Rafael Mulero

Chemically modified solid state nanopores for high throughput nanoparticle separation

The separation of biomolecules and other nanoparticles is a vital step in several analytical and diagnostic techniques. Towards this end we present a solid state nanopore-based set-up as an efficient separation platform. The translocation of charged particles through a nanopore was first modeled mathematically using the multi-ion model and the surface charge density of the nanopore membrane was identified as a critical parameter that determines the selectivity of the membrane and the throughput of the separation process. Drawing from these simulations a single 150 nm pore was fabricated in a 50 nm thick free-standing silicon nitride membrane by focused-ion-beam milling and was chemically modified with (3-aminopropyl)triethoxysilane to change its surface charge density. This chemically modified membrane was then used to separate 22 and 58 nm polystyrene nanoparticles in solution. Once optimized, this approach can readily be scaled up to nanopore arrays which would function as a key component of next-generation nanosieving systems.

Nanopore-Based Devices for Bioanalytical Applications

With over a decade passed since the first reported use of a Staphylococcal α-hemolysin pore to study single molecules of single-stranded DNA, research in the field of nanopores has advanced rapidly. We discuss the technological progression of nanopore-based devices from the initial use of α-hemolysin pores to the advent of solid-state nanopores to the burgeoning of organic-inorganic hybrid pores driven by the desire to achieve fast and inexpensive DNA sequencing. Additional nanopore-based efforts are also discussed that study other classes of molecules, such as proteins. We discuss the use of nanopores for protein folding and binding analysis. In addition to single-molecule analysis, we report on the introduction of nanopore arrays on thin film membranes for ultrafiltration. Owing to their reduced spatial dimensionality, such membranes offer greater control over how the pores interact with analytes thus leading to very efficient separation. With several technical hindrances yet to be overcome, the devices we report are still works in progress. The realization of these devices will enhance laboratory processes by permitting superior spatial and temporal analytical resolution at the single-molecule level resulting in laboratory capacities of great impact.

Low aspect ratio micropores for single-particle and single-cell analysis

This paper describes microparticle and bacterial translocation studies using low aspect ratio solid‐state micropores. Micropores, 5 μm in diameter, were fabricated in 200 nm thick free‐standing silicon nitride membranes, resulting in pores with an extremely low aspect ratio, nominally 0.04. For microparticle translocation experiments, sulfonated polystyrene microparticles and magnetic microbeads in size range of 1–4 μm were used. Using the microparticle translocation characteristics, we find that particle translocations result in a change only in the pores geometrical resistance while the access resistance remains constant. Furthermore, we demonstrate the ability of our micropore to probe high‐resolution shape information of translocating analytes using concatenated magnetic microspheres. Distinct current drop peaks were observed for each microsphere of the multibead architecture. For bacterial translocation experiments, nonflagellated Escherichia coli (strain HCB 5) and wild type flagellated Salmonella typhimurium (strain SJW1103) were used. Distinct current signatures for the two bacteria were obtained and this difference in translocation behavior was attributed to different surface protein distributions on the bacteria. Our findings may help in developing low aspect ratio pores for high‐resolution microparticle characterization and single‐cell analysis.

Ultra-Fast Low Concentration Detection of Candida Pathogens Utilizing High Resolution Micropore Chips

Although Candida species are the fourth most common cause of nosocomial blood stream infections in the United States, early diagnostic tools for invasive candidemia are lacking. Due to an increasing rate of candidemia, a new screening system is needed to detect the Candida species in a timely manner. Here we describe a novel method of detection using a solid-state micro-scale pore similar to the operational principles of a Coulter counter. With a steady electrolyte current flowing through the pore, measurements are taken of changes in the current corresponding to the shape of individual yeasts as they translocate or travel through the pore. The direct ultra-fast low concentration electrical addressing of C. albicans has established criteria for distinguishing individual yeast based on their structural properties, which may reduce the currently used methods’ complexity for both identification and quantification capabilities in mixed blood samples.

High Throughput Nanofluidic Architectures for Nanoparticle Separation

High blood cholesterol levels and associated complications are a major health concern the world over and current techniques to deal with this, especially low density lipoprotein (LDL) apheresis, have significant room for improvement. We had previously reported a proof of concept technique that relies on silicon nitride based solid state nanopores to separate high density lipoprotein (HDL) like particles from LDL like particles. A mathematical model to describe the setup is reported. This model revealed that charge density of the pore surface was critical in determining the efficiency of separation. Accordingly we chemically modified our nanopores with (3-aminopropyl)-triethoxysilane (APTES) to achieve more efficient, high throughput particle separation. Such a technique could make it possible to develop safe and affordable LDL apheresis devices in the future.Copyright

Synthetic Nanoscale Architectures for Lipoprotein Separation

Current low density lipoprotein (LDL) apheresis procedures are expensive and time consuming. We report here a novel technique to detect and separate nanoparticles using solid state nanopores. Our technique relies on the resistive pulse phenomenon used in coulter counters. We used a 150nm diameter nanopore to detect nanoparticles that closely resembled HDL and LDL in terms of their size and surface charge. Statistical analysis of the translocation data revealed that our setup preferentially allowed the particles resembling HDL to pass thorough while restricting the translocation of the particles that resembled LDL.Copyright

Probing Bacterial Flagellar Polymorphism in Various Fluidic Environments Using Solid-State Sub-Micropores

A novel method for the detection of an assortment of environmental conditions in a microfluidic system using bacterial flagella and submicro-scale solid state pores is presented. Differences in various environmental conditions stimulate the polymorphic helix structure of Salmonella typhimurium flagella to transform to its lowest energetic conformation. By measuring the ionic current blockage (resistive pulse) as flagella electrophoretically translocate a submicro-scale pore, detection of the polymorphic state of flagella corresponding to the conditions of the environmental stimuli is possible. We test the viability of this method using purified depolymerized and repolymerized S. Typhimurium flagella and a high resolution electrical signal readout sub-micropore-based detection system.Copyright

High Resolution Detection and Configuration of Bacteria Using Microscale Pores

A novel method for detecting and configuring bacteria using a micro-scale pore is presented. The method distinguishes between different species of bacteria by measuring the ionic current blockage (resistive pulse) as bacteria electrophoretically translocate the micro-pore. Both wild-type flagellated (HCB 33) and non-flagellated Escherichia coli (HCB 5), and Polystyrene microbeads were used in this study to demonstrate the efficacy of this method. High resolution electrical signal readout enabled discrimination of the orientation of both non-flagellated and peritrichously flagellated bacteria as they move through the solid-state pore.Copyright

Fractal Geometry Based Model for a Musical Cymbal Design and Rapid Manufacturing

This paper aims at an equation based simulation, CAD modeling and manufacturing of a fractal surface embedded on a musical cymbal. This study is essentially a proof-of-concept of a new method of complex-surface characterization, design and manufacturing using an equation-based approach. A cymbal shape was chosen to carry the fractal profile because generally most musical cymbals have an inherent broken surface pattern that is created to enhance the resulting musical quality, and hence it would be a topic of further study to characterize the musical notes emanating from a fractal surface on this cymbal. A fractal surface cymbal model was developed; a point cloud representation of the cymbal was generated from Matlab; a surface model was built and processed in image data processing software Imageware; a solid model was completed in CAD software PRO/E; a rapid prototype of the fractal surface cymbal was fabricated; and MasterCam software was used to generate CNC codes and simulate the CNC machining process. The effect of the variation of the parameters of the equation based surface is also shown by varying the topothesy and fractal parameter of the surface. Finally, the equation based method is used to generate other complex surfaces such as Cassini surface and the Bohemian Dome which are then solidified and prepared of manufacture.Copyright