Effect of Organic Cation Adsorption on Ion-Transport Selectivity in a Cation-Permselective Nanopore Membrane.

A key knowledge gap in the emerging field of nanofluidics concerns how the ionic composition and ion-transport properties of a nanoconfined solution differ from those of a contacting bulk solution. We and others have been using potentiometric concentration cells, where a nanopore or nanotube membrane separates salt solutions of differing concentrations to explore this issue. The membranes studied contained a fixed pore/tube wall anionic charge, which ideally would prohibit anions and salt from entering the pore/tube-confined solution. We have been investigating experimental conditions that allow for this ideally permselective cation state to be achieved. Results of potentiometric investigations of a polymeric nanopore membrane (10 ± 2 nm-diameter pores) with anionic charge due to carbonate are presented here. While studies of this type have been reported using alkaline metal and alkaline earth cations, there have been no analogous studies using organic cations. This paper uses a homologous series of tetraalkylammonium ions to address this knowledge gap. The key result is that, in contrast to the inorganic cations, the ideal cation-permselective state could not be obtained under any experimental conditions for the organic cations. We propose that this is because these hydrophobic cations adsorb onto the polymeric pore walls. This makes ideality impossible because each adsorbed alkylammonium must bring a charge-balancing anion, Cl-, with it into the nanopore solution. The alkylammonium adsorption that occurred was confirmed and quantified by using surface contact angle measurements.

Biological nanopores elucidate the differences between isomers of mercaptobenzoic-capped gold clusters.

Identification of isomers using traditional mass spectroscopy methods has proven an interesting challenge due to their identical mass to charge ratios. This proves particularly consequential for gold clusters, as subtle variations in the ligand and cluster structure can have drastic effects on the cluster functionalization, solubility, and chemical properties. Biological nanopores have proven an effective tool in identifying subtle variations at the single molecule limit. This paper reports on the ability of an α-hemolysin (αHL) pore to differentiate between para-, meta-, and ortho- (p-, m-, and o-, respectively) mercaptobenzoic acid ligands attached to gold clusters at the single cluster limit. Detecting differences between p-MBA and m-MBA requires pH-dependent studies that illustrate the role inter-ligand binding plays in stabilizing m-MBA-capped clusters. Additionally, this paper investigates the difference in behavior for these clusters when isolated, and when surrounded by small ligand-Au complexes (AunLm, n = 0, 1, 2… and m = 1, 2,…) that are present following cluster synthesis. It is found that continuous exposure of clusters to freely diffusing ligand complexes stabilizes the clusters, while isolated clusters either disintegrate or exit the nanopore in seconds. This has implications for long term cluster stability.

Chemoresponsive Nanofluidic Pump That Turns Off in the Presence of Lead Ion.

There are many applications that require the integration of a pump and a chemical sensor so that the solution being pumped can be analyzed in real time for a specific chemical species and the flow adjusted according to the measured concentration of that species. We describe here an alternative strategy: a chemoresponsive pump where a single device acts as both the sensor and pump simultaneously. We demonstrate this concept with a nanofluidic Pb2+-responsive pump that uses electroosmotic flow as the pumping technology, and a Pb2+-binding ionophore that allows the device to selectively respond to Pb2+. The pump yields high flow rates at low Pb2+ concentrations (<1 µM), but flow rate decreases with concentrations above this threshold and ultimately goes to zero at concentrations above 100 µM.

The Effect of Voltage Charging on the Transport Properties of Gold Nanotube Membranes.

Porous membranes are used in chemical separations and in many electrochemical processes and devices. Research on the transport properties of a unique class of porous membranes that contain monodisperse gold nanotubes traversing the entire membrane thickness is reviewed here. These gold nanotubes can act as conduits for ionic and molecular transports through the membrane. Because the tubes are electronically conductive, they can be electrochemically charged by applying a voltage to the membrane. How this "voltage charging" affects the transport properties of gold nanotube membranes is the subject of this Review. Experiments showing that voltage charging can be used to reversibly switch the membrane between ideally cation- and anion-transporting states are reviewed. Voltage charging can also be used to enhance the ionic conductivity of gold nanotube membranes. Finally, voltage charging to accomplish electroporation of living bacteria as they pass through gold nanotube membranes is reviewed.

Nanomaterial Preparation by Extrusion through Nanoporous Membranes.

Template synthesis represents an important class of nanofabrication methods. Herein, recent advances in nanomaterial preparation by extrusion through nanoporous membranes that preserve the template membrane without sacrificing it, which is termed as "non-sacrificing template synthesis," are reviewed. First, the types of nanoporous membranes used in nanoporous membrane extrusion applications are introduced. Next, four common nanoporous membrane extrusion strategies: vesicle extrusion, membrane emulsification, precipitation extrusion, and biological membrane extrusion, are examined. These methods have been utilized to prepare a wide range of nanomaterials, including liposomes, emulsions, nanoparticles, nanofibers, and nanotubes. The principle and historical context of each specific technology are discussed, presenting prominent examples and evaluating their positive and negative features. Finally, the current challenges and future opportunities of nanoporous membrane extrusion methods are discussed.

Voltage-Rectified Current and Fluid Flow in Conical Nanopores.

Ion current rectification (ICR) refers to the asymmetric potential-dependent rate of the passage of solution ions through a nanopore, giving rise to electrical current-voltage characteristics that mimic those of a solid-state electrical diode. Since the discovery of ICR in quartz nanopipettes two decades ago, synthetic nanopores and nanochannels of various geometries, fabricated in membranes and on wafers, have been extensively investigated to understand fundamental aspects of ion transport in highly confined geometries. It is now generally accepted that ICR requires an asymmetric electrical double layer within the nanopore, producing an accumulation or depletion of charge-carrying ions at opposite voltage polarities. Our research groups have recently explored how the voltage-dependent ion distributions and ICR within nanopores can induce novel nanoscale flow phenomena that have applications in understanding ionics in porous materials used in energy storage devices, chemical sensing, and low-cost electrical pumping of fluids. In this Account, we review our most recent investigations on this topic, based on experiments using conical nanopores (10-300 nm tip opening) fabricated in thin glass, mica, and polymer membranes. Measurable fluid flow in nanopores can be induced either using external pressure forces, electrically via electroosmotic forces, or by a combination of these two forces. We demonstrate that pressure-driven flow can greatly alter the electrical properties of nanopores and, vice versa, that the nonlinear electrical properties of conical nanopores can impart novel and useful flow phenomena. Electroosmotic flow (EOF), which depends on the magnitude of the ion fluxes within the double layer of the nanopore, is strongly coupled to the accumulation/depletion of ions. Thus, the same underlying cause of ICR also leads to EOF rectification, i.e., unequal flows occurring for the same voltage but opposite polarities. EOF rectification can be used to electrically pump fluids with very precise control across membranes containing conical pores via the application of a symmetric sinusoidal voltage. The combination of pressure and asymmetric EOF can also provide a means to generate new nanopore electrical behaviors, including negative differential resistance (NDR), in which the current through a conical pore decreases with increasing driving force (applied voltage), similar to solid-state tunnel diodes. NDR results from a positive feedback mechanism between the ion distributions and EOF, yielding a true bistability in both fluid flow and electrical current at a critical applied voltage. Nanopore-based NDR is extremely sensitive to the surface charge near the nanopore opening, suggesting possible applications in chemical sensing.

Low-Voltage Flow-Through Electroporation in Gold-Microtube Membranes.

Electroporation is used to create pores within the membrane of living cells in order to deliver a substance, for example, a gene, into the cytoplasm. To achieve the high electric field gradients required to porate the membrane, current electroporation devices deliver voltage pulses in the kV range to the cell medium. We describe a new device based on gold-microtube membranes that can accomplish electroporation with voltage pulses that are orders of magnitude smaller, ≤5 V. This is possible because the voltage pulses are applied to the gold microtubes resulting in large electric field gradients down the length of the tubes. We used COMSOL simulations to calculate the electric field gradients, and these theoretical results were compared with known experimental values required to electroporate Escherichia coli. We developed two fluorescence-based methods to demonstrate successful electroporation of E. coli. The percentages of electroporated bacteria were found to be more than an order of magnitude higher than obtained with a commercial electroporator, although the voltage employed was 500 times lower. Furthermore, this microtube membrane device is flow through and is therefore capable of continuous, as opposed to batch-wise, electroporation and cell analysis. Cell throughput of >30 million cells per min, higher than any previously reported device, were obtained.

Preparation of mineralized nanofibers: collagen fibrils containing calcium phosphate.

We report a straightforward, bottom-up, scalable process for preparing mineralized nanofibers. Our procedure is based on flowing feed solution, containing both inorganic cations and polymeric molecules, through a nanoporous membrane into a receiver solution with anions, which leads to the formation of mineralized nanofibers at the exit of the pores. With this strategy, we were able to achieve size control of the nanofiber diameters. We illustrate this approach by producing collagen fibrils with calcium phosphate incorporated inside the fibrils. This structure, which resembles the basic constituent of bones, assembles itself without the addition of noncollagenous proteins or their polymeric substitutes. Rheological experiments demonstrated that the stiffness of gels derived from these fibrils is enhanced by mineralization. Growth experiments of human adipose derived stem cells on these gels showed the compatibility of the fibrils in a tissue-regeneration context.

The Ionic Composition and Chemistry of Nanopore-Confined Solutions.

There is increasing interest in understanding the properties of solutions confined within nanotubes and synthetic or biological nanopores. How the ionic content of a nanopore-confined solution differs from that of a contacting bulk salt solution is of particular importance, for example, to water desalinization, industrial electrolysis, and all living systems. This paper explores ionic content, ionic interactions, and ion-transport properties of solutions confined within the 10 nm diameter pores of a synthetic polymer membrane. The membrane has a fixed negative pore-wall and surface charge due to ionizable carbonate groups. As a result, under some conditions, the nanopore-confined solution contains only cations and no anions or salt present in a contacting solution, ideal cation permselectivity. This anion- and salt-rejecting ability varies greatly with the cation of the salt, a result that is in contradiction to the prevailing model for permselectivity in nanopores. The extant model fails because it does not account for specific chemical interactions between the cation and the carbonate groups. The nature of these ion-selective interactions is discussed here.

Drug-delivery strategies by using template-synthesized nanotubes.

Encapsulating drugs within hollow nanotubes offers several advantages, including protection from degradation, the possibility of targeting desired locations, and drug release only under specific conditions. Template synthesis utilizes porous membranes prepared from alumina, polycarbonate, or other materials that can be dissolved under specific conditions. The method allows for great control over the lengths and diameters of nanotubes; moreover, tubes can be constructed from a wide variety of tube materials including proteins, DNA, silica, carbon, and chitosan. A number of capping strategies have been developed to seal payloads within nanotubes. Combining these advances with the ability to target and internalize nanotubes into living cells will allow these assemblies to move into the next phase of development, in vivo experiments.

General method for producing organic nanoparticles using nanoporous membranes.

Two liquids are separated by a nanoporous membrane and one liquid is made to flow into the other, causing nanoparticles to be formed at the exits of the nanopores. In particular, we report the generation of nanoparticles of the biodegradable polysaccharide polymer chitosan by placing the chitosan in a low pH aqueous solution that is flowed into a high pH aqueous solution. The size of the nanoparticles (5-20 nm) can be roughly controlled by choosing the size of the nanopores and the pumping rate. In addition, it is possible to load the chitosan nanoparticles with drug molecules, which is demonstrated by incorporation of up to 3.3% rhodamine 6G molecules in the chitosan nanoparticles.

Electroosmotic flow rectification in pyramidal-pore mica membranes.

We demonstrate here a new electrokinetic phenomenon, Electroosmotic flow (EOF) rectification, in synthetic membranes containing asymmetric pores. Mica membranes with pyramidally shaped pores prepared by the track-etch method were used. EOF was driven through these membranes by using an electrode in solutions on either side to pass a constant ionic current through the pores. The velocity of EOF depends on the polarity of the current. A high EOF velocity is obtained when the polarity is such that EOF is driven from the larger base opening to the smaller tip opening of the pore. A smaller EOF velocity is obtained when the polarity is reversed such that EOF goes from tip to base. We show that this rectified EOF phenomenon is the result of ion current-rectification observed in such asymmetric-pore membranes.

An adsorption-based model for pulse duration in resistive-pulse protein sensing.

We have been investigating an electrochemical single-molecule counting experiment called nanopore resistive-pulse sensing. The sensor element is a conically shaped gold nanotube embedded in a thin polymeric membrane. We have been especially interested in counting protein molecules using these nanotube sensors. This is accomplished by placing the nanotube membrane between two electrolyte solutions, applying a transmembrane potential difference, and measuring the resulting ionic current flowing through the nanopore. In simplest terms, when a protein molecule enters and translocates the nanopore, it transiently blocks the ion current, resulting in a downward current pulse. We have found that the duration of such current-pulses are many orders of magnitude longer than the electrophoretic transport time of the protein through the nanotube detection zone. We develop here a simple model that accounts for this key, and previously explained, observation. This model assumes that the protein molecule engages in repeated adsorption/desorption events to/from the nanotube walls as it translocates through the detection zone. This model not only accounts for the long pulse duration but also for the triangular shape of the current pulse and the increase in the standard deviation of the pulse duration with increasing protein size. Furthermore, the results of our analyses are in general agreement with results obtained from other investigations of protein adsorption to surfaces. This includes the observations that smaller proteins stick more readily to the surface but remain adsorbed for shorter times than larger proteins. In addition, the sticking probabilities calculated from our data are in general agreement with results obtained from other methods.

Laser desorption ionization mass spectrometry on silicon nanowell arrays.

This paper describes a new technique for fabrication of nanostructured porous silicon (pSi) for laser desorption ionization mass spectrometry. Porous silicon nanowell arrays were prepared by argon plasma etching through an alumina mask. Porous silicon prepared in this way proved to be an excellent substrate for desorption/ionization on silicon (DIOS) mass spectrometry (MS) using adenosine, Pro-Leu-Gly tripeptide, and [Des-Arg(9)]-bradykinin as the model compounds. It also allows the analyses of complex biological samples such as a tryptic digest of bovine serum albumin and a carnitine standard mixture. Nanowell array surfaces were also used for direct quantification of the illicit drug fentanyl in red blood cell extracts. This method also allows full control of the surface features. MS results suggested that the pore depth has a significant effect on the ion signals. Significant improvement in the ionization was observed by increasing the pore depth from 10 to 50 nm. These substrates are useful for laser desorption ionization in both the atmospheric pressure and vacuum regimes.

Low-Voltage Flow-Through Electroporation Membrane and Method.

Electroporation uses high electric field gradients to create pores within the membrane of living cells in order to deliver a substance, for example a gene, into the cytoplasm. To achieve such gradients, current electroporation devices deliver voltage pulses in the kV range to the cell medium. We describe here a new device based on gold-microtube membranes that can accomplish electroporation with voltage pulses that are orders of magnitude smaller, 4 V. The percentages of electroporated bacteria were found to be more than an order of magnitude higher than obtained with a commercial electroporator.

Chemical Sensing and Chemoresponsive Pumping with Conical-Pore Polymeric Membranes.

Synthetic membranes containing asymmetrically shaped pores have been shown to rectify the ionic current flowing through the membrane. Ion-current rectification means that such membranes produce nonlinear current-voltage curves analogous to those observed with solid-state diode rectifiers. In order to observe this ion-current rectification phenomenon, the asymmetrically shaped pores must have pore-wall surface charge. Pore-wall surface charge also allows for electroosmotic flow (EOF) to occur through the membrane. We have shown that, because ion-current is rectified, EOF is likewise rectified in such membranes. This means that flow through the membrane depends on the polarity of the voltage applied across the membrane, one polarity producing a higher, and the opposite producing a lower, flow rate. As is reviewed here, these ion-current and EOF rectification phenomena are being used to develop new sensing technologies. Results obtained from an ion-current-based sensor for hydrophobic cations are reviewed. In addition, ion-current and EOF rectification can be combined to make a new type of device-a chemoresponsive nanofluidic pump. This is a pump that either turns flow on or turns flow off, when a specific chemical species is detected. Results from a prototype Pb2+ chemoresponsive pump are also reviewed here.

Controlling the length of conical pores etched in ion-tracked poly(ethylene terephthalate) membranes.

An etching procedure that allows for reproducible control of the length of conically shaped pores etched into poly(ethylene terephthalate) (PET) membranes is developed. At the lower etch temperature used (20 degrees C), the length of the pore is found to be linearly related to etch time. At the higher etch temperature (30 degrees C) the etch rate is five times faster and the pores quickly propagate through the entire thickness of the PET membrane. Hence, the lower etch temperature is best for controlling the pore length. Pores etched at this temperature are used to prepare arrays of gold cones where the length of the cones is controlled from 1 to 10 microm. The track-etch rates and the radial-etch rates at both of the etch temperatures used are also reported.

A method for reproducibly preparing synthetic nanopores for resistive-pulse biosensors.

There is increasing interest in using nanopores in synthetic membranes as resistive-pulse sensors for biomedical analytes. Analytes detected with prototype artificial-nanopore biosensors include drugs, DNA, proteins, and viruses. This field is, however, currently in its infancy. A key question that must be addressed in order for such sensors to progress from an interesting laboratory experiment to practical devices is: Can the artificial-nanopore sensing element be reproducibly prepared? We have been evaluating sensors that employ a conically shaped nanopore prepared by the track-etch method as the sensor element. We describe here a new two-step pore-etching procedure that allows for good reproducibility in nanopore fabrication. In addition, we describe a simple mathematical model that allows us to predict the characteristics of the pore produced given the experimental parameters of the two-step etch. This method and model constitute important steps toward developing practical, real-world, artificial-nanopore biosensors.

Developing synthetic conical nanopores for biosensing applications.

In this review we bring together recent results from our group focused towards the development of biosensors from single conically-shaped artificial nanopores. The nanopores, used in the work presented here, were prepared using the track-etch process. The fabrication of track-etched conical nanopores has been optimized to allow for single nanopores with reproducible dimensions to be prepared. We have also demonstrated techniques that allow for easy and controllable manipulation of nanopore geometry (e.g., cone angle). We will consider the ion transport properties of the conical nanopores and factors that affect these properties. Methods for introducing functions that mimic biological ion channels, such as voltage-gating, into these nanopores will also be addressed. Three prototype sensors developed from single conical nanopores will be presented. In the first two sensors, the single conical nanopores function as resistive-pulse sensors and detect the presence of analytes as current-blockade events in the ion current. The third sensor functions in an on/off mode, much like a ligand-gated ion channel. In the presence of a target analyte, the ion current permanently shuts off.

Biofunctionalization and capping of template synthesized nanotubes.

Using alumina templates both nanotubes (open on both ends) and nano test tubes (open on only one end) have been synthesized from many different materials and these have great potential as delivery vehicles for biomedical applications. This review focuses on our recent results directed towards developing "smart" nanotubes for biomolecule delivery applications. While intensive efforts have focused on spherical nanoparticles that are easier to make, cylindrical particles or nanotubes offer many advantages. First, the tunable alumina template allows one to dictate both the pore diameter and length of the nanotube. In addition, template synthesized nanotubes can be differentially functionalized on their inner and outer surfaces. This review highlights these advantages in the contexts of drug extraction and antibody-antigen interactions, the synthesis of protein nanotubes, and recent advances in covalently capped ("corked") nanotubes designed to prevent premature payload leakage. Though diverse applications for nanotubes have already been discovered, many new and exciting paths await exploration.