Measurement of particle charge

Abstract

A method of determining a charge of at least one test particle (as herein defined), comprising: applying one of an electric current or a voltage across an aperture connecting two chambers, whereby the chambers are at least partially filled with electrolyte and whereby the at least one test particle is suspended in the electrolyte of at least one of the chambers; measuring a value indicative of the other of the electric current or voltage across the aperture; determining a time interval between a first and a second point in time, the second point in time corresponding to a point in time when the measured current or voltage has reached a specific proportion of the measured current or voltage at the first point in time; and determining the charge of the at least one test particle by: determining a value indicative of an electrical velocity component of a total velocity of at least one calibration particle having a known charge, taking into account that the total velocity of the at least one calibration particle comprises a non zero-convective velocity component and the electrical velocity component; determining a value indicative of an electrical velocity component of a total velocity of the at least one test particle, taking into account that the total velocity of the at least one test particle comprises a non-zero convective-velocity component and the electrical velocity component; and using the determined values indicative of the electrical velocity components of the test particle and the calibration particle to calibrate the quantitative relationship between the charge of the at least one test particle and the determined time interval.

Claims

1. A method of determining a charge of at least one test particle, comprising: applying one of an electric current or a voltage across an aperture connecting two chambers, whereby the chambers are at least partially filled with an electrolyte and whereby the at least one test particle is suspended in the electrolyte of at least one of the chambers, wherein the aperture has a geometry that does not need to be known; measuring a value indicative of the other of the electric current or the voltage across the aperture; determining a time interval between a first and a second point in time, the second point in time corresponding to a point in time when the measured current or the measured voltage has reached a specific proportion of the measured current or the measured voltage at the first point in time; and determining the charge of the at least one test particle by: determining a value indicative of an electrical velocity component of a total velocity of at least one calibration particle having a known charge, taking into account that the total velocity of the at least one calibration particle comprises a non-zero convective velocity component and the electrical velocity component; determining a value indicative of an electrical velocity component of a total velocity of the at least one test particle, taking into account that the total velocity of the at least one test particle comprises a non-zero convective velocity component and the electrical velocity component; and using the determined values indicative of the electrical velocity components of the at least one test particle and the at least one calibration particle to calibrate a quantitative relationship between the charge of the at least one test particle and the determined time interval.

2. The method of claim 1, wherein determining the value indicative of the electrical velocity component of the total velocity of the at least one calibration particle comprises determining a value indicative of the total velocity of the at least one calibration particle and a value indicative of the non-zero convective velocity component of the at least one calibration particle, and subtracting the value indicative of the non-zero convective velocity component from the value indicative of the total velocity.

3. The method of claim 2, further comprising determining a further time interval for the at least one calibration particle, wherein the further time interval is a time interval between a third and a fourth point in time, the fourth point in time corresponding to a point in time when the measured current or the measured voltage for the at least one calibration particle has reached a specific proportion of the measured current or the measured voltage at the third point in time.

4. The method of claim 3, wherein determining the value indicative of the electrical velocity component of the total velocity of the at least one calibration particle comprises determining a respective further time interval at each of a plurality of applied voltages.

5. The method of claim 4, further comprising determining the value indicative of the electrical velocity component of the at least one calibration particle based on: the plurality of applied voltages, and the respective further time intervals, or values derived therefrom, at each of the plurality of applied voltages.

6. The method of claim 4, further comprising determining the slope of a curve fitted to a plot of the inverse of the respective further time intervals against the plurality of applied voltages, said slope being indicative of the electrical velocity component per unit voltage for the at least one calibration particle.

7. The method of claim 3, wherein determining the value indicative of the non-zero convective velocity component of the at least one calibration particle comprises measuring a respective further time interval at each of a plurality of applied voltages.

8. The method of claim 7, wherein determining the value indicative of the non-zero convective velocity component of the at least one calibration particle comprises using an extrapolation based on: the plurality of applied voltages, and the respective further time intervals, or values derived therefrom, at each of the plurality of applied voltages.

9. The method of claim 7, wherein determining the value indicative of the non-zero convective velocity component of the at least one calibration particle comprises plotting the inverse of the further time intervals against the plurality of applied voltages, and extrapolating a line defined by the plot to a point where the voltage is zero.

10. The method of claim 3, wherein determining the value indicative of the non-zero convective velocity component of the at least one calibration particle comprises measuring a respective further time interval at each of a plurality of applied pressure values, wherein the applied pressure is a pressure that is externally applied to at least one of the two chambers to change or establish a pressure differential across the aperture.

11. The method of claim 10, further comprising: determining, for the at least one calibration particle, a value indicative of a convective velocity per unit pressure based on the applied pressure values; and determining the value indicative of the non-zero convective velocity component of the at least one calibration particle based on the value indicative of the convective velocity per unit pressure, the applied pressure, and an inherent pressure head, wherein the inherent pressure head is a contribution to the pressure differential across the aperture that results from a difference in height between the electrolyte in the two chambers.

12. The method of claim 11, wherein determining the value indicative of the non-zero convective velocity component of the at least one calibration particle further comprises: plotting the inverse of the respective further time intervals against the plurality of applied pressures; determining the slope of said plot, said slope being indicative of the convective velocity per unit pressure; and determining the value indicative of the non-zero convective velocity component as the product of the convective velocity per unit pressure and the sum of the applied pressure and the inherent pressure head.

13. The method of claim 2, further comprising determining the charge of the at least one test particle by: determining a value indicative of the total velocity of the at least one test particle using the value indicative of the total velocity of the at least one calibration particle and the determined time interval for the at least one test particle; determining a value indicative of the non-zero convective velocity component of the at least one test particle as the product of a convective velocity per unit pressure and the sum of an applied pressure and an inherent pressure head; determining the value indicative of the electrical velocity component of the at least one test particle by subtracting the value indicative of the non-zero convective velocity component of the at least one test particle from the value indicative of the total velocity of the at least one test particle; determining a zeta potential of the at least one test particle using the value indicative of the electrical velocity component of the at least one calibration particle, the value indicative of the electrical velocity component of the at least one test particle, a zeta potential of the aperture, and a zeta potential of the at least one calibration particle, wherein the zeta potential of the at least one calibration particle is determined using the known charge of the at least one calibration particle; and determining the charge of the at least one test particle using the determined zeta potential of the at least one test particle.

14. The method of claim 13, further comprising determining the charge of the at least one test particle by averaging a plurality of zeta potential values determined for a plurality of specific proportions.

15. The method of claim 1, wherein the value indicative of the electrical velocity component and the non-zero value indicative of the non-zero convective velocity component of the total velocity of the at least one calibration particle are determined independent of each other.

16. The method of claim 1, wherein the specific proportion is set based on an average total velocity of the at least one test particle relative to a data sampling rate.

17. The method of claim 16, wherein: when the average total velocity of the at least one test particle is greater than a predetermined threshold, the specific proportion is set to a first value; and when the average total velocity of the at least one test particle is less than the predetermined threshold, the specific proportion is set to a second value; wherein the first value is smaller than the second value.

18. The method of claim 1, wherein the at least one test particle and the at least one calibration particle are suspended in the same electrolyte and are analyze in the same experiment, or wherein the at least one test particle is a biological particle, the method further comprising determining a zeta potential of the aperture before and after measurement of the at least one test particle.

19. The method of claim 1, wherein the total velocity of the at least one calibration particle is a sum of the non-zero convective velocity component and the electrical velocity component.

20. The method of claim 1, wherein the total velocity of the at least one test particle is a sum of the non-zero convective velocity component and the electrical velocity component.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is a schematic comparison of the blockade shapes resulting from a larger and smaller particle travelling at the same speed, for a generally conical aperture.

(2) FIG. 2 is a schematic comparison of the blockade shapes resulting from a larger and smaller particle travelling at the same speed, for a different aperture shape.

(3) FIG. 3 is a plot of Average (Vx)tot (i.e., 1/Tx) against bias voltage V for calibration particles (Example 1).

(4) FIG. 4 is a plot of Calculated (Vx)c as a function of % of dR max for calibration particles, from 10% to 40% of dR max (Example 1).

(5) FIG. 5 is a plot of zeta potential vs size for multiple applied pressures, showing repeatable results.

(6) FIG. 6 is a plot of zeta potential vs size for multiple applied bias voltages, showing repeatable results.

(7) FIG. 7 is a plot of zeta potential vs size for highly charged (Carboxylated) and less charged (Plain) PS particles.

(8) FIG. 8 is a plot of zeta potential vs size illustrating the detection of the surface modification of liposomes via detection of a change in zeta potential.

(9) FIG. 9 is a plot of zeta potential vs size illustrating the measurement of a suspension of liposomes spiked with carboxylated calibration particles.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS OF THE INVENTION

(10) Certain embodiments of the invention involve the use of the apparatus disclosed in WO2013017671, the entire contents of which are hereby incorporated by reference.

(11) In certain embodiments of the invention, use is made of a nanopore or micropore of fixed or variable geometry, formed in an impermeable membrane. The exact geometry and dimensions of this pore may be known but typically the exact geometry and dimensions of the pore would be unknown.

(12) The membrane is fitted into apparatus which allows a well (or reservoir or chamber) of electrolyte to be placed on either side of the membranethe wells are connected to each other only through the pore itself. In one form, the aperture or pore may be provided as a (small) hole in a sheet, the sheet forming (part of) a wall between the two chambers and thereby separating the chambers from each other. The apparatus contains at least one electrode in contact with each of the electrolyte wells such that a user defined bias voltage can be applied across the pore and the resulting electric current through the pore can be monitored. In certain embodiments of the invention a user defined electric current may instead be applied through the pore and the resulting voltage across the pore may be monitored.

(13) The apparatus includes a means to vary the pressure applied to either one or both of the electrolyte wells such that a user defined differential pressure (Pap) can be applied in either direction across the pore, resulting in a convective flow of electrolyte from one well to the other or vice versa.

(14) The inherent pressure head of the system (Pin) due to gravitational forces and/or surface tension is known and consistent from measurement to measurement (typically achieved by using the same volume of fluid).

(15) The objects passing through the pore for analysis can consist of any material including solids (e.g. carbon, silica, polymers, metals), biological particles (e.g. viruses, bacteria, microvesicles, liposomes, cells), liquids (e.g. emulsions) or gases (e.g. nanobubbles). In the preferred form solid calibration particles (e.g. carboxylated polystyrene) are used. Objects passing through the pore are therefore referred to as particles below.

(16) The method allows for quantitative measurement of chemical/biochemical reactions on particle surfaces that alter particle zeta potential. The capacity to detect such changes in particle zeta potential forms the basis of pore based diagnostic assays. Surface reactions may include the specific binding of aptamers, proteins, antibodies, lectins, DNA, RNA, other chemical or biochemical reagents to the surface of the particle. The particle may include cells, extracellular vesicles, subcellular complexes, liposomes, synthetic particles, but not limited to these. The reaction may be quantified by comparative measurement of particle zeta potential before and after incubation of the particle with the surface modifier for a period of time under appropriate conditions or the reaction process may be monitored continuously in real time.

(17) When each particle passes through the pore, there is a resultant resistive pulse or blockade. For objects with a small aspect ratio (largely spherical), the general shape of any given blockade is determined by the shape of the pore; this general shape is stretched in magnitude (height) and duration (width) depending on the size and the speed of each particle.

(18) FIG. 1 illustrates the passage of a larger particle (top half of FIG. 1) and a smaller particle (bottom half of FIG. 1) travelling through a pore at the same speed, with corresponding plots of electrical resistance against time showing the resulting blockade shapes.

(19) The method identifies the point of greatest resistance in the signal trace (the blockade peak), which in the preferred pore setup is close to the beginning of the blockade. For each blockade, the time at which the peak occurs is defined as t.sub.100 (time at 100% of peak magnitude) and the maximum magnitude of the pulse (relative to the local baseline resistance) is recorded as dR max.

(20) In the simplest embodiment, the blockade is divided into n sections, and the duration from t.sub.100 is recorded for each section. In the example shown in FIG. 1 there are n=4 sections: 40%, 30%, 20% and 10% of dR max. The duration from t.sub.100 to each of these sections is defined at t.sub.40, t.sub.30, t.sub.20 and t.sub.10.

(21) By working with relative magnitudes for each blockade, the difference in particle size can be eliminated from the charge analysis calculations. When the proportional blockade magnitude is equal for any given particles, those particles are at the same position in the pore. The relative velocity of those particles can therefore be directly derived by comparing the time it has taken for the particles to get to that point from t.sub.100.

(22) Particles travelling through a pore with a net pressure and voltage bias will have three velocity components: 1. Convectionthe pressure driven flow of fluid will carry particles with it 2. Electrophoresisthe particle charge will cause the particles to move through the surrounding fluid towards the oppositely charged electrode 3. Electro-osmosisthe surface charge of the pore membrane (typically negative) attracts a higher density of oppositely charged (typically positive) ions to be present in the vicinity of the pore. These positive ions move towards the negative electrode and carry water molecules with them to create a plug flow of fluid.

(23) The magnitude of the convection component increases linearly with applied pressure, and the magnitudes of the electrical components increase linearly with applied voltage.

(24) Combining the Electrical Forces

(25) Electrophoresis and electro-osmosis are opposing forces if the particle charge (Zeta Potential .sub.p) has the same polarity as the membrane charge (Zeta Potential .sub.m). Their relative velocity components per mV of Zeta Potential are equal and opposite, so if the Zeta Potential of both the particle and the pore are known (as is the case when using calibration particles), the velocity components can be simplified to two: 1. Convection velocity or v.sub.c 2. Electrical velocity or v.sub.e obtained by setting the effective Zeta Potential of the pore to zero and setting the net Zeta Potential of the particle as follows:
.sub.net=.sub.p.sub.mEquation A:

(26) The relative velocity (v.sup.i.sub.x).sub.tot for a particle i at any given point in space x within the pore (reached after time T.sup.i.sub.x) is given by:

(27) $\begin{array}{cc}{\left(v_x^i\right)}_{\mathrm{tot}}=\frac{x}{T_x^i}=\frac{{}_0^{T_x^i}{v}^i\left(t\right)dt}{T_x^i}.& \mathrm{Equation}B\end{array}$

(28) Please note that x is set equal to 0 at the pore entrance. v.sub.tot is the sum of the time averaged electrical and convection velocity components (Equation C). Electrical and convection time averaged velocities are calculated in a similar way as v.sub.tot in equation B, by substituting v.sub.tot with v.sub.e or v.sub.c. Time averaged velocities are related with inverted times through Equation B.
(v.sub.x.sup.i).sub.tot=(v.sub.x.sup.i).sub.e+(v.sub.x.sup.i).sub.cEquation C:

(29) It should be noted that this equation can be applied at any given reference point x in the porefor the simple worked example the surface charge of the particle is calculated at each of the four reference points t.sub.40, t.sub.30, t.sub.20, and t.sub.10 and the answers are averaged. More advanced analysis methods can be applied to improve the data quality, as detailed below.

(30) The inverse times 1/T.sup.i.sub.x are averaged over N calibration particles (typically 200), as shown in Equation D.

(31) $\begin{array}{cc}\frac{1}{\mathrm{Tx}}=\underset{i=1}{\overset{N}{.Math.}}\frac{1}{T_x^i}/N& \mathrm{Equation}D\end{array}$

(32) In the same way (v.sup.i.sub.x).sub.tot are averaged over N calibration particles:
(v.sub.x).sub.tot=.sub.i=1.sup.N(v.sub.x.sup.i).sub.tot/NEquation E:
(v.sup.i.sub.x).sub.tot is proportional to 1/T.sup.i.sub.x (Equation B). For the purpose of the calculation and from here on (v.sup.i.sub.x).sub.tot is set equal to 1/T.sup.i.sub.x, with T.sup.i.sub.x being the duration the particle i takes, from entering the pore to the position x within the pore.

(33) Equation E also applies for v.sub.e and v.sub.v and hence Equation F applies:
(v.sub.x).sub.tot=(v.sub.x).sub.e+(v.sub.x).sub.cEquation F:
Calibrating the Pore and Quantifying the Convection Component

(34) Quantifying the convection component v.sub.c in Equation F will leave an electrical component v.sub.e, that can be used to calculate the surface charge density and zeta potential of the particle.

(35) Charged calibration particles of closely controlled diameter and known Zeta Potential .sub.p are typically used to calibrate the pore. A number of calibration particles (typically more than 200 per analysis) are analysed in the pore at a number of applied voltages V (e.g. V=0.3 V, 0.5 V and 0.7 V), and the average (v.sub.x).sub.tot is calculated by averaging (v.sup.i.sub.x).sub.tot over. N particles (see Equation E).

(36) The change in (v.sub.x).sub.tot with voltage is entirely due to the (v.sub.x).sub.e term, since the pressure is unchanged between the three measurements.

(37) (v.sub.x).sub.c can be calculated in several ways:

(38) Method 1: (v.sub.x).sub.tot is plotted against voltage and the defined line is extrapolated back to V=0, at which point there is no electrical force on the particles and hence no electrical velocity contribution ((v.sub.x).sub.e=so (v.sub.x).sub.c=(v.sub.x).sub.tot.

(39) Method 2: (v.sub.x).sub.c of the calibration is determined by measuring the calibration at various pressures and plotting (v.sub.x).sub.tot vs pressure. The slope of the (v.sub.x).sub.tot vs P linear curve is proportional to the relative convective velocity per unit pressure v.sub.x.sup.P which is defined as:
v.sub.x.sup.P=(v.sub.x).sub.c(P.sub.in+P.sub.ap)Equation G:

(40) P.sub.in is the inherent pressure head (a known constant for any given equipment setup) and P.sub.ap the applied pressure.

(41) v.sub.x.sup.P can be used to calculate the convection velocity (v.sub.x).sub.c at any pressure or vacuum setting P.sub.ap.(v.sub.x).sub.c and (v.sub.x).sub.e can be either determined together (by calculating one and subtracting that from (Vx)tot to determine the other) or calculated independently by application of a number of pressures and voltages (by plotting 1/T.sup.i.sub.x vs P and 1/T.sup.i.sub.x, vs V, respectively). The latter method may give better measurement repeatability when sample and calibration particles are analysed over a wide range of pressures and voltages. In particular, by calculating the two components separately it is possible to more accurately assess the velocity component when it is dominant, and similarly with the electrical component. When one component is dominant the value calculated for the other component may be less accurate. If the settings (voltage and pressure) are adjusted to make one force (convection or electrical force) dominant over the other, then it is possible to calculate that force and corresponding velocity component more accurately. The settings can then be adjusted to make the other force dominant, to more accurately calculate that other force and corresponding velocity component. This method may avoid the magnification of measurement errors of the non-dominant component as the relative contribution of the forces varies. For example, the convection velocity component may make up only a small fraction of the total velocity at the voltage sweep calibration settings, so large errors may be present in the calculated convective velocity per unit pressure. When a large pressure or vacuum is applied that error may be greatly magnified. Therefore, measurements may be taken at a second pressure value, and the flow per unit pressure can thus be calculated independently. This may result in a small discrepancy at the voltage sweep calibration point, but gives improved stability for applying a range of pressures and vacuums.

(42) Quantifying the Electrical Component for the Calibration Particles

(43) Knowing the average (v.sub.x).sub.tot and average (v.sub.x).sub.c, average (v.sub.x).sub.e can now be calculated for the calibration particles (Equation F), using the measurements taken at any of the non-zero voltages V. The relative electrical velocity per unit voltage v.sub.x.sup.V can be calculated as follows:

(44) $\begin{array}{cc}{v}_x^V=\frac{{\left(v_x\right)}_e}{V}& \mathrm{Equation}H\end{array}$

(45) v.sub.x.sup.V can be used to calculate the theoretical (v.sub.x).sub.e Cal of the calibration particles' at any voltage V.

(46) Calculating Zeta Potential of Sample Particles

(47) Particles with the same Zeta Potential have the same surface charge density (assuming that the Smoluchovski approximation applies which for particles of interest in physiological buffers is typically the case), and particles with the same surface charge density have the same electrophoretic mobility under an applied voltage, independent of particle diameter.

(48) The Zeta Potential of each sample (unknown) particle is calculated as follows:

(49) Calibration particles are cleaned out of the pore and fluid wells and sample particles are analysed in the same electrolyte at the same pore stretch (for a flexible pore). Applied pressure and voltage may differ from the calibration run.

(50) The convection velocity (v.sub.x).sub.c Sample for the sample particle measurement pressure is the product of v.sub.x.sup.P and the net pressure (P.sub.inP.sub.ap).sub.Sample(Equation G).

(51) The electrical velocity of the sample, particle i (v.sup.i.sub.x).sub.e Sample is the measured (v.sup.i.sub.x).sub.tot Sample minus the calculated (v.sup.i.sub.x).sub.c Sample (Equation F).

(52) The theoretical electrical velocity of the calibration particles (v.sub.x).sub.e Cal is calculated at the sample V.sub.Sample setting (Equation H) and the two velocities are compared as follows:

(53) $\begin{array}{cc}\frac{{\left(v_x^i\right)}_{e\mathrm{Sample}}}{{\left(v_x\right)}_{e\mathrm{cal}}}=\frac{{}_{x\mathrm{net}\mathrm{Sample}}^i}{{}_{\mathrm{net}\mathrm{Cal}}}& \mathrm{Equation}I\end{array}$

(54) .sub.net Sample can be calculated because the three other terms are known. The Zeta Potential of each sample particle can now be calculated using equation A:
.sub.p Sample=.sub.net Sample+.sub.mEquation J:

(55) The zeta potential of each sample particle i can be calculated from the ratio of the electrical velocities of sample and calibration (Equation I). The zeta potential of each sample particle i is given by averaging respective zeta potential values, calculated at positions x.

(56) $\begin{array}{cc}\begin{array}{c}{}_{\mathrm{Sample}}^i=\frac{\underset{x}{\overset{}{.Math.}}{}_{x,\mathrm{Sample}}^i}{\underset{x}{\overset{}{.Math.}}}\\ =\frac{\begin{array}{c}\underset{x}{\overset{}{.Math.}}\left({\left(v_x^i\right)}_{\mathrm{Sample}}-v_x^P*{\left(P_{\mathrm{in}}+P_{\mathrm{ap}}\right)}_{\mathrm{Sample}}\right)/\\ \left({\left(v_x^V\right)}_{\mathrm{Cal}}*V_{\mathrm{Sample}}\right)\end{array}}{\underset{x}{\overset{}{.Math.}}}*\\ {}_{\mathrm{net}\mathrm{Cal}}+{}_m\end{array}& \mathrm{Equation}K\end{array}$
Maximising Information Content from the Data

(57) The simplest embodiment of the method analyses pre-defined points on each blockade (40% to 10% of the blockade peak magnitude dR max in the example above).

(58) In reality the data stream is neither pure nor continuous. Depending on a number of factors including the nanopore size and shape, the applied pressure, the sampling frequency of the electronics and the signal to noise ratio of the blockades, certain sections of each analysed blockade will contain more information than others.

(59) Referring to the blockade shape in FIG. 1, the accuracy of information extracted from each blockade depends primarily on the accuracy of: 1. Identifying the blockade maximum dR max (or a distinct datum feature for alternative pore geometries see FIG. 2) 2. Identifying the time of dR max (t100 or another distinct datum feature FIG. 2) 3. Identifying t40, t30, t20, t10 (or other distinct features on the blockade FIG. 2)

(60) A particle travelling very fast will move a long way between data sampling points. It is therefore likely that the very peak of the blockade Will fall between sample points and will not be correctly identified, leading to an error in the value of both dR max and t100. An error in t100 will generate a large percentage error in the calculated t90, for example, but a much smaller percentage error in t10 so the trailing end of the blockade may give better quality information than the area around the peak.

(61) A particle travelling slowly will not move a long way between data sampling points, so the identified blockade peak dR max and time t100 are likely to be relatively accurate. In this case, better information may be obtained at t90 than at t10, due to the (9) higher signal to noise ratio around the blockade peak. When noise is a high percentage of the measured signal, the calculated time to reach the designated blockade height (e.g. t10) becomes less accurate.

(62) Information quality can be improved in a number of ways, including but not limited to the following: 1. Adjust the applied pressure and voltage to optimise the blockade profiles for each sample. This could either be done by feedback to the user to make system adjustments, or fully automated. An optimised system would aim for blockades with a number of sample points around the peak area, but even the least charged particle must still travel through the pore in the same direction as the calibration particles. 2. There will be variation in size and surface charge within the sample even when the system settings have been optimised, so optimising the information extracted from faster and smaller blockades is still critical: a. Using the known profile of the blockade from the calibration particle measurements and extrapolating from points further down the blockade, the peak dR max and the time t100 for each sample blockade can be calculated more accurately b. Using similar extrapolation at the tailing end of the blockade will reduce the impact of noise on the measured resistance. 3. Identify the section of each blockade that will give optimum information quality by calculating the timing errors and noise errors across the whole blockade. 4. Implement signal to noise improvements by, for example: a. Modifying the system electronics to allow application of a larger voltage for a given pore size. b. Implement algorithms that allow signal information to be extracted from within the noise floor. Such algorithms are well known in the art.
Application of the Method to Alternative Geometries

(63) The principles of the method can be applied to pores of any geometry, by identifying one or more distinct features in the blockade profile that allow the relative velocities of different particles to be calculated. The example in FIG. 2 shows a pore with a different profile.

(64) With the pore shape in FIG. 2 there are four distinct features that could be used to calculate relative times Tn. For example, the time difference between the two maxima could be used:
Tn=TcTbEquation M:

EXAMPLE 1

(65) This example shows the use of calibration particles to establish v.sub.x.sup.P and v.sub.x.sup.e Measurements of the calibration particles were made at three different applied voltages; 0.4V, 0.5V, and 0.6V. The results are displayed in FIG. 3 and FIG. 4. The measurement details are:

(66) NanoporeIzon NP200

(67) Carboxylated polystyrene calibration particles CPC200 Izon Tris Buffer 0.1 M KCl Applied Voltages (V): 0.4 V, 0.5 V and 0.6 V Applied Pressure (Pap): 0 mm H.sub.2O Inherent Pressure (Pin): 4.7 mm H.sub.2O Particle Count369 at V=0.4 V, 619 at V=0.5 V, 596 at V=0.6 V

(68) At V=0 V there are no electrical forces so (v.sub.x).sub.tot=(v.sub.x).sub.c: (v.sub.x).sub.c 40=2806 s.sup.1 and v.sub.x.sup.P.sub.40=(2806/4.7)=597 s.sup.1 (v.sub.x).sub.c 30=1672 s.sup.1 and v.sub.x.sup.P.sub.30=(1672/4.7)=356 s.sup.1 (v.sub.x).sub.c 20=1131 s.sup.1 add v.sub.x.sup.P.sub.20=(1131/4.7)=241 s.sup.1 (v.sub.x).sub.c 10=626 s.sup.1 and v.sub.x.sup.P.sub.10=(626/4.7)=84 s.sup.1
(v.sub.x).sub.c for the calibration particles can be calculated at any of the applied voltages, for example (v.sub.x).sub.c 40 is calculated below at all three voltages:

(69) TABLE-US-00001 V (v.sub.x).sub.tot 40 (v.sub.x).sub.c 40 (v.sub.x).sub.e 40 (s.sup.1) = v.sub.x.sup.e.sub.40 (s.sup.1) = (V) (s.sup.1) (s.sup.1) (v.sub.x).sub.tot 40 (v.sub.x).sub.c 40 (v.sub.x).sub.e 40/V 0.4 6224 2806 3418 8454 0.5 7166 2806 4360 8720 0.6 7946 2806 5140 8567 Average: 8580

EXAMPLE 2

(70) This example demonstrates measuring the sample at multiple pressures. This illustrates the fact that system pressure can be varied without having any material effect on the analysis result as can be seen in FIG. 5. The Measurement details are: Calibration (and Sample) Particlescarboxylated polystyrene CPC200 NanoporeIzon NP200 Electrolyte Izon Tris Buffer 0.1 M KCl Applied Voltage (V): 0.8 V Applied Pressure (Pap): 0 mm H.sub.2O to 8 mm H.sub.2O in steps of 2 mm (P0 to P8) Inherent Pressure (Pin): 4.7 mm H.sub.2O System calibrated with ZERO applied pressure

(71) It can be seen from FIG. 5 that the measured mean Zeta potential does not change significantly with applied pressure. This result is obtained using v.sub.x.sup.P against calibration particles measured at zero applied pressure. Pulse height is slightly truncated as applied pressure increases, leading to a reduced mean diameter value.

EXAMPLE 3

(72) This example demonstrates measuring the sample at multiple bias voltages. This illustrates the fact that applied bias can be varied without having any material effect on the analysis result as can be seen in FIG. 6. The Measurement details are: Calibration (and Sample) Particlescarboxylated polystyrene CPC200 NanoporeIzon NP200 ElectrolyteIzon Tris Buffer 0.1 M KCl Applied Pressure (Pap): 0 mm H.sub.2O Inherent Pressure (Pin): 4.7 mm H.sub.2O Applied Voltage (V): 0.8 V for calibration System calibrated with ZERO applied pressure

(73) It can be seen from FIG. 6 that the measured mean Zeta potential does not change significantly with applied voltage. This result is obtained using v.sub.x.sup.e against calibration particles measured at 0.8 V and zero applied pressure.

EXAMPLE 4

(74) This example demonstrates measuring charged and uncharged particles of different diameter. The measurement details are: Calibration Particlescarboxylated polystyrene, mean diameter 118 nm NanoporeIzon NP100 ElectrolytePBS 0.25 M Applied Pressure (Pap): 0 mm H.sub.2O calibration Inherent Pressure (Pin): 4.7 mm H.sub.2O Applied Voltage (V): 0.66 Vcalibration and sample

(75) It can be seen from FIG. 7 that the plain NIST traceable polystyrene particles (CPN100 and CPN150) are clearly distinguished from the carboxylated polystyrene CPC70, Bangs 6569 and CPC100 particles.

EXAMPLE 5

(76) This example demonstrates the measurement of particle zeta potential after deliberate surface modification to demonstrate detection of specific surface targets. This example shows the binding of a protein (CD63) to a liposome that was modified to bind CD63. FIG. 8 shows the liposomes before and after binding of CD63. A shift in zeta potential toward a more positive value is observed upon binding of protein, which was expected as the protein is slightly positively charged in experimental conditions. In FIG. 8, it can be seen that the liposomes before protein binding form a distinct group from the liposomes with surface-bound protein. This illustrates analyses that can, be used to detect particular targets in the sample. FIG. 8 also shows a control particle set of known, negative charge, which validates the results.

EXAMPLE 6

(77) This example demonstrates running an analysis whereby calibration particles (of a known Zeta Potential) are added to the sample fluid. FIG. 9 shows the measurement of lecithin liposomes with calibration particles added as a sample spike run within the sample set. The two similarly sized particle sets (Liposomes and calibration particles) can be clearly distinguished based on their surface charge, and the accuracy of the data can be benchmarked by the Zeta Potential result for the calibration particles (which have a known average Zeta Potential of 20 mV in this electrolyte).

(78) General Remarks

(79) As set out in the present specification, according to embodiments of the present invention a number of values (such as pressure, time, voltage, current etc.) are measured, determined, calculated or otherwise derived, as well as processed, plotted, stored or otherwise used. It will be understood that, where appropriate (and whether or not specifically mentioned) a reference to such values may include a reference to values derived therefrom.

(80) Although the invention has been described in terms of preferred embodiments as sei forth above, it should be understood that these embodiments are illustrative only and that the claims are not limited to those embodiments. Those skilled in the art will be able to make modifications and alternatives in view of the disclosure which are contemplated as falling within the scope of the appended claims. Each feature disclosed or illustrated in the present specification may be incorporated in the invention, whether alone or in any appropriate combination with any other feature disclosed or illustrated herein.