore with a low limit of detection.

Emerging infectious diseases, cancer, and other diseases are quickly tested mainly via immune reactions based on specific molecular recognition between antigens and antibodies. By changing the diameter of solid-state pores, biomolecules of various sizes can be rapidly detected at the single-molecule level. The combination of immunoreactions and solid-state pores paves the way for an efficient testing method with high specificity and sensitivity. The challenge in developing this method is achieving quantitative analysis using solid-state pores. Here, we demonstrate a method with a low limit of detection for testing tumor markers using a combination of immunoreactions and solid-state pore technology. Quantitative analysis of the mixing ratio of two and three beads with different diameters was achieved with an error rate of up to 4.7%. The hybrid solid-state pore and immunoreaction methods with prostate-specific antigen (PSA) and anti-PSA antibody-modified beads achieved a detection limit of 24.9 fM PSA in 30 min. The hybrid solid-state pore and immunoreaction enabled the rapid development of easy-to-use tests with lower limit of detection and greater throughput than commercially available immunoassay for point-of-care testing.

Dielectric Coatings for Resistive Pulse Sensing Using Solid-State Pores.

The present study reports on the systematic characterization of the effectiveness of dielectric coating to tailor capture-to-translocation dynamics of single particles in solid-state pores. We covered the surface of SiNx membranes with SiO2, HfO2, Al2O3, TiO2, or ZnO, which allowed us to change the ζ-potential at the pore wall, reflecting the isoelectric points of these coating materials. Resistive pulse measurements of negatively charged polystyrene beads elucidated more facile electrophoretic capture of the particles and slower translocation motions in the channel under more negative electric potential at the oxide surface. These findings provide a guide to engineer pore wall surface for optimizing the translocation dynamics for efficient sensing of particles and molecules.

Back-Side Polymer-Coated Solid-State Nanopore Sensors.

We systematically investigated the influence of polymer coating on temporal resolution of solid-state nanopores. We fabricated a Si3N4 nanopore integrated with a polyimide sheet partially covering the substrate surface. Upon detecting the nanoparticles dispersed in an electrolyte buffer by ionic current measurements, we observed a larger resistive pulse height along with a faster current decay at the tails under larger coverage of the polymeric layer, thereby suggesting a prominent role of the water-touching Si3N4 thin film as a significant capacitor serving to retard the ionic current response to the ion blockade by fast translocation of particles through the nanopores. From this, we came up with back-side polymer-coated chip designs and demonstrated improved pore sensor temporal resolution by developing a nanopore with a thick polymethyl-methacrylate layer laminated on the bottom surface. The present findings may be useful in developing integrated solid-state nanopore sensors with embedded nanochannels and nanoelectrodes.

Electric field interference and bimodal particle translocation in nano-integrated multipores.

Parallel integration of multiple channels is a fundamental strategy for high-throughput particle detection in solid-state nanopores wherein understanding and control of crosstalk is an important issue for the post resistive pulse analysis. Here we report on a prominent effect of cross-channel electric field interference on the ionic current blockade by nanoparticles in nano-spaced pore arrays in a thin Si3N4 membrane. We systematically investigated the variations in resistive pulse profiles in multipore systems of various inter-channel distances. Although each pore acted independently when they were formed at excessively far distances, we observed significant cross-pore electrostatic interactions under close-integration that led the multipores to virtually act as a single-pore of equivalent area. As a result of the interference, the resistive pulse height demonstrated bimodal distributions due to the pronounced particle trajectory-dependence of the ionic blockade effects. Most importantly, the overcrowded multi-channel structure was found to deliver significant crosstalk with serious degradation of the sensor sensitivity to particle sizes. The present results provide a guide to design multipore structures regarding the trade-off between the detection throughput and sensor sensitivity.

Silicon substrate effects on ionic current blockade in solid-state nanopores.

We investigated the roles of silicon substrate material compositions in ionic current blockade in solid-state nanopores. When detecting single nanoparticles using an ionic current in a Si3N4 nanopore supported on a doped silicon wafer, resistive pulses were found to be blunted significantly via signal retardation due to predominant contributions of large capacitance at the ultrathin membrane. Unexpectedly, in contrast, changing the substrate material to non-doped silicon led to the sharpening of the spike-like signal feature, suggesting a better temporal resolution of the cross-channel ionic current measurements by virtue of the thick intrinsic semiconductor layer that served to diminish the net chip capacitance. The present results suggest the importance of the choice of Si compositions regarding the capacitance effects to attain better spatiotemporal resolution in solid-state nanopore sensors.

Particle Capture in Solid-State Multipores.

Utilization of multiple-channel structure is a promising way of accomplishing high-throughput detections of analytes in solid-state pore sensors. Here we report on systematic investigation of particle capture efficiency in Si3N4 multipore systems of various array configurations. We demonstrated enhanced detection throughput with increasing numbers of pore channels in a membrane. Meanwhile, we also observed significant contributions of the interchannel crosstalk in closely integrated multipores that tended to deteriorate throughput performance by causing shrinkage of the absorption zone via the interference-derived weakening of the electric field around the pore orifice. At the same time, the interference-derived electric field distributions were also found to diminish the electroosmotic contributions to the particle capture efficiency. The present findings can be useful in designing pore arrays with optimal throughput performance.

Association between the catechol-O-methyltransferase polymorphism Val158Met and Alzheimer's disease in a Japanese population.

OBJECTIVE: Catechol-O-methyltransferase (COMT) plays an important role in dopamine degradation, which is associated with the pathophysiology of Alzheimer's disease (AD) and alcoholism. A functional COMT polymorphism, Val158Met (rs4680 G > A), affects the onset of AD and is associated with alcohol dependence through dopamine receptor sensitivity in the prefrontal cortex. METHODS: The aim of this case-control study (398 cases and 149 controls) was to investigate whether Val158Met polymorphism influences the onset of AD stratified according to alcohol consumption and apolipoprotein E (APOE) status. We also used single photon-emission computed tomography (SPECT) to analyse 26 patients with AD with the polymorphism. RESULTS: As a function of APOE status, the genotypic frequencies of rs4680 in patients with AD did not differ from those in controls. We detected a significant association between high alcohol consumption in patients with AD (HAC-AD group) and the polymorphism in genotypic and allelic frequencies. Logistic regression analyses demonstrated that the presence of the APOE genotype with rs4680 increased the risk for HAC-AD synergistically. Hyperperfusion in the right sub-lobar insula of patients with the G/G genotype was found compared with that of patients with the G/A genotype. SPECT studies showed a relationship between the polymorphism and compensatory reactions for dysfunctions of dopaminergic neurotransmission in AD pathophysiology. CONCLUSION: Although genetic association between the polymorphism and the onset of AD in a Japanese population were not observed, the polymorphism affected the risk for HAC-AD.

Polymorphisms in the aldehyde dehydrogenase 2 and dopamine ß hydroxylase genes are not associated with Alzheimer's disease.

Since ethanol and its metabolite, acetaldehyde, are directly neurotoxic, alcohol intake could affect the development of Alzheimer's disease (AD). Mitochondrial aldehyde dehydrogenase 2 (ALDH2) metabolizes acetaldehyde into acetate and also protects against oxidative stress, playing an important role in the development of AD. The activity of dopamine ß hydroxylase (DBH) is reduced in the hippocampus and neocortex in the AD brain. DBH is also involved in the pathophysiology of alcoholism. The aim of this study was to investigate whether polymorphisms of both ALDH2 and DBH genes were associated with AD. ALDH2*2 and two functional single nucleotide polymorphisms (SNPs) of the DBH gene were analyzed using a case-control study design. Our case-control data set consisted of 201 AD patients and 130 age-matched controls. We also analyzed stratifying by alcohol consumption and apolipoprotein E (APOE) genotypes. There were no associations between the SNPs studied here and the onset of AD. No synergetic associations were found among the SNPs, APOE and the risk for AD. Although high alcohol consumption AD (HAC-AD) patients were analyzed in detail, the current three SNPs were not related with HAC-AD. ALDH2*2 and functional SNPs of the DBH gene did not modify the risk for AD. Since our data set was constructed only with AD in a Japanese population, further detailed genetic analyses with other ethnic groups would be needed.

Changes in Mini-Mental State Examination score in Alzheimer's disease patients after stopping habitual drinking.

BACKGROUND: Globally, Alzheimer's disease (AD) is becoming an increasing problem as the population ages, and the effects of lifestyle factors on cognitive decline need to be better understood. This study examined the effects of alcohol abstinence on cognitive decline in AD. METHODS: Cognitive function after alcohol abstinence was retrospectively reviewed in AD patients (high and low alcohol consumption groups) and then compared with an alcohol-naïve AD group. The alcohol-naïve AD group included 18 outpatients with no history of habitual drinking. The alcohol-abstinence AD group included 20 outpatients who stopped drinking after their diagnoses. The latter group was classified into high and low groups depending on the amount of they drank before abstinence. Cognitive function was evaluated with the Mini-Mental State Examination at baseline, 6 months, and 12 months. For statistical analyses, a repeated measures, two-factor anova and post-hoc multiple comparisons were performed using the Bonferroni method. RESULTS: There was a significant effect of time on Mini-Mental State Examination score, but there was no difference in the baseline scores of the alcohol-naïve and alcohol-abstinence AD groups. The score was significantly lower at 6 and 12 months than at baseline in the alcohol-naïve group, but no significant difference was seen in the alcohol-abstinence group. There was a significant interaction between time and alcohol consumption subgroup on the score, with no difference in baseline score between the low and high consumption groups. The score was significantly lower only in the high consumption group at 12 months. CONCLUSIONS : In AD patients with a history of habitual drinking, abstinence was effective for reducing cognitive decline during the clinical course. However, such an effect was not seen in patients who had consumed high amounts of alcohol before diagnosis of AD.