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.

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.

Resistive-pulse detection of short dsDNAs using a chemically functionalized conical nanopore sensor.

AIMS: To develop nanopore resistive-pulse sensors for the detection of short (50 base-pair [bp] and 100 bp) DNAs. MATERIALS & METHODS: Conically shaped nanopores were chemical etched into polyethylene terphthalate membranes. The as-etched membrane had anionic carboxylate sites on the pore walls. Neutral and hydrophilic ethanolamine functional groups were attached to these carboxylate sites using well-established EDC (1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride) chemistry. RESULTS & DISCUSSION: The ethanolamine-functionalized pores were used to detect 50 and 100 bp DNAs via the resistive-pulse method. The resistive-pulse signature produced by the 50 bp DNA could be distinguished from that of the 100 bp DNA with these sensors. CONCLUSIONS: Attachment of ethanolamine to the carboxylate groups on the pore wall lowered the anionic charge density on the wall. This mitigated the problem of electrostatic rejection of the anionic DNAs from the pore and enabled the detection of these DNA analytes.

Resistive-pulse studies of proteins and protein/antibody complexes using a conical nanotube sensor.

There is increasing interest in using nanopores in synthetic membranes as resistive-pulse sensors for molecular and macromolecule analytes. In general, this method entails measuring current pulses associated with translocation of the analyte through the nanopore sensor element. A key challenge for this sensing paradigm is building selectivity into the protocol so that the current pulses for the target analyte can be distinguished from current pulses for other species that might be present in the sample. We show here that this can be accomplished with a protein analyte by adding to the solution an antibody that selectively binds the protein. We demonstrate this concept using bovine serum albumin (BSA) and a Fab fragment from a BSA-binding polyclonal antibody. Because the complex formed upon binding of the Fab to BSA is larger than the free BSA molecule, the current-pulse signature for the BSA/Fab complex can be easily distinguished from the free BSA. Furthermore, the BSA/Fab pulses can be easily distinguished from the pulses obtained for the free Fab and from pulses obtained for a control protein that does not bind to the Fab. Finally, we also show that the current-pulse signature for the BSA/Fab complex can provide information about the size and stoichiometry of the complex.