Decreasing the Limits of Detection and Analysis Time of Ion Current Rectification Biosensing Measurements via a Mechanically Applied Pressure Differential.

Improving on the analytical capabilities of a measurement is a fundamental challenge with all assays, particularly decreasing the limit of detection while maintaining a practical associated analysis time. Of late, ion current rectification (ICR) biosensing measurements have received a great deal of attention as an analyte-specific, label-free assay. In ICR biosensing, a nanopore coated with an analyte specific binding molecule (e.g., an antibody, an aptamer, etc.) is used to detect a target analyte based on the ability of the target analyte to alter the ICR response of the nanopore upon it binding to the aperture interior. This binding changes the local surface charge and/or size of the nanopore aperture, thus altering its ICR response in a time dependent manner. Here, we report the ability to enhance the transport of a target analyte molecule to and through the aperture of an antibody modified glass nanopore membrane (AMGNM) with the application of a mechanically applied pressure differential. We demonstrate that there is an optimal pressure that balances the flux of the target analyte through the AMGNM aperture with its ability to be bound and detected. Applying the optimal pressure differential allows for picomolar concentrations of the cleaved form of synaptosomal-associated protein 25 (cSNAP-25) to be detected within the same analysis time as micromolar concentrations detected without the use of the pressure differential. The methodology presented here significantly expands the utility of ICR biosensing measurements for detecting low-abundance biomolecules by lowering the limit of detection and reducing the associated analysis time.

Antigen detection via the rate of ion current rectification change of the antibody-modified glass nanopore membrane.

Ion current rectification (ICR), defined as an increase in ion conduction at a given polarity and a decrease in ion conduction for the same voltage at the opposite polarity, i.e., a deviation from a linear ohmic response, occurs in conical shaped pores due to the voltage dependent solution conductivity within the aperture. The degree to which the ionic current rectifies is a function of the size and surface charge of the nanopore, with smaller and more highly charged pores exhibiting greater degrees of rectification. The ICR phenomenon has previously been exploited for biosensing applications, where the level of ICR for a nanopore functionalized with an analyte-specific binding molecule (e.g., an antibody, biotin, etc.) changes upon binding its target analyte (e.g., an antigen, streptavidin, etc.) due to a resulting change in the size and/or charge of the aperture. While this type of detection measurement is typically qualitative, for the first time, we demonstrate that the rate at which the nanopore ICR response changes is dependent on the concentration of the target analyte introduced. Utilizing a glass nanopore membrane (GNM) internally coated with a monoclonal antibody specific to the cleaved form of synaptosomal-associated protein 25 (cSNAP-25), creating the antibody-modified glass nanopore membrane (AMGNM), we demonstrate a correlation between the rate of ICR change and the concentration of introduced cSNAP-25, over a range of 500 nM-100 µM. The methodology presented here significantly expands the applications of nanopore ICR biosensing measurements and demonstrates that these measurements can be quantitative in nature.

Monitoring the escape of DNA from a nanopore using an alternating current signal.

We present the use of an alternating current (AC) signal as a means to monitor the conductance of an alpha-hemolysin (alphaHL) pore as a DNA hairpin with a polydeoxyadenosine tail is driven into and released from the pore. Specifically, a 12 base pair DNA hairpin attached to a 50-nucleotide poly-A tail (HP-A(50)) is threaded into an alphaHL channel using a DC driving voltage. Once the HP-A(50) molecule is trapped within the alphaHL channel, the DC driving voltage is turned off and the conductance of the channel is monitored using an AC voltage. The escape time, defined as the time it takes the HP-A(50) molecule to transport out of the alphaHL channel, is then measured. This escape time has been monitored as a function of AC amplitude (20 to 250 mV(ac)), AC frequency (60-200 kHz), DC drive voltage (0 to 100 mV(dc)), and temperature (-10 to 20 degrees C), in order to determine their effect on the predominantly diffusive motion of the DNA through the nanopore. The applied AC voltage used to monitor the conductance of the nanopore has been found to play a significant role in the DNA/nanopore interaction. The experimental results are described by a one-dimensional asymmetric periodic potential model that includes the influence of the AC voltage. An activation enthalpy barrier of 1.74 x 10(-19) J and a periodic potential asymmetry parameter of 0.575 are obtained for the diffusion at zero electrical bias of a single nucleotide through alphaHL.

Sensitivity and signal complexity as a function of the number of ion channels in a stochastic sensor.

Alternating current, phase-sensitive stochastic detection using between 1 and 26 alpha-hemolysin ion channels reconstituted in a lipid bilayer, suspended over a 160-nm-radius orifice glass nanopore, is reported. As predicted by the binomial distribution, simultaneous analyte detection at large numbers of channels is effectively zero, independent of the number of ion channels. The results indicate that alphaHL channels are noninteracting and that significant gains in sensitivity are possible without sacrificing the simplicity of single-molecule detection strategies.

AC conductance of transmembrane protein channels. The number of ionized residue mobile counterions at infinite dilution.

Simultaneous measurements of the AC and DC conductances of alpha-hemolysin (alphaHL) ion channels and outer membrane protein F (OmpF) porins in dilute ionic solutions is described. AC conductance measurements were performed by applying a 10 mV rms AC voltage across a suspended planar bilayer of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine in the absence and presence of the protein and detecting the AC current response using phase-sensitive lock-in techniques. The conductances of individual alphaHL channels and OmpF porins were measured in symmetric KCl solutions containing between 5 and 1000 mM KCl. The AC and DC conductances of each protein were in agreement for all solution conditions, demonstrating the reliability of the AC method in single-channel recordings. Linear plots of conductance versus bulk KCl concentration for both proteins extrapolate to significant nonzero conductances (0.150 +/- 0.050 nS and 0.028 +/- 0.008 nS for OmpF and alphaHL, respectively) at infinite KCl dilution. The infinite dilution conductances are ascribed to mobile counterions of the ionizable residues within the protein lumens. A method of analyzing the plots of conductance vs KCl concentration is introduced that allows the determination of the concentration of mobile counterions associated with ionizable groups without knowledge of either the protein geometry or the ion mobilities. At neutral pH, an equivalent of 3 mobile counterions (K+ or Cl-) is estimated to contribute to the conductivity of the alphaHL channel.

What controls open-pore and residual currents in the first sensing zone of alpha-hemolysin nanopore? Combined experimental and theoretical study.

The electrophoretic transport of single-stranded DNA through biological nanopores such as alpha-hemolysin (αHL) is a promising and cost-effective technology with the potential to revolutionize genomics. The rational design of pores with the controlled polymer translocation rates and high contrast between different nucleotides could improve significantly nanopore sequencing applications. Here, we apply a combination of theoretical and experimental methods in an attempt to elucidate several selective modifications in the pore which were proposed to be central for the effective discrimination between purines and pyrimidines. Our nanopore test set includes the wild type αHL and six mutants (E111N/M113X/K147N) in which the cross-section and chemical functionality of the first constriction zone of the pore are modified. Electrophysiological recordings were combined with all-atom Molecular Dynamics simulations (MD) and a recently developed Brownian Dynamics (BROMOC) protocol to investigate residual ion currents and pore-DNA interactions for two homo-polymers e.g. poly(dA)40 or poly(dC)40 blocking the pore. The calculated residual currents and contrast in the poly(dA)40/poly(dC)40 blocked pore are in qualitative agreement with the experimental recordings. We showed that a simple structural metric allows rationalization of key elements in the emergent contrast between purines and pyrimidines in the modified αHL mutants. The shape of the pore and its capacity for hydrogen bonding to a translocated polynucleotide are two essential parameters for contrast optimization. To further probe the impact of these two factors in the ssDNA sensing, we eliminated the effect of the primary constriction using serine substitutions (i.e. E111S/M113S/T145S/K147S) and increased the hydrophobic volume of the central residue in the secondary constriction (L135I). This pore modification sharply increased the contrast between Adenine (A) and Cytosine (C).

Creating a Single Sensing Zone within an Alpha-Hemolysin Pore Via Site Directed Mutagenesis.

Although significant progress has recently been made towards realizing the goal of direct nanopore based DNA sequencing [1], there are still numerous hurdles that need to be overcome. One such hurdle associated with the use of the biological nanopore α-hemolysin (αHL) is the fact that the wild type channel contains three very distinct recognition or sensing regions within the ß-barrel [2, 3], making identification of the bases residing within or moving through the pore very difficult. Through site directed mutagenesis, we have been able to selectively remove one of two sensing regions while simultaneously enhancing the third. Our approach has led to the creation of αHL pores containing single sensing zones and provides the basis for engineering αHL pores suitable for direct DNA sequencing.

Simultaneous alternating and direct current readout of protein ion channel blocking events using glass nanopore membranes.

Alternating current (ac) phase-sensitive detection is used to measure the conductance of the ion channel alpha-hemolysin (alphaHL), while simultaneously applying a direct current (dc) bias to electrostatically control the binding affinity and kinetics of charged molecules within the protein lumen. Ion channel conductance was recorded while applying a 10-20 mV rms, 1-2 kHz bias across a single alphaHL protein inserted in a 1,2-diphytanoyl-sn-glycero-3-phosphocholine lipid bilayer that is suspended across the orifice (100-500 nm radius) of a glass nanopore membrane. Step changes in the ac ion channel conductance with a temporal response (t(10-90)) of 1.5 ms and noise amplitude of approximately 2 pA were obtained using a low-noise potentiostat and a lock-in amplifier. These conditions were used to monitor the reversible and stochastic binding of heptakis-(6-O-sulfo)-beta-cyclodextrin and a nine base pair DNA hairpin molecule to the ion channel. Alternating current methodology allows the binding kinetics and affinity between the protein ion channel and analyte to be investigated as a function of the dc bias, including ion channel conductance measurements in the absence of a dc bias.

Single ion-channel recordings using glass nanopore membranes.

Protein ion-channel recordings using a glass nanopore (GNP) membrane as the support structure for lipid bilayer membranes are presented. The GNP membrane is composed of a single conical-shaped nanopore embedded in a approximately 50 microm-thick glass membrane chemically modified with a 3-cyanopropyldimethylchlorosilane monolayer to produce a surface of intermediate hydrophobicity. This surface modification results in lipid monolayer formation on the glass surface and a lipid bilayer suspended across the small orifice (100-400 nm-radius) of the GNP membrane, while allowing aqueous solutions to fully wet the glass nanopore. The GNP membrane/bilayer structures, which exhibit ohmic seal resistances of approximately 70 GOmega and electrical breakdown voltages of approximately 0.8 V, are exceptionally stable to mechanical disturbances and have lifetimes of at least 2 weeks. These favorable characteristics result from the very small area of bilayer (10(-10)-10(-8) cm(2)) that is suspended across the GNP membrane orifice. Fluorescence microscopy and vibrational sum frequency spectroscopy demonstrate that a lipid monolayer forms on the 3-cyanopropyl-dimethylchlorosilane modified glass surface with the lipid tails oriented toward the glass. The GNP membrane/bilayer structure is well suited for single ion-channel recordings. Reproducible insertion of the protein ion channel, wild-type alpha-hemolysin (WTalphaHL), and stochastic detection of a small molecule, heptakis(6-O-sulfo)-beta-cyclodextrin, are demonstrated. In addition, the insertion and removal of WTalphaHL channels are reproducibly controlled by applying small pressures (-100 to 350 mmHg) across the lipid bilayer. The electrical and mechanical stability of the bilayer, the ease of which bilayer formation is achieved, and the ability to control ion-channel insertion, coupled with the small bilayer capacitance of the GNP membrane-based system, provide a new and nearly optimal system for single ion-channel recordings.

Bench-top method for fabricating glass-sealed nanodisk electrodes, glass nanopore electrodes, and glass nanopore membranes of controlled size.

A simple benchtop method of fabricating glass-sealed nanometer-sized Au and Pt disk electrodes, glass nanopore electrodes, and glass nanopore membranes is reported. The synthesis of all three structures is initiated by sealing the tips of electrochemically sharpened Au and Pt microwires into glass membranes at the end of a soda lime or lead glass capillary. Pt and Au nanodisk electrodes are obtained by hand polishing using a high-input impedance metal oxide semiconductor field effect transistor (MOSFET)-based circuit to monitor the radius of the metal disk. Proper biasing of the MOSFET circuit, based on a numerical analysis of the polishing circuit impedance, allows for the reproducible fabrication of Pt disk electrodes of radii as small as 10 nm. Significantly smaller background currents in voltammetric measurements are obtained using lead glass capillaries, a consequence of the lower mobility of Pb(2+) (relative to Na(+)) in the glass matrix. Glass nanopore electrodes and glass nanopore membranes are fabricated, respectively, by removal of part or all of the metal sealed in the glass membranes. The nanostructures are characterized by atomic force microscopy, steady-state voltammetry, and ion conductivity measurements.

Ionic conductivity of the aqueous layer separating a lipid bilayer membrane and a glass support.

The in-plane ionic conductivity of the approximately 1-nm-thick aqueous layer separating a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) bilayer membrane and a glass support was investigated. The aqueous layer conductivity was measured by tip-dip deposition of a POPC bilayer onto the surface of a 20- to 75-microm-thick glass membrane containing a single conical-shaped nanopore and recording the current-voltage (i-V) behavior of the glass membrane nanopore/POPC bilayer structure. The steady-state current across the glass membrane passes through the nanopore (45-480 nm radius) and spreads radially outward within the aqueous layer between the glass support and bilayer. This aqueous layer corresponds to the dominant resistance of the glass membrane nanopore/POPC bilayer structure. Fluorescence recovery after photobleaching measurements using dye-labeled lipids verified that the POPC bilayer maintains a significant degree of fluidity on the glass membrane. The slopes of ohmic i-V curves yield an aqueous layer conductivity of (3 +/- 1) x 10(-3) Omega(-1) cm(-1) assuming a layer thickness of 1.0 nm. This conductivity is essentially independent of the concentration of KCl in the bulk solution (10-4 to 1 M) in contact with the membrane. The results indicate that the concentration and mobility of charge carriers in the aqueous layer between the glass support and bilayer are largely determined by the local structure of the glass/water/bilayer interface.