EP2986975A1 - Controlling nanobubble and nanoparticle dynamics in conical nanopores - Google Patents

Abstract

Systems and methods for detect, manipulate and characterize nanobubbles and nanoparticles in solution. These comprise use of a conical-shaped nanopore having a nanoparticle or nanobubble sensing zone configured to be in communication with a saline solution, and having a half-cone angle sufficiently small to provide for significant channel-like character at the nanopore. An electrode(s) is used to apply a voltage/potential across the nanopore to provide electrophoretic force (EPF) across the nanopore, the nanopore is subjected to an electroosmotic force (EOF), pressure is applied/adjusted across the nanopore, and wherein adjusting at least one parameter selected from the group consisting of EPF, EOF, and pressure across the nanopore, provides for fine control of particle or nanobubble translocation velocities across the sensing zone of the nanopore (e.g., by shifting the zero velocity point to an applied voltage/potential to provide for an acceptable signal-to-noise ratio).

Description

CONTROLLING NANOBUBBLE AND NANOP ARTICLE DYNAMICS IN

CONICAL NANOPORES

CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No.

61/812,791, filed April 17, 2013, the contents of which are incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

Aspects of the invention generally relate to systems and methods for manipulating and characterizing nanobubbles and nanoparticles in solution, and more particularly related to systems and methods comprising use of conical-shaped nanopores (e.g., glass, or other suitable material) having a sensing zone with a half-cone angle sufficiently small to provide for significant channel-like character at the nanopore, wherein adjusting at least one parameter selected from electrophoretic force (EPF), electroosmotic force (EOF), and pressure across the nanopore, provides for fine control of nanoparticle or nanobubble translocation velocities across the sensing zone of the nanopore (e.g., by shifting the zero velocity point to an applied voltage/potential to provide for an acceptable signal-to-noise ratio) to provide methods detecting, manipulating and characterizing nanobubbles and nanoparticles in solution.

BACKGROUND OF THE INVENTION

Characterization of the geometry, charge, and dynamic properties of individual nanoscale objects in bulk solution presents a significant challenge, particularly for objects at the lower end of the scale. Transmission electron microscopy (TEM) does not assess particles in bulk solution, and dynamic light scattering (DLS) does not provide information about individual particles. Furthermore, these techniques are prone to artifacts (Domingos, R., et al, Environ. Sci. Technol 43:7277, 2009). By contrast, nanopores provide a method that measures individual nanoscale particles in bulk solution as well as providing information about particle charge. Recent adaptations of the Coulter-counter technique to the nanoscale range have been used as a label-free method for studying biological molecules, especially DNA, and nanoparticles having a variety of compositions and surface charges (DeBlois, R. W., et al., Journal of Colloid and Interface Science 61 :323-335, 1977). In these techniques, an electrical potential difference is applied between the electrodes on the two sides of a nanopore. Nanoparticles passing through the pore cause a brief decrease in the electrical current plotted as a function of time. The duration, magnitude, and shape of these current-time profiles provide a wealth of information about the variety of forces that act on the nanoparticles as they pass through the pore (Domingos, R., et al, Environ. Sci. Technol 43:7277, 2009; and Lan, W.-J., et al, Anal. Chem 83:3840-3847, 2011). However, large particle velocities can limit/exclude the application of nanopore techniques to a significant portion of the nanoscale range.

Reliable detection and characterization of small nanoparticles is limited by Electronic filtering, which for typical bandwidths of 10kHz leads to underestimation of peak heights for detectable particles and can even entirely miss particles below 40 nm for certain pore geometries (Lan, W.-J., et al, Anal. Chem 83:3840-3847, 2011). Innovative attempts to overcome the problem of excessive translocation speed include chemical modification of pores (Wu, H.-C, et al, J. Am. Chem. Soc 129: 16142-16148, 2007) and variations in pore size (Wanunu, M., et al, Biophysical Journal 95:4716-4725, 2008), shape (Wanunu, M., et al, Nature Nanotech 5:807-814, 2010), salt concentration, temperature, and solution viscosity (Fologea, D., et al, Nano Letters 5: 1734-1737, 2005), as well as employing repeated measurements of individual particles (Berge, L. I., et al, Review of Scientific Instruments 60:2756, 1989; and Gershow & Golovchenko, Nature Nanotech 2:775-779, 2007). By varying pH to adjust the difference in zeta potential between the particle and the pore, Firnkes et al. were able to manipulate the effective velocity of a single protein and reverse the translocation driving force from electrophoretic to electroosmotic (Firnkes, M., et al, Nano Letters 10:2162-2167, 2010). While this method provides an important step forward in controlling particle speed, significant diffusion rates across the 10-nm wide pore yet reduce signal fidelity.

Cylindrical carbon nanotubes (Lan, W.-J., et al, Anal. Chem 83:3840-3847, 2011) and glass nanochannels (Ito, T., et al, Anal. Chem 75:2399-2406, 2003) have been used to characterize 60-nm and 40-nm particles, respectively, but measurement of smaller particles was hindered by low signal-to-noise ratios. By contrast, focusing of the sensing zone in conical nanopores to a much smaller volume imparts many advantages including high signal-to-noise ratios and asymmetric peak shapes, which provide information about translocation direction (Wu, H.-C, et al, J. Am. Chem. Soc 129: 16142-16148, 2007). Recently, Vogel et al. reported a method for characterizing the surface charge of 200-nm particles based upon resistive pulse sensing in conical nanopores under variable pressure (Vogel, R., et al, Anal. Chem 84:3125-3131, 2012). The elastomeric pores used in these studies have the advantage that they can be dynamically varied in size; however, the hydrophobic nature of this pore material may lead to undesirable interactions with hydrophobic analytes and solvents other than water. By contrast, the hydrophilic surfaces of silicon nitride (SiN) and glass nanopores (GNPs) are often desirable for studies involving both hydrophobic and hydrophilic analytes. SiN-based nanopores are frequently used since they have the advantage that one can readily measure their pore size during manufacturing. Despite this advantage, SiN chip type pores have limited usefulness for nanoparticles below 40 nm due to noise problems, and their manufacturing process is complex and expensive.

By contrast, simple and inexpensive methods exist for producing GNPs that can detect molecules as small as 1.5 nm (Gao, C, et al, Anal. Chem 81 :80-86, 2009; and Li, G.-X., et al, Chinese Journal of Analytical Chemistry 38: 1698-1702, 2010). In addition to hydrophilicity, GNPs have numerous advantages compared to other types of pores in terms of exceptional electrical properties for high bandwidth measurements, ability to withstand high pressure, compatibility with optical measurements, chemical stability, and the possibility to modify their surface with a variety of functional groups. Gao et al. reduced particle velocities sufficiently to detect 10-nm gold nanoparticles by producing GNPs near the threshold at which the particle could pass through (Gao, C, et al., Anal. Chem 81 :80-86, 2009). Though inadequate for general control of particle dynamics, this approach did provide a method for determining pore size, which was not possible using electron microscopy (Gao, C, et al, Anal. Chem 81 :80-86, 2009; and Li, G.-X., et al, Chinese Journal of Analytical Chemistry 38: 1698-1702, 2010).

Nanobubbles. There is no information or suggestion in the art as to whether nanopores (e.g., GNPs) would have any utility for characterization of the geometry, charge, and dynamic properties of nanobubbles of any size.

SUMMARY OF THE INVENTION

Aspects of the present invention relate to controlling the forces acting on a nanoparticle or nanobubble as it passes through a nanopore.

In certain aspects, the velocity of a nanoparticle (e.g., Au nanoparticle) or nanobubble can be controlled over 3 orders of magnitude by balancing the pressure, electrophoretic and electroosmotic forces acting on it.

According to particular aspects, the nanoparticle or nanobubble velocity can be controlled with high precision by adjusting the voltage across the nanopore, allowing observation of nanoparticle or nanobubble translocation that would normally be too rapid to be observed by applying either an electrical or pressure force alone.

According to additional aspects, cancellation of the electrical and pressure forces allows observation of the random motion of a nanoparticle or nanobubble as it moves through the nanopore.

According to further aspects, these fundamental results are unprecedented, and have substantial utility in applications for studying particle or nanobubble dynamics, and in the analysis of nanoparticles and nanobubbles, and the conclusions provide for a wide range of applications for characterization of nanoparticles, nanobubbles and macromolecules.

According to particular aspects, the threshold condition is further investigated, with demonstrated control of velocities over three orders of magnitude for exemplary nanoparticles (e.g., 8-nm nanoparticles) or nanobubbles in glass nanopores (GNPs).

Additional aspects provide a rationale for controlling nanoparticle or nanobubbles dynamics by balancing the pressure, electrophoretic (EPF), and electroosmotic (EOF) forces (Figure 1), wherein this balance of three forces provides for previously unattainable control over particle dynamics in a conical pore.

In particular aspects, fine control of particle velocities is provided by taking advantage of electroosmosis and by applying a constant pressure to shift the zero velocity point to a potential with acceptable signal-to-noise ratio.

Further aspects provide finite element analysis (FEA) simulations that model the experimental results.

Additional aspects provide insights into pore geometry, spatial distribution of nanoparticle and nanobubble velocities within the pore, and the influence of both the nanoparticle or nanobubble surface, and the pore surface charge densities.

Preferred aspects provide systems and methods for detecting, manipulating and characterizing nanobubbles and nanoparticles in solution, comprising: providing a saline solution having nanoparticles or nanobubbles; providing a conical-shaped nanopore having a nanopore diameter, a proximal end, a distal end in communication with the saline solution, and having a nanoparticle or nanobubble sensing zone between the proximal and the distal ends, and wherein the sensing zone of the conical-shaped nanopore has a half- cone angle sufficiently small to provide for significant channel-like character at the nanopore; applying, using an electrode, a voltage/potential across the nanopore to provide an electrophoretic force (EPF) across the nanopore; subjecting the nanopore to an electroosmotic force (EOF); applying a pressure across the nanopore; and adjusting at least one parameter selected from EPF, EOF, and pressure across the nanopore, to provide for fine control of particle or nanobubble translocation velocities across the sensing zone of the nanopore (e.g., by shifting the zero velocity point to an applied voltage/potential to provide for an acceptable signal-to-noise ratio), to provide a method for detecting, manipulating and characterizing nanobubbles and nanoparticles in solution.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a diagram showing, according to particular exemplary aspects of the present invention, driving forces that, on balance, determine the velocity of nanoparticle translocation through an exemplary glass conical nanopore.

Figures 2A and 2B are, according to particular exemplary aspects, illustrations of the tip of a glass micropipette pulled, using a programmable micropipette puller, to form a narrow ~1-μιη opening that was melted to produce a terminal bulb enclosing a cone- shaped cavity (A). The terminal bulb was sanded and briefly melted with a microforge to form a flattened geometry (dashed lines delineate the outlines of the original bulb shown in (A)). Ag/AgCl electrodes were placed across the unopened pore and hydrofluoric acid etchant was used as the external solution to form a nano-scale pore in the sanded and remelted tip (B). Pore formation was detected by a spike in the current.

Figure 3 shows, according to particular exemplary aspects, three Scanning electron microscope (SEM) images of the type of nanopore used in this study (this particular pore is larger than the ones used to detect 8-nm nanoparticles). A pipette having a resistance of 39 ΜΩ measured in 1.0 M NaCl was sputtered with an ~7 nm thick layer of gold prior to SEM imaging. The arrow in the lowest panel marks the location of the nanopore, which has a diameter of ~70 nm. Although these images show an external well surrounding the actual pore opening, we believe that, due to its large width, the well does not influence the nanopore-sensing zone.

Figures 4A and 4B show, according to particular exemplary aspects, current versus time (i-t) traces used to determine when the Au nanoparticle size exceeds or is just at the threshold of the pore size.

Figures 5A, 5B, 5C, and 5D show, according to particular exemplary aspects, i-t traces showing a single nanoparticle repeatedly going in and out of a nanopore as the applied potential is reversed. Figures 6A and 6B show, according to particular exemplary aspects, nanoparticle translocation velocity vs. applied voltage under -0.047 atm (A) and -0.35 atm pressure (B).

Figure 7 illustrates, according to particular exemplary aspects, the contributions to the effective velocity made by the applied pressure, electrophoresis (v = ε/τ\ξ_ρΆΐύ(:ι_βΕ) and electroosmosis (v = -ε/τ ξ_ρ0ΙβΕ) for the experiments represented in Figure 6 A.

Figures 8A-8C illustrate, according to particular exemplary aspects, (A) simulated velocity profile for a nanoparticle (ζ = -15 mV) in a 0.2 M NaCl solution, at 0.35 atm pressure and applied voltages between 100 and 500 mV corresponding to the turquoise lines in Figure 6B and Figure 8C. (B) and (C) are plots of particle velocities corresponding to the data in Figures 6 A and 6B, respectively. The data point colors and symbols follow the same scheme used to plot experimental data in Figures 6 A and 6B. Parameters and other details of the finite-element simulation are presented in the SI file.

Figure 8D illustrates, according to particular exemplary aspects, the contributions to the effective velocity made by the applied pressure, electrophoresis (v = e/T| paiticieE) and electroosmosis (v = -ε/τ ξ_ρ0ΙβΕ) for a ζ = -15 mV nanoparticle.

Figures 9A and 9B show, according to particular exemplary aspects, forward and reverse translocation of three nanoparticles as a function of the applied pressure (current versus time traces for three nanoparticles reversing direction due to applied pressure).

Figure 10 shows a simulated potential profile generated by a -9 mC/m² charged Au nanoparticle with a diameter of 8 nm.

Figure 11 shows geometry and boundary conditions for the finite-element simulation in a 0.1 M or 0.2 M NaCl solution and P = 0.35 atm.

Figure 12 shows geometry and boundary conditions for the finite-element simulation in 1M NaCl and 0.047 atm.

DETAILED DESCRIPTION OF THE INVENTION

Conical nanopores are a powerful tool for characterizing nano-scale particles and even small molecules. Although the technique provides a wealth of information, such pores are limited in their ability to investigate particle dynamics below ~40 nm due to high particle velocities through a very short sensing zone, and nothing is known or suggested in the art about whether conical nanopores would be efficacious characterizing nanobubbles (e.g., air, oxygen or other gas(es) nanobubbles). Particular aspects provide methods for detecting, manipulating and characterizing nanobubbles and nanoparticles in solution, comprising balancing electrokinetic forces acting on nanoparticles (e.g., 8 nm or in the range of, for example 3 to 40 nm diameter ) by application of pressure to provide for sufficient slowing of the particle velocity to enable detection and characterization.

According to additional aspects, nanoparticles having different zeta potentials were studied in conical nanopores by varying salt concentration, applied pressure, and potential to reveal the point at which forces are balanced and the particles reverse direction through the nanopore, and adjusting these conditions allows the characterization of nano particles and their dynamics down to the smallest end of the nanoscale range.

According to preferred aspects, the methods are applicable for detecting, manipulating and characterizing nanobubbles in solution.

According to particular aspects, the velocity of 8-nm nanoparticles (e.g., 8 nm, or in the range of X to Y nm) can be controlled in glass conical nanopores by applying pressure and varying voltage to balance electrophoretic (EPF) and electroosmotic (EOF) forces.

Figure 1 is a diagram showing, according to particular exemplary aspects of the present invention, driving forces that on balance determine the velocity of particle (or nanobubble) translocation through an exemplary conical nanopore. For example, during translocation experiments, as described herein, positive potentials applied to an electrode within the pipette and negative pressures applied within the pipette both tend to draw negatively charged particles inward from the external solution. The applied potential also induces a counteracting electroosmotic force that tends to drive particles out of the pipette into the external solution. The balance of these different forces determines the velocity of particle translocations. This fine control allows characterization of particles in a previously unattainable size range, and is also applicable to nanobubbles, including very small nanobubbles (e.g., less than lOOnm, less than 50nm, less than lOnm, less than 5nm, and less than 2 nm in diameter). EXAMPLE 1

(Materials and Methods)

Materials. Spherical gold nanoparticles (diameter: 8 nm ± 7%, SD, measured by TEM) conjugated with a carboxy methyl polymer were purchased from Nanopartz, Inc. (Love land, CO). Zeta potentials were measured as -51 mV and -15 mV (Nanopartz) and as -52 mV and -22 mV (Particle Characterization Laboratories, Inc., Novato, CA) in deionized water, and as -38 mV and -12 mV in 0.1 NaCl PBS pH 7.4 plus 0.1% Triton X- 100 (Particle Characterization Laboratories, Inc.). Attempts to measure zeta potentials at higher salt concentrations yielded irreproducible values. The particles are denoted as -51 mV and -15 mV in the text even though ζ values are lower in salt solutions. Other materials included borosilicate glass micropipettes (OD: 1.5 mm, ID: 0.86 mm, Length: 10 cm, Sutter Instruments), hydrofluoric acid (48%), ammonium fluoride solution (~40%>), ammonium fluoride etching mixture (AF 875-125, Sigma), pH 7.4 phosphate buffered saline (PBS) 10X (Invitrogen), 3M 12 micron Lapping Film (Ted Pella), Triton-XlOO (Amresco), 0.25 mm Ag wire (World Precision Instruments), household bleach (5%> hyprochorite) and sodium bicarbonate (Costco). Solutions were filtered through Millex- GP, 0.22 μιη, polyethersulfone filters (Millipore).

Pipettes. Pipettes were pulled with a Model P-1000 Flaming/Brown micropipette puller (Sutter Instruments) to a ~1 μιη opening. Pulled pipette tips were then melted with a butane hand torch (flame tip positioned -5 mm from the tip) for -130 ms as the pipettes rotate by on a turntable at 3.5 cm/s. Sanding of the resulting terminal bulb was carried out by hand prior to microforge heating, which involved placing the pipette tip within a Ω- shaped platinum-iridium alloy filament (5 mm x 5 mm) heating element made from a 5- mm wide platinum/iridium strip for -400 ms. Pipette tips were initially imaged using an inverted Olympus 1X50 microscope, and then a few were selected for imaging with a FEI Sirion SEM.

Glass nanopore fabrication. Borosilicate glass micropipettes were heated at 600°C for 12 h and then immediately sealed at both ends. After being pulled to -1 μιη opening, they were kept under a stream of dry nitrogen until the sharp tip was melted. The terminal bulb inclosing a conical cavity was then sanded to a flattened tip using fine sandpaper followed by microforge heating. Just prior to etching, the other end of the pipette was opened, fire polished, and backfilled with 1.0 M NaCl. Ag/AgCl electrodes where prepared by immersing an Ag wire in bleach for -15 min prior to experiments, and were placed inside multiple pipettes connected in parallel as well as the etchant solution (a 1 :2 dilution of 48% hydrofluoric acid in a -40% ammonium fluoride solution). Pore formation was indicated by a jump in current measured using a Princeton Applied Research 2273 PARS TAT potentiostat operating in current vs. time mode with 250 mV applied potential. Pipette tips were immediately dipped into 3.0 M KOH for 10 s and transferred to a 1.0 M NaCl solution for current measurements. Pores having resistances between 100 and 200 ΜΩ were routinely made in this way, etched to larger sizes as needed by dipping briefly (15 s) into a 1 :20 dilution of Ammonium fluoride etching mixture (AF 875-125), and repeating the etching process until threshold translocations no longer occurred.

Resistive Pore Sensing Measurements and Data Analysis. Pipettes were placed into a BNC style electrode holder that allowed for application of pressure within the pipette (Warner Instruments), and current measurements made using a HEKA EPC-10 amplifier at a cutoff frequency of 3 kHz applied with a three-pole Bessel low-pass filter. PATCHMASTER data acquisition software was used to initially analyze and export current-time traces. A custom VBA Excel program was used to determine translocation peak parameters such as peak position, height, and width at half-height as a function of applied voltage. Each peak was inspected manually to ensure accurate measurements; in general, resistive pulses having a signal-to-noise ratio of less than 7: 1 and/or a base width of less than 1 ms were excluded.

Finite-element simulations. The finite-element simulations were performed using

COMSOL Multiphysics 3.5 (Comsol, Inc.) on a high performance desktop PC.

EXAMPLE 2

(Micropipette-based Glass Nanopores were fabricated) Glass micropipettes were pulled to a ~1-μιη opening and the pipette tips were then melted with a butane hand torch to produce a terminal bulb (Figure 2A), similar to the method of Gao et al.¹³ When applying etchant to the terminal bulb of a micropipette to expose the enclosed cavity, there is a tendency for a sharpened tip to form,¹⁵ which can result in pore formation at points other than the tip. Wax coating of the sides of the tip has been used to avoid this problem,¹³ but we found that we can exert better control over the etching process by taking steps to produce a flattened tip geometry prior to etching. To achieve this (Figure 2B), the pipettes were sanded and the tip briefly remelted. Although sanding alone can be used to produce nanopores,¹⁴ we have found inconsistent results with pores produced in this way. Speculating that sanding may introduce small cracks, we chose to stop sanding well before opening the pore. A setup with hydrofluoric acid etchant as the external solution was used to form a nano-scale pore in the sanded and remelted tip. Pore formation was detected by a spike in the current.

Specifically, Figures 2A and 2B are, according to particular exemplary aspects, illustrations of the tip of a glass micropipette pulled, using a programmable micropipette puller, to form a narrow ~1-μιη opening that was melted to produce a terminal bulb enclosing a cone-shaped cavity (A). The terminal bulb was sanded and briefly melted with a microforge to form a flattened geometry (dashed lines delineate the outlines of the original bulb shown in (A)). Ag/AgCl electrodes were placed across the unopened pore and hydrofluoric acid etchant was used as the external solution to form a nano-scale pore in the sanded and remelted tip (B). Pore formation was detected by a spike in the current.

EXAMPLE 3

(Exemplary Micropipette-based Glass Nanopores were imaged) Glass micropipettes were prepared by a modification of the method described by

Gao et al (Anal. Chem 81 :80— 86, 2009) (Figures 2A and 2B). Determining the size of GNPs is not simple. Indeed, others have reported being unable to image similar nanopores in the tip of glass pipettes using SEM (Vogel, R., et al, Anal. Chem 84:3125-3131, 2012; and Gao, C, et al, Anal. Chem 81 :80— 86, 2009). In particular aspects, for example, the pore size of one of our larger (70-nm) pores (Figure 3) was measured. Based on the microscopic images (Figures 2A and 2B) and the characteristic asymmetric translocation profiles and FEA simulations, vide infra, the inner pore geometry is conical, with a ~2° cone angle (White & Bund, Langmuir 24:2212-2218, 2008).

Specifically, Figure 3 shows, according to particular exemplary aspects, three Scanning electron microscope (SEM) images of the type of nanopore used in this study (this particular pore is larger than the ones used to detect 8-nm nanoparticles). A pipette having a resistance of 39 ΜΩ measured in 1.0 M NaCl was sputtered with an ~7 nm thick layer of gold prior to SEM imaging. The arrow in the lowest panel marks the location of the nanopore, which has a diameter of ~70 nm. Although these images show an external well surrounding the actual pore opening, we believe that, due to its large width, the well does not influence the nanopore-sensing zone.

EXAMPLE 4

(Nanoparticles were detected at the Threshold of the Pore Size) Identifying the size of a particle at the threshold of passing through the pore provides an alternative to SEM imaging for sizing micropipette-based GNPs. Experiments were performed to detect nanoparticle translocations using exemplary 8-nm diameter carboxy methyl polymer-coated Au nanoparticles having small standard deviation in size (± 0.6 nm), at a typical concentration of 200 nM in a 1.0 M NaCl solution. Current vs. time (i-t) traces were recorded while a positive potential was applied to an Ag/AgCl wire electrode within the micropipette relative to the external solution. Very small pores (having a resistance between 100 and 200 ΜΩ, measured in 1.0 M NaCl) were initially produced, and repeatedly widened with dilute etchant until we detected pressure driven nanoparticle translocations (e.g., triggered by applying negative pressure within the pipette).

This approach enabled us to detect cases in which square blocks were terminated with a sharp spike at the end as illustrated by the 17 pA current block in Figure 4B, which ends with a 70 pA peak before returning to the base current. Since this terminal spike is large and has characteristics of a typical translocation, it likely represents a particle passing through the pore after an initial partial blockade of the opening. Vercoutere et al. observed similar long shallow blockades caused by individual hairpin DNA molecules prior to a rapid deep blockage indicating translocation of the DNA through an a- hemolysin pore (Vercoutere, W., et al, Nature Biotechnology 19:248-252, 2001). Though the geometrical considerations for gold nanoparticles are much simpler, it is possible that the particle coating requires time to compress in order for the particle to fit through the pore at the threshold size. Gao et al., also used the threshold condition to estimate the size of their pores using DNA, 10-nm gold nanoparticles, and even single molecules of β- cyclodextrin, based on simple square-shaped blocks lacking a terminal spike (Gao, C, et al, Anal. Chem 81 :80-86, 2009). Based upon repeated observations of this kind, it is concluded that the occurrence of square blockages without a sharp spike at the end represent transient blockages, Figure 4A, of the nanopore orifice by the Au nanoparticles, but without successful translocation.

Figures 4A and B show, according to particular exemplary aspects, current versus time (i-t) traces used to determine when the Au nanoparticle size exceeds or is just at the threshold of the pore size. In these experiments, 8-nm Au nanoparticles (ζ = -51 mV) were placed in the external solution, and a pressure of -0.5 atm and voltage of 250 mV were applied to drive the particles into the nanopore. (A) Square-shaped blockades of widely varying duration are observed when the particle size exceeds the pore size. The current within these blocks sometimes increases briefly, as seen at 0.59 s and 0.68 s, but eventually returns to the base current level as seen in the dashed oval in (A) (the trace on the right is an expansion of this region). (B) When the particle is at the threshold of the pore size it will eventually p ass through the pore accompanied by a large current spike (dashed oval in (B)). Note that this current spike (expanded on the right) has the asymmetric shape characteristic of a typical translocation through a conical pore. The 1.0 M NaCl solution was buffered at pH 7.4 with 7 mM Na₂HP0₄, 21 mM KH₂P0₄, and contained 0.1% Triton X- 100. EXAMPLE 5

(Particle Capture and Release by Applied Pressure was demonstrated) According to particular aspects, applying pressure within the pipette offers considerable control over nanoparticle translocation, including the ability to draw individual particles into the pore and push them out again repeatedly, as illustrated in Figures 9 A and 9B. Because the quasi-triangular peak shape depends on the direction of translocation, these experiments provide confirmation that the present exemplary pores are conically shaped and open inwardly. Observations of particle reversal with application of pressure have been used to measure the size of individual particles depending upon their recapture probability (Lan & White, ACS nano 2012, 6, 1757-1765, 2012). In the present experiment, the distinct differences seen in translocation shape for individual particles reflect the acute sensitivity of this technique to monitor subtle nanopore/nanoparticle characteristics that are most likely based on geometrical and charge interactions.

Figures 9A and 9B show, according to particular exemplary aspects, forward and reverse translocation of three nanoparticles as a function of the applied pressure (e.g., current versus time (i-t) traces for three nanoparticles reversing direction due to applied pressure). A nanopore having a resistance of 117 ΜΩ measured in 1.0 M NaCl was used to observe 8-nm gold nanoparticles under constant applied potential (250 mV). In (A), three particles enter the pore between 1.2 and 1.6 s as negative pressure (-0.25 atm) is applied to the pipette. A pore block between 1.8 and 2.8 s is removed by applying positive pressure (0.5 atm), pushing the three particles back out of the pipette between 3.1 and 3.3 s. A negative pressure (-0.25 atm) is then applied at 4.5 s to draw the three particles back through the nanopore between 5 s and 7 s. Although the standard deviation in the particle size distribution was only ± 0.6 nm, distinct peak shapes seen in the i-t expansions (B) reflect subtle differences in the particle sizes, and allow identification of individual particles. The applied positive pressure between 3.1 and 3.3 s was greater than the applied negative pressures resulting in increased translocation velocity and therefore narrower peak widths.

EXAMPLE 6 (Particles were Captured and Released by Applied Potential)

According to additional aspects, Figures 5A-5D demonstrate that varying the applied potential can also be used to drive particles into and out of a pore repeatedly.

Figures 5A, 5B, 5C, and 5D illustrate, according to particular exemplary aspects, i- t traces showing a single nanoparticle repeatedly going in and out of a nanopore as the applied potential is reversed. A voltage square wave oscillating at 10 Hz between +1000 mV and -1000 mV is shown in (A) with circled portions indicating where translocations occur in the experimental traces (B) (resistive pulses in the i-t trace shown in (B)). The i-t traces in (B) are clipped to show just the relevant 50-ms portions of the square wave where translocations occur. In (D), a particle reverses direction (single nanoparticle passing back and forth through the pore orifice) for a square wave oscillating at 3 Hz between only +525 and +225 mV (C). Both solutions contained 8-nm gold nanoparticles (ζ = -51 mV) in 1.0 M NaCl PBS pH 7.4 plus 0.1% Triton X-100. Particle concentration for the experiment in (B), equals 50 nM, and in (D) equals 320 nM.

In the experimental results shown in Figures 5A and 5B, no pressure was applied to the pipette, but instead the particle motion followed a 10 Hz square wave varying between +1.0 V and -1.0 V. The four occasions of a particle going into and out of the pore were preceded and followed by several seconds without any particle translocations, indicating that repeated translocations of a single particle were observed. Voltage switching experiments have been used to recapture individual DNA strands (Gershow & Golovchenko, Nature Nanotech 2:775-779, 2007). The results shown in Figures 5C and 5D demonstrate that even a small change in the amplitude of applied potential (between only +225 mV to +525 mV) is sufficient to drive particles into and out of a pore.

According to further aspects, note that the resistive pulse asymmetry in these experiments is opposite from what would be expected based upon electrophoresis, implying that electroosmosis is the dominant force.

EXAMPLE 7

(Nanoparticle Dynamics were controlled by Applied Pressure and Applied Potential; taking advantage of electroosmosis and by applying a constant pressure to shift the zero velocity point to a potential with acceptable signal-to-noise ratio) Either electrophoresis or applied pressure alone has typically been used as the driving force for moving nanoparticles through a nanopore and demonstrated in the previous section. Decreasing the particle translocation velocity by lowering voltage has limitations, however, because the signal-to-noise ratio is reduced dramatically as the voltage decreases. Here we report fine control of particle velocities by taking advantage of electroosmosis and by applying a constant pressure to shift the zero velocity point to a potential with acceptable signal-to-noise ratio (Figures 6A and 6B).

Figures 6A and 6B show, according to particular exemplary aspects, nanoparticle translocation velocity vs. applied voltage under -0.047 atm (A) and -0.35 atm pressure (B). The solution conditions are for (A): 1.0 M NaCl, open and filled red triangles (ζ=-51 mV) and open and filled blue circles (ζ=-15 mV), and for (B): 0.2 M NaCl: open orange triangles (ζ=-51 mV) and open blue circles (ζ=-15 mV); 0.1 M NaCl: open black triangles (ζ=-51 mV) and open black circles (ζ=-15 mV); all solutions were buffered at pH 7.4 with 7 mM Na₂HP0₄, 21 mM KH₂P0₄, and contained 0.1% TritonX-100. Filled and open symbols in (A) represent two consecutive dataset collected under identical conditions. Dashed lines through data points represent second order polynomial fits. Representative i- t traces for particular translocations at different voltages are shown. The insets illustrate current-time (i-t) traces for particular data points; the scales for the traces in (A) and (B) are indicated in the upper right of each panel.

In this experiment, translocation velocities were assumed to be proportional to the inverse of the peak width at half height, with positive values indicating translocations into the pipette. Negative pressures indicate fluid flow into the pipette; positive voltages are measured relative to the external solution (see Figure 1). At the outset of the experiment, the majority of 8-nm gold particles were outside of the pipette, except for a small number of particles that had been pulled into the pipette under vacuum just prior to the experiment. The pipette was then subjected to a constant negative pressure (-0.047 atm in Figure 6 A and -0.35 atm in Figure 6B) and +500 mV. Both of these forces should act to drive negatively charged particles into the pipette, and yet the particle translocation profiles clearly indicated that nanoparticles were expelled from the pipette.

According to particular aspects, this is explained by the presence of a large electroosmotic flow (EOF) that overpowers both the applied pressure and the electrophoretic forces (EPF) acting on the particles under these conditions (Figure 1).

With reference to Fig. 6, as the potential was ramped down to +100 mV over the course of 5 minutes, the EOF decreased at a faster rate than the EPF and driving forces acting on the particle were balanced at a characteristic transition voltage that was determined by the zeta potential of the particles. Particle velocities, measured as the peak widths at half height, were markedly reduced at this transition. Of the 1,890 translocations shown in Figures 6A and 6B, thirteen had peak widths greater than 20 ms and two were as large as -200 ms. For the slowest translocations, the negation of all particle driving forces allowed us to see the effects of Brownian motion as the particle flickered in and around the sensing zone (blue trace inset in Figure 6A). This is in sharp contrast to the outer portions of the figure where peak widths were < 0.2 ms, representing an increase in particle velocity of 3 orders of magnitude. The limited number of translocations near 200 - 300 mV is a consequence of the diminishing particle rate of entry near the transition voltage and of the small number of particles initially inside the pipette, the particles were eventually exhausted as the potential was decreased from 500 mV to the transition voltage. Below this voltage, particles were drawn into the pipette from the external solution, as the combined EPF and applied pressure force became larger than the EOF.

Figure 7 illustrates the contributions to the effective velocity made by the applied pressure, electrophoresis (v = e/T| _particieE) and electroosmosis (v = -ε/τ ξ_ρ0ΙβΕ) for the experiments represented in Figure 6A. Specifically, Figure 7 schematically shows, according to particular exemplary aspects, controlling nanoparticle velocity in conical nanopores. The experiments presented in Figure 6 A are explained in terms of force contributions to the effective velocities on each type of nanoparticle, where the voltage- dependent peak widths presented in Figures 6A and 6B result from the summed

contributions of different forces acting on the charged nanoparticle. The applied pressure (-0.047 atm) remains constant throughout all measurements, but the particle-dependent electrophoretic and particle-independent electroosmotic forces change at different rates with varying voltage. As a result, the ζ=-51 mV particles reach a minimum velocity near 300 mV while the ζ=-15 mV reach a minimum velocity near 200 mV. EXAMPLE 8

(Factors Governing Particle Velocity)

In resistive pulse sensing, particle velocities are governed by the relative strengths of the EPF, EOF, and applied pressure. While EPF is a function of the charge of the particle, the EOF is only dependent upon the charge of the pore, and therefore the two forces increase with the applied voltage at different rates. Furthermore, these forces have different dependencies on pore geometry. Increasing the channel-like character of conical pores spreads the electric field over a larger sensing zone, which would be expected to reduce EPF. By contrast, increased pore channel length has been observed to increase the EOF. ' Though the geometry of the GNPs used in this study are conical, they have significant channel-like character due to the small cone angle (~2°; e.g., selected from among: 2° ± 0.1° ; 2° ±0.2° ; 2° ±0.3° ; 2° ±0.4° ; 2° ±0.5° ; 2° ±0.6° ; 2° ±0.7° ; 2° ±0.8° ; 2° ±0.9 ; 2° ±1.0°), and, according to particular aspects of the present invention, this is important in achieving the delicate balance of the forces controlling particle dynamics.

Without pressure, the minimum in particle velocity occurs at zero voltage. By applying suitable pressure, we are able to shift the minimum velocity point to a voltage range that is convenient, and that has substantial utility for measurements. Thus, for a particular pipette, we applied a pressure necessary to place the transition voltage in this range; that is, the voltage at which particle velocities are minimized due to equivalence of the forces drawing particles into the pore (primarily the EPF and fluid flow caused by applied pressure) and those driving particles out of the pore (primarily the EOF). Firnkes et al. were able to balance the EPF and EOF by finding a pH at which the zeta potential of the pore and the molecule studied were equal.¹⁰ However, simply eliminating the driving force does not allow for general control of particle dynamics. For the conical pores used in this study, the EOF appears to increase with voltage at a greater rate than the EPF, and we observed translocations in the opposite direction of electrophoresis under atmospheric conditions. Zhang et al. also demonstrated DNA translocating in the opposite direction of electrophoresis and attributed this to a large EOF.¹¹ In the experiments in Figures 6A and 6B we took advantage of the large change in EOF with respect to voltage, and were able to control the entire range of particle velocities from near zero to the limit of the electronic bandwidth filtering of the amplifier (10 kHz), in both the inward and outward direction and between +100 and +500mV. EXAMPLE 9

(The Effects of Salt Concentration and Particle Charge on Nanoparticle Dynamics) Experiments in 0.1M and 0.2M NaCl solutions required a much larger applied pressure (-0.35 atm) than those in 1M NaCl (-0.047 atm); the data are presented on a separate graph to accommodate a sufficiently wide range of particle velocities (Figure 6B). The need for higher pressure would be expected from the longer Debye lengths at lower salt concentrations, which generate significantly larger EOFs along the pore surface.

Since the EOF at the transition voltage is larger than the applied pressure, we can say that pressures greater than 0.3 atm are generated in 0.2M NaCl at 250 mV, and in 0.1M NaCl comparable pressures are generated at 180 mV. By contrast, Takamura et al. reported the fabrication of "extremely high pressure" electroosmotic pumps of 0.05 atm under tens of volts.²³

Examining the velocities of differently charged particles at a particular

combination of salt concentration and applied voltage reveals the effect of particle charge. Under these conditions, the applied pressure and EOF are identical, and therefore the remaining electrophoretic force decreases the velocity of negative particles moving out of the pore and increases their velocities as they move in. This explains why the velocity trend lines for the more highly charged (ζ = -51 mV) particles were always above those for the less highly charged (ζ = -15 mV) particles (Figures 6 A and 6B). It should be noted that the 0.1M NaCl velocities fall below the 0.2M NaCl due to an increased EOF and not because of charge effects.

EXAMPLE 10

(Factors Affecting Resistive Pulse Peak Shape)

It is well known that the path of a particle through a conical nanopore determines the shape of a resistive pulse event. Inhomogeneity of the electric field within the sensing zone due to a stronger field near pore walls has been shown to cause as much as a 15% deviation in peak amplitude for particles that do not travel straight through the center of the pore (off-axial translocations).²⁰ Interaction of particles with pore walls can also lengthen translocation times, a factor that must be taken into account for analysis based upon peak widths.⁹ The ability to slow particle velocity to a degree achieved in our experiments allows a closer examination of the factors that affect translocation kinetics. This is illustrated by the insets in Figures 6A and 6B, which demonstrate clear peak shape differences during the course of translocations. In particular, we have observed the steep side of a typical asymmetric translocation exhibiting biphasic character to differing degrees (compare the rightmost inset translocation with both the second and the fifth from right). These stages of resistance change may be explained by contributions from an inhomogeneous electric field, pore wall interactions, diffusion, and/or possibly a second EOF that arises from the double layer associated with the particle itself. An additional complicating factor would be if our pores were not entirely smooth throughout the sensing zone, although the observation of numerous "ideally shaped" translocations²¹ argues against this possibility. EXAMPLE 11

(V elocity Measurement Limitations)

Although there was considerable data scatter, the general trend was reproducible across two independent experiments carried out under identical conditions (Figure 6A, opened and closed symbols). The experiments in Figure 6A were carried out with the same pipette (having a resistance of 110 ΜΩ at 1M NaCl), and those in Figure 6B were all carried out with a different pipette. Some of the data scatter at highest and lowest applied potential is based on limitations in our ability to accurately measure peak width for the fastest moving particles (thus the digitization seen on the right of Figure 6B). Slow moving particles also involve scatter, presumably because additional surface forces acting on the particles become significant under these conditions. The data scatter is particularly severe when the salt concentration is <0.2M NaCl, mostly due to the relatively poor signal-to-noise. The experiments under low salt conditions (0.1 - 0.2M NaCl) were done with a pore within 10 ΜΩ (measured at 1M NaCl) of the threshold size in order to maximize the amplitude of the resistive pulses, particularly near 100 mV. The observation of several near zero velocity events that do not fall in line with the data trend likely indicates particles that interacted strongly with the pore wall, because fluid flow was not fast enough to deter physisorption.

One additional source of apparent data scatter is cross contamination between experiments. For example, the ζ = -15 mV data shown in Figure 6A was collected prior to the ζ = -51 mV data shown in the same figure, and despite efforts to thoroughly rinse the pipette between experiments the red triangles falling in line with the ζ = -15 mV data likely indicates the presence of residual ζ = -15 mV particles. This assumption is supported the fact that the signals show the opposite peak symmetry at the transition voltage of the ζ = -51 mV particles. This is demonstrated by the lowest red inset in

Figure 6A, which suggests that two particles, one with ζ = -15 mV and one with ζ= -51 mV, are crossing the pore in different directions at the same applied potential.

In summary, according to particular aspects, the dynamics of 8-nm nanoparticle translocations, and nanobubble translocations, through micropipette GNPs can be controlled, and we demonstrated control over the interplay of electrophoretic (EPF), electroosmotic (EOF) and pressure forces by balancing translocation velocity as a function of particle charge, salt concentration, and applied pressure. Detection and characterization of nanoparticles and nanobubbles has a growing number of applications across different disciplines, from research and diagnostics, drug delivery, detection of nanoparticle waste released by industrial nanotechnology applications, and bio-sensing. Overcoming the problem of excessive particle or bubble velocities through appropriate choice of nanopores and observation parameters is an important step toward applying these technologies.

Controlling nanoparticle and nanobubble dynamics allows nanopore sensing to advance from mere detection of nanoparticles into the realm of nanoparticle characterization in a previously unattainable range.

EXAMPLE 12

{Finite Element Analysis (FEA) Simulations were performed)

Finite-element simulations using COMSOL Multiphysics were performed to provide a more quantitative description of the experimental results at each of the salt concentrations studied. We used a quasi-steady method which assumes that the fluid and particle are in equilibrium.^20"22 Based on the assumption that the sum of the hydrodynamic drag and electrokinetic forces on the nanoparticle (or nanobubble) are zero, the velocity of the particle or bubble may be iteratively determined using the Newton-Raphson method to solve the following equations from an appropriate initial guess.

A quasi-steady force balance is expressed as:

Ftotai = FH + FE = 0 (1) where F_H and F_E are hydrodynamic force and electrokinetic force exerted on the particle, respectively. These forces are given by eqs (2) and (3):

where T_H and T_E are the hydrodynamic stress tensor and the Maxwell stress tensor, respectively, n is the unit normal vector, and S represents the surface of nanoparticle.

The Navier-Stokes equation describes the laminar flow of the incompressible fluid.

uVu = -(-V/? + //V²u - ( Σζ_Λ)νΦ) (4)

P i In eq 4, u and Φ are the local position-dependent fluid velocity and potential, d and are concentration and charge of species i in solution, p is the pressure and F is Faraday's constant. The solution density p = 1000 kg/m³ and the dynamic viscosity η = 0.001 Pa*s, respectively, correspond approximately to the aqueous solution. The particle velocity u corresponds to the boundary velocity between the particle surface and surrounding fluid, eq 4.

The ion distribution and potential profile in the system are modeled by the Nernst- Planck-Poisson equations as below:

Fz.

^{Ji = " VCi "} RJ ^{CiVO + CiU (5)}

In eq 5, J_z and are the ion flux vector and diffusion coefficient of species i in solution, respectively. D_Na ⁺ = 1.33 x 10^~9 m²/s and Dei = 2.03 x 10^~9 m²/s. The absolute temperature T= 298 K, and the gas constant R = 8.314 J/K. £ is the dielectric constant of 78.

Figures 8A-8C present results of the FEA simulations corresponding to the experiments in Figures 6 A and 6B. Figure 8 A shows velocity profiles and streamlines along the pore axis corresponding to the experimental conditions (zeta potential ζ = -15 mV, 0.2 M NaCl and 0.35 atm external pressure) in Figure 6B (light blue line). Using a cone angle of 1.87°, the general trends seen in the experiment were reproduced, with particles entering the pore at lOOmV, exiting the pore at 500mV, and a crossover point occurring at -200 mV (simulated) and -250 mV (experimental).

In Figures 8B and 8C, simulation parameters were varied to reproduce the velocity trends seen in Figures 6B and 6A, respectively, for the differently charged particles at varying salt concentrations. A better quantitative match with experimental results is seen at the lower salt concentrations (Figure 8B). Specifically, the same velocity trends are seen as particle charge and the ionic strength of the solution are varied, with velocity reversal occurring in the applied voltage between 100 and 500 mV range. At higher salt concentration (Figure 8C) the agreement with the experimental measurements is weaker, but still qualitatively capture the trend in the experimental results. Given the approximations in the modeling parameters and the uncertainty in the nanopore geometry, the governing equations employed in the FEA simulations provide a very satisfactory description of the particle motion. Figure 8D illustrates, according to particular exemplary aspects, the contributions to the effective velocity made by the applied pressure, electrophoresis (v = e/r| particieE) ^and electroosmosis (v = for a ζ = -15 mV nanoparticle.

Figures 9A and 9B show forward and reverse translocation of three nanoparticles as a function of the applied pressure. A nanopore having a resistance of 117 ΜΩ measured in 1 M NaCl was used to observe 8-nm diameter Au nanoparticles at constant applied potential (250 mV). In (A), three particles enter the pore between 1.2 and 1.6 s as negative pressure (-0.25 atm) is applied to the pipette. A pore block between 1.8 and 2.8 s is removed by applying a positive pressure (0.5 atm), pushing the three particles out of the pipette between 3.1 and 3.3 s. A negative pressure (-0.25 atm) is then applied at 4.5 s to draw the three particles back through the nanopore between 5 s and 7 s. Although the standard deviation in the particle size distribution was only ±0.6 nm, distinct peak shapes seen in the i-t expansions shown in (B) reflect subtle differences in the particle sizes, and allow identification of individual particles. The applied positive pressure was greater than the applied negative pressures resulting in increased translocation velocity and therefore narrower peak widths.

Surface charge density of the Au nanoparticle estimated from the zeta potential in an extremely diluted electrolyte solution.

The effective surface charge of the Au nanoparticles was estimated by finite- element simulation, assuming that the simulated surface potential is equal to the measured zeta potential. Experimentally, the zeta potential of nanoparticle was measured in deionized (DI) water which contains ~10^"7 M hydroxide (OFT) and hydronium ion (H₃O ) due to water's self-dissociation. Considering trace ions remain in the DI water, the electrolyte was set as 10^"6 M KC1 in the simulation. An arbitrary surface charge density was initially set on the Au nanoparticle surface, and then Poisson and Nernst-Planck equations were iteratively solved to obtain a surface charge density value, which yields a surface potential within 10% of the measured zeta potential. A surface charge density of -3 and -9 mC/m² were obtained which produces a simulated surface potential of -17.0 and - 51.0 mV, respectively, compared with measured -15 and -51 mV. Figure 10 shows the simulated potential profile generated by a -9 mC/m² charged gold nanoparticle with a diameter of 8 nm.

The geometry and boundary conditions for a simulation of the particle velocity in 0.1 and 0.2 MNaCl solutions.

In the simulation, 0.35 atm pressure and -0.1 to -0.5 V voltage were applied across the nanopore, corresponding to experimental values. An 8-nm diameter gold nanoparticle was placed at the nanopore orifice, z = 0 and r = 0, whose surface charge density was chosen as -3 or -9 mC/m², corresponding to a zeta potential of -15 and -51 mV. The determination of the nanoparticle surface charge density was detailed in SI 2 above. A mesh size < 0.5 nm was used at the nanopore 's charged surface (red line highlighted) as well as the nanoparticle surface, which is sufficient to resolve the electrical double layer.

The nanopore surface charge density and geometry were estimated based on the nanopore ion current and ion current rectification ratio, defined as the ratio of currents at - 500 and 500 mV (inside vs. outside nanopore). In 0.1 M NaCl, a nanopore surface charge density of -4 mC/m² produces a simulated rectification ratio of -1.13 while the experimental value is -1.2; the simulated current at 500 mV is 550 pA, while the experimental value is 600 pA. Figure 11 shows the geometry, mesh and boundary conditions used in the simulation.

The geometry and boundary conditions for a simulation of the particle velocity in 1 MNaCl solution.

The boundary conditions and mesh setting were the same as in the example of Figure 11, except that the pressure was decreased to 0.047 atm and the bulk salt concentration was increased to 1 M, corresponding to experimental parameters.

The same method to determine surface charge and geometry as in the example of Figure 11, which is based on the ion current rectification ratio, was not employed here, because 1 M NaCl screens the surface charge and almost eliminate the ion rectification. However, we can obtain additional information about the nanopore geometry from the resistive pulse current blockage. The experimental result shows that at 1 M NaCl and 500 mV, the resistive pulse blockage is -70 nA which is only twice that of the resistive pulse blockage at 0.1 M NaCl and 500 mV applied voltage (see Figures 6A and 6B), less than the expected -10 fold difference. One possible reason is that the nanopore employed at the higher NaCl concentration is slightly larger than the one used in the lower NaCl concentration experiment, leading to a smaller blockage. Also, to achieve a similar reversal velocity profile between 100 and 500 mV as shown in Figures 6A and 6B, the surface charge density was varied and finally chosen as -5.6 mC/m². The increase of surface charge density from -4 mC/m² at 0.1M NaCl to -5.6 mC/m² at 1M NaCl is also justified by Grier et al., who found that a higher concentrated salt solution enhanced the dissociation of surface silanol group, leading to a surface charge density increase.¹ Figure 12 shows the geometry, mesh, and boundary conditions used in the simulation.

EXAMPLE 13

(Systems for detecting, manipulating and characterizing gas nanobubbles and

nanoparticles in solution)

Certain embodiments provide systems (e.g., apparatus, devices and the like, including computerized, software augmented or driven systems) for detecting, manipulating and characterizing nanobubbles and nanoparticles in solution.

Particular aspects provide a system or device for detecting, manipulating and characterizing nanobubbles and nanoparticles in solution, comprising: a conduit for saline solution configured to be placed in communication with a source of saline solution (e.g., having nanoparticles or nanobubbles); a conical-shaped nanopore (e.g., conical-shaped glass nanopore) having a nanopore diameter, a proximal end, a distal end in communication with the conduit for saline solution, the conical-shaped nanopore having a nanoparticle or nanobubble sensing zone between the proximal and the distal ends, and wherein the sensing zone of the conical-shaped nanopore has a half-cone angle sufficiently small to provide for significant channel-like character at the nanopore; electrodes (e.g., an electrode pair) positioned on opposite sides of the conical-shaped nanopore, the electrodes in communication with a voltage/potential source, and configured to provide for application of a voltage/potential across the nanoparticle or nanobubble sensing zone between the proximal and the distal ends to provide an electrophoretic force (EPF) across the nanopore; a nanopore/electrode holder in communication with a source of pressure, and configured to provide for application of pressure within the conical-shaped nanopore; a current measuring component (e.g., a suitable amplifier) configured to be in operative communication with computer-implemented data acquisition software suitable to analyze and export current-time traces, and optionally, computer-implemented software suitable to determine translocation peak parameters such as peak position, height, and width at half- height as a function of applied voltage; and control means for adjusting at least one parameter selected from EPF, electroosmottic force EOF, and pressure across the nanopore, to provide for fine control of particle or nanobubble translocation velocities across the sensing zone of the nanopore (e.g., by shifting the zero velocity point to an applied voltage/potential to provide for an acceptable signal-to-noise ratio), to provide a method for detecting, manipulating and characterizing nanobubbles and nanoparticles in solution.

Though the geometry of the GNPs used in this study are conical, they have significant channel-like character due to the small cone angle (~2°; e.g., selected from among: 2° ± 0.1° ; 2° ±0.2° ; 2° ±0.3° ; 2° ±0.4° ; 2° ±0.5° ; 2° ±0.6° ; 2° ±0.7° ; 2° ±0.8° ; 2° ±0.9 ; 2° ±1.0°), and, according to particular aspects of the present invention, this is important in achieving the delicate balance of the forces controlling particle dynamics.

Incorporation by Reference. All of the above U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications and non- patent publications referred to in this specification and/or listed in the Application Data Sheet, are incorporated herein by reference, in their entirety.

It should be understood that the drawings and detailed description herein are to be regarded in an illustrative rather than a restrictive manner, and are not intended to limit the invention to the particular forms and examples disclosed. On the contrary, the invention includes any further modifications, changes, rearrangements, substitutions, alternatives, design choices, and embodiments apparent to those of ordinary skill in the art, without departing from the spirit and scope of this invention, as defined by the following claims. Thus, it is intended that the following claims be interpreted to embrace all such further modifications, changes, rearrangements, substitutions, alternatives, design choices, and embodiments.

The foregoing described embodiments depict different components contained within, or connected with, different other components. It is to be understood that such depicted architectures are merely exemplary, and that in fact many other architectures can be implemented which achieve the same functionality. In a conceptual sense, any arrangement of components to achieve the same functionality is effectively "associated" such that the desired functionality is achieved. Hence, any two components herein combined to achieve a particular functionality can be seen as "associated with" each other such that the desired functionality is achieved, irrespective of architectures or intermedial components. Likewise, any two components so associated can also be viewed as being "operably connected", or "operably coupled", to each other to achieve the desired functionality.

While particular embodiments of the present invention have been shown and described, it will be obvious to those skilled in the art that, based upon the teachings herein, changes and modifications may be made without departing from this invention and its broader aspects and, therefore, the appended claims are to encompass within their scope all such changes and modifications as are within the true spirit and scope of this invention. Furthermore, it is to be understood that the invention is solely defined by the appended claims. It will be understood by those within the art that, in general, terms used herein, and especially in the appended claims (e.g., bodies of the appended claims) are generally intended as "open" terms (e.g., the term "including" should be interpreted as "including but not limited to," the term "having" should be interpreted as "having at least," the term "includes" should be interpreted as "includes but is not limited to," etc.). It will be further understood by those within the art that if a specific number of an introduced claim recitation is intended, such an intent will be explicitly recited in the claim, and in the absence of such recitation no such intent is present. For example, as an aid to understanding, the following appended claims may contain usage of the introductory phrases "at least one" and "one or more" to introduce claim recitations. However, the use of such phrases should not be construed to imply that the introduction of a claim recitation by the indefinite articles "a" or "an" limits any particular claim containing such introduced claim recitation to inventions containing only one such recitation, even when the same claim includes the introductory phrases "one or more" or "at least one" and indefinite articles such as "a" or "an" (e.g., "a" and/or "an" should typically be interpreted to mean "at least one" or "one or more"); the same holds true for the use of definite articles used to introduce claim recitations. In addition, even if a specific number of an introduced claim recitation is explicitly recited, those skilled in the art will recognize that such recitation should typically be interpreted to mean at least the recited number (e.g., the bare recitation of "two recitations," without other modifiers, typically means at least two recitations, or two or more recitations). Accordingly, the invention is not limited except as by the appended claims.

REFERENCES; incorporated by reference herein in their respective entireties:

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Claims

1. A method for detecting, manipulating and characterizing nanobubbles and nanoparticles in solution, comprising:

providing a saline solution having nanoparticles or nanobubbles;

providing a conical-shaped nanopore having a nanopore diameter, a proximal end, a distal end in communication with the saline solution, and having a nanoparticle or nanobubble sensing zone between the proximal and the distal ends, and wherein the sensing zone of the conical-shaped nanopore has a half-cone angle sufficiently small to provide for significant channel-like character at the nanopore;

applying, using an electrode, a voltage/potential across the nanopore to provide an electrophoretic force (EPF) across the nanopore;

subjecting the nanopore to an electroosmotic force (EOF);

applying a pressure across the nanopore; and

adjusting at least one parameter selected from EPF, EOF, and pressure across the nanopore, to provide for fine control of particle or nanobubble translocation velocities across the sensing zone of the nanopore by shifting the zero velocity point to an applied voltage/potential to provide for an acceptable signal-to-noise ratio, to provide a method for detecting, manipulating and characterizing nanobubbles and nanoparticles in solution.

2. The method of claim 1, wherein adjusting the EOF comprises adjusting at least one variable selected from salt concentration, applied voltage/potential, and pH.

3. The method of claim 1, wherein the pressure across the nanopore is maintained at a constant value, and wherein the applied voltage/potential across the nanopore is adjusted.

4. The method of claim 1, wherein the applied voltage/potential across the nanopore is maintained at a constant value, and wherein the pressure across the nanopore is adjusted.

5. The method of claim 1, wherein adjusting at least one parameter selected from EPF and EOF, comprises adjusting the charge or zeta potential of the nanoparticles or nanobubbles.

6. The method of claim 1, wherein a nanoparticle or nanobubble is translocated across the sensing zone of the nanopore to provide for resistive pulse detection, and characterization of the nanoparticle or nanobubble.

7. The method of claim 6, wherein translocation of the nanoparticle or nanobubble provides for determining/measuring the size of the nanoparticle or nanobubble.

8. The method of claim 1, wherein the half-cone angle of the conical nanopore has a value in the range of 0.1 degrees to 4 degrees, to provide for achieving the delicate balance of the forces controlling the particle dynamics.

9. The method of claim 8, wherein the half-cone angle of the conical nanopore has a value in the range of about 2 degrees, to provide for achieving the delicate balance of the forces controlling the particle dynamics.

10. The method of claim 1, wherein the nanoparticles or nanobubbles have a diameter of less than 8 nM, less than 10 nM, less than 20 nM, less than 30 nM, less than 40 nM, less than 50 nM, or less than 100 nm.

11. The method of claim 10, wherein the nanoparticles or nanobubbles have a diameter in the range of 8 nM to 10 nM, of 8 nM to 20 nM, of 8 nM to 30 nM, of 8 nM to 40 nM, of 8 nM to 50 nM, or of 8 nM to 100 nM.

12. The method of claim 1, comprising translocation of the nanoparticle or nanobubble is in the inward and/or outward direction.

13. The method of claim 1, wherein the applied voltage/potential, in one direction or another (+ or -), is between 100 and 500mV, optimally between -lOOmV and 900mV, optimally between -250mV and 900mV, optimally at about 250mV.

14. The method of claim 1, comprising conducting the method using a device subjected to an external pressure, to provide for modulating the diameter of the nanobubbles.

15. The method of claim 14, wherein the external pressure is in the range of between

16. The method of claim 15, wherein the external pressure is about 200 psi.

17. The method of claim 1, wherein the salt concentration is between 100 mM and 1 M, between 300 mM and 1 M, between 500 mM and 1 M, optimally about 150 mM, optimally about 300 mM, optimally at from 150 to about 300 mM sodium chloride.

18. The method of claim 1 , wherein the nanobubbles are oxygen nanobubbles.

19. The method of claim 1, wherein the saline solution is R S60 as known in the art.

20. The method of claim 17, wherein the salt concentration is between 100 and 300 mM, optimally at about 150 mM.

21. A system or device for detecting, manipulating and characterizing nanobubbles and nanoparticles in solution, comprising:

a conduit for saline solution configured to be placed in communication with a source of saline solution (e.g., having nanoparticles or nanobubbles);

a conical-shaped nanopore (e.g., conical-shaped glass nanopore) having a nanopore diameter, a proximal end, a distal end in communication with the conduit for saline solution, the conical-shaped nanopore having a nanoparticle or nanobubble sensing zone between the proximal and the distal ends, and wherein the sensing zone of the conical- shaped nanopore has a half-cone angle sufficiently small to provide for significant channel-like character at the nanopore;

electrodes (e.g., an electrode pair) positioned on opposite sides of the conical- shaped nanopore, the electrodes in communication with a voltage/potential source, and configured to provide for application of a voltage/potential across the nanoparticle or nanobubble sensing zone between the proximal and the distal ends to provide an electrophoretic force (EPF) across the nanopore;

a nanopore/electrode holder in communication with a source of pressure, and configured to provide for application of pressure within the conical-shaped nanopore; a current measuring component (e.g., a suitable amplifier) configured to be in operative communication with computer-implemented data acquisition software suitable to analyze and export current-time traces; and

control means for adjusting at least one parameter selected from EPF, electroosmottic force EOF, and pressure across the nanopore, to provide for fine control of particle or nanobubble translocation velocities across the sensing zone of the nanopore (e.g., by shifting the zero velocity point to an applied voltage/potential to provide for an acceptable signal-to-noise ratio), to provide a method for detecting, manipulating and characterizing nanobubbles and nanoparticles in solution.

22. The system or device of claim 21, further comprising computer- implemented software suitable to determine translocation peak parameters such as peak position, height, and width at half-height as a function of applied voltage.