Molecular transport of proteins through nanoporous membranes fabricated by interferometric lithography.

Millimeter sized arrays of uniformly-distributed nanopores (180-220 nm) were created in thin (200 nm) silicon nitride membranes using interferometric lithography. Molecular transport properties of the fabricated devices were investigated experimentally and compared with those of state-of-the-art polycarbonate track etched membranes. Two similarly-sized proteins, bovine serum albumin (BSA) and bovine hemoglobin (BHb), were used as permeates in the transport experiments. Up to 40 fold higher pore fluxes were achieved with unmodified silicon nitride membranes relative to thicker commercial nanoporous membranes. Similarly, in mixed protein experiments, ∼5.0 and 1.9 fold higher BSA and BHb selectivities were obtained with fabricated thin membranes at pH 4.7 and 7.0, respectively, relative to the commercial nanoporous membranes.

Mesoscale simulations of biomolecular transport through nanofilters with tapered and cylindrical geometries.

Molecular transport properties in short cylindrical and pyramidal nanopores are investigated by mesoscale dissipative particle dynamics simulations. We examine the effect of pore geometry, size, flow direction, particle diameter and electrostatic forces on membrane flux, selectivity and fouling. Biomolecules of various sizes are represented by spherical particles as they move through nanopores. The highest molecular concentration in pores is obtained with a cylindrical geometry, whereas the lowest concentration is obtained with a pyramidal geometry when the molecular transport direction is from large to small pore opening. This reveals a higher tendency for fouling in cylindrical pores relative to pyramidal pores. In general, increasing pore size and decreasing molecular diameter increase diffusion and fluxes, as expected, and the highest fluxes are achieved when the molecule is in neutral state. For large, short pores, higher diffusion rates are achieved with a cylindrical geometry compared to a pyramidal geometry. For pore: particle diameter ratios below 10, highly restricted motion is observed. In the presence of electrostatic forces, the molecular separation potential of pyramidal pores is 1.5× higher relative to short cylindrical pores, although the diffusion rate with cylindrical pores is 1.8× higher. Finally, we demonstrate that decreasing the pore size by a factor of 1.2 can reduce the pore molecular concentration by at least a factor of 3 for all pore types. This finding is consistent with a surprising recent experimental study in which larger ceramic pores were observed to foul much faster than smaller pores.

The role of pore geometry in single nanoparticle detection.

We observe single nanoparticle translocation events via resistive pulse sensing using silicon nitride pores described by a range of lengths and diameters. Pores are prepared by focused ion beam milling in 50 nm-, 100 nm-, and 500 nm-thick silicon nitride membranes with diameters fabricated to accommodate spherical silica nanoparticles with sizes chosen to mimic that of virus particles. In this manner, we are able to characterize the role of pore geometry in three key components of the detection scheme, namely, event magnitude, event duration, and event frequency. We find that the electric field created by the applied voltage and the pore's geometry is a critical factor. We develop approximations to describe this field, which are verified with computer simulations, and interactions between particles and this field. In so doing, we formulate what we believe to be the first approximation for the magnitude of ionic current blockage that explicitly addresses the invariance of access resistance of solid-state pores during particle translocation. These approximations also provide a suitable foundation for estimating the zeta potential of the particles and/or pore surface when studied in conjunction with event durations. We also verify that translocation achieved by electro-osmostic transport is an effective means of slowing translocation velocities of highly charged particles without compromising particle capture rate as compared to more traditional approaches based on electrophoretic transport.

Polystyrene particles reveal pore substructure as they translocate.

In this article, we report resistive-pulse sensing experiments with cylindrical track-etched PET pores, which reveal that the diameters of these pores fluctuate along their length. The resistive pulses generated by polymer spheres passing through these pores have a repeatable pattern of large variations corresponding to these diameter changes. We show that this pattern of variations enables the unambiguous resolution of multiple particles simultaneously in the pore, that it can detect transient sticking of particles within the pore, and that it can confirm whether any individual particle completely translocates the pore. We demonstrate that nonionic surfactant has a significant impact on particle velocity, with the velocity decreasing by an order of magnitude for a similar increase in surfactant concentration. We also show that these pores can differentiate by particle size and charge, and we explore the influence of electrophoresis, electroosmosis, and pore size on particle motion. These results have practical importance for increasing the speed of resistive-pulse sensing, optimizing the detection of specific analytes, and identifying particle shapes.

Rapid-viability PCR method for detection of live, virulent Bacillus anthracis in environmental samples.

In the event of a biothreat agent release, hundreds of samples would need to be rapidly processed to characterize the extent of contamination and determine the efficacy of remediation activities. Current biological agent identification and viability determination methods are both labor- and time-intensive such that turnaround time for confirmed results is typically several days. In order to alleviate this issue, automated, high-throughput sample processing methods were developed in which real-time PCR analysis is conducted on samples before and after incubation. The method, referred to as rapid-viability (RV)-PCR, uses the change in cycle threshold after incubation to detect the presence of live organisms. In this article, we report a novel RV-PCR method for detection of live, virulent Bacillus anthracis, in which the incubation time was reduced from 14 h to 9 h, bringing the total turnaround time for results below 15 h. The method incorporates a magnetic bead-based DNA extraction and purification step prior to PCR analysis, as well as specific real-time PCR assays for the B. anthracis chromosome and pXO1 and pXO2 plasmids. A single laboratory verification of the optimized method applied to the detection of virulent B. anthracis in environmental samples was conducted and showed a detection level of 10 to 99 CFU/sample with both manual and automated RV-PCR methods in the presence of various challenges. Experiments exploring the relationship between the incubation time and the limit of detection suggest that the method could be further shortened by an additional 2 to 3 h for relatively clean samples.

A sample-in-answer-out instrument for the detection of multiple respiratory pathogens in unprepared nasopharyngeal swab samples.

Multiplex RT-PCR suspension array assays provide a powerful tool for identifying the causative agent(s) of respiratory infections. These assays are time consuming and laborious on a time-per-sample basis if only a few samples require processing. To address this shortcoming and provide an automated solution for fast detection and identification of viral pathogens, we developed the first automated multiplex RT-PCR suspension array instrument capable of handling unprepared clinical samples. The instrument requires less than 3 minutes of hands-on time for a result generated in approximately 2.5 hours. In analytical studies, the instrument performed as well as manually performed assays. The performance of the instrument and loaded multiplex viral detection assay was then tested using unprepared nasopharyngeal samples. The instrument-performed assay detected 61 of 71 RSV positive samples, for a sensitivity of 85.9%. Adenovirus (n = 5) and influenza B (n = 3) were less prevalent in the sample set, but detected to similar levels, 80% and 75%, respectively. The same sample set was also tested using FDA approved immuno-assay rapid tests, and the instrument was found to be more sensitive than the rapid tests with the sole exception being influenza A (n = 16), which was poorly detected due to significant sequence mismatches between the influenza A primer/probe set included in the multiplex mixture and the circulating influenza A strains. Overall, these data demonstrate the developed prototype platform performs multiplex array assays as well as hand-performed assays, and that the instrument's sensitivity and specificity are dictated by the quality of the loaded multiplex assay.

Hydrogen production by a hyperthermophilic membrane-bound hydrogenase in water-soluble nanolipoprotein particles.

Hydrogenases constitute a promising class of enzymes for ex vivo hydrogen production. Implementation of such applications is currently hindered by oxygen sensitivity and, in the case of membrane-bound hydrogenases (MBHs), poor water solubility. Nanolipoprotein particles (NLPs) formed from apolipoproteins and phospholipids offer a novel means of incorporating MBHs into a well-defined water-soluble matrix that maintains the enzymatic activity and is amenable to incorporation into more complex architectures. We report the synthesis, hydrogen-evolving activity, and physical characterization of the first MBH-NLP assembly. This may ultimately lead to the development of biomimetic hydrogen-production devices.

Multiplexed reverse transcriptase PCR assay for identification of viral respiratory pathogens at the point of care.

We have developed a nucleic acid-based assay that is rapid, sensitive, and specific and can be used for the simultaneous detection of five common human respiratory pathogens, including influenza virus A, influenza virus B, parainfluenza virus types 1 and 3, respiratory syncytial virus (RSV), and adenovirus groups B, C, and E. Typically, diagnosis on an unextracted clinical sample can be provided in less than 3 h, including sample collection, preparation, and processing, as well as data analysis. Such a multiplexed panel would enable rapid broad-spectrum pathogen testing on nasal swabs and therefore allow implementation of infection control measures and the timely administration of antiviral therapies. We present here a summary of the assay performance in terms of sensitivity and specificity. The limits of detection are provided for each targeted respiratory pathogen, and result comparisons were performed on clinical samples, our goal being to compare the sensitivity and specificity of the multiplexed assay to the combination of immunofluorescence and shell vial culture currently implemented at the University of California-Davis Medical Center hospital. Overall, the use of the multiplexed reverse transcription-PCR assay reduced the rate of false-negative results by 4% and reduced the rate of false-positive results by up to 10%. The assay correctly identified 99.3% of the clinical negatives and 97% of the adenovirus, 95% of the RSV, 92% of the influenza virus B, and 77% of the influenza virus A samples without any extraction performed on the clinical samples. The data also showed that extraction will be needed for parainfluenza virus, which was only identified correctly 24% of the time on unextracted samples.

Pore conductivity control at the hundred-nanometer scale: an experimental and theoretical study.

We report on the observation of an unexpected mechanism that controls conductivity at the 100-nm scale on track-etched polycarbonate membranes. Transport measurements of positively charged methyl viologen performed by absorption spectroscopy under various pH conditions demonstrate that for 100-nm-diameter pores at pH 2 conductivity is blocked, while at pH 5 the ions move through the membrane according to diffusion laws. An oppositely charged molecular ion, naphthalene disulfonate, in the same membrane, shows the opposite trend: diffusion of the negative ion at pH 2 and very low conductivity at pH 5. The influence of parameters such as ionic strength and membrane surface coating are also investigated. A theoretical study of the system shows that at the 100-nm scale the magnitude of the electric field in the vicinity of the pores is too small to account for the experimental observations; rather, it is the surface trapping of the mobile ion (Cl- or Na+) that gives rise to the observed control of the conductivity. This surprising effect has potential applications for high-throughput separation of large molecules and bio-organisms.

Hydrolysis of acetylcholinesterase inhibitors--organophosphorus acid anhydrolase enzyme immobilization on photoluminescent porous silicon platforms.

We report on the immobilization of an OPAA enzyme on luminescent porous silicon devices, and on the utilization of this new platform to hydrolyze p-nitrophenyl-soman.

Functionalized silicon membranes for selective bio-organism capture.

Membranes with various pore size, length, morphology and density have been synthesized from diverse materials for size-exclusion-based separation. An example is the sterilization of intravenous lines by exclusion of bacteria and viruses using polyvinylidene fluoride membranes with 0.1-microm-diameter pores. Chemically specific filtration has recently been addressed for small molecules. Nevertheless, specific bio-organism immobilization and detection remains a great technical challenge in many biomedical applications, such as decontamination or analysis of air and liquids such as drinking water and body fluids. To achieve this goal, materials with controlled pore diameter, length and surface chemistry are required. In this letter, we present the first functionalized silicon membranes and demonstrate their ability to selectively capture simulated bio-organisms. These extremely versatile and rigid devices open the door to a new class of materials that are able to recognize the external fingerprints of bio-organisms-such as size and outer membrane proteins-for specific capture and detection applications.

New method for attachment of biomolecules to porous silicon.

Biomolecules have been attached to porous silicon by a new linking method that forms a direct Si-C bond on the surface and retains the photoluminescence of the porous silicon.