Nanopore-based determination of protein charge, shape, volume, rotational diffusion coefficient, and dipole moment
View Patent ↗Physical parameters of macromolecules are determined by measuring electrical current I over time for translocation events as the macromolecules in solution move between two liquid compartments that are separated by and fluidically coupled through a synthetic nanopore. Values of charge, volume, shape, rotational diffusion coefficient, ad dipole moment are derived from the measurements.
1. A method of deriving values for physical parameters of a particle, comprising:
(a) measuring electrical current I or equivalent electrical parameter as a function of time between two liquid compartments separated by and fluidically coupled through a synthetic nanopore upon translocation of the particle and a plurality of other particles in a plurality of translocation events from one liquid compartment through the synthetic nanopore to the other liquid compartment;
(b) detecting translocation events by recognizing a change in current ΔI relative to a baseline current;
(c) collecting values of intra-event values ΔI(t);
(d) for the plurality of translocation events, finding a value ΔI max of a maximum change in current and a value ΔI min of a minimum change in current;
(e) deriving a volume Λ of the particle and a length to diameter ratio m of the particle from the distribution of values of ΔI max from the translocation events; and
(f) deriving one or more of a rotational diffusion coefficient D R and a dipole moment μ of the particle from the volume Λ of the particle, the length to diameter ratio m of the particle, and the intra-event values ΔI(t).
2. A method according to claim 1 , wherein step (a) comprises measuring an equivalent electrical parameter selected from conductivity, resistivity, resistance, conductance, or voltage between the two compartments.
3. A method according to claim 1 , wherein step (a) comprises measuring the current between the two liquid compartments.
4. The method according to claim 1 , comprising maintaining a voltage between an anode in the one liquid compartment and a cathode in the other, and measuring the current over time as perturbations arising from the passage of individual particles through the synthetic nanopore.
5. The method according to claim 1 , wherein in step (b) a translocation event is detected when a transient decreases in current is greater than 5 times the standard deviation of the measured current.
6. The method according to claim 1 , wherein the particle is a macromolecule.
7. The method according to claim 6 , wherein the particle is a protein.
8. The method according to claim 7 , wherein the protein is a spherical protein.
9. A method of detecting the presence of a target macromolecule in a fluid composition, the method comprising determining one or more parameters selected from a volume Λ, a length to diameter ratio m, a rotational diffusion coefficient D R and a dipole moment μ of a macromolecule in the fluid composition by a method according to claim 6 , comparing the determined parameter or parameters to corresponding known parameters of the target macromolecule, and determining presence of the target macromolecule in the fluid composition if the parameters of the macromolecule match the parameters of the target macromolecule.
10. A method for determining one or more of a volume Λ, a length to diameter ratio m, a rotational diffusion coefficient D R and a dipole moment μ of a macromolecule in a fluid composition comprising an electrolyte, comprising
causing macromolecules in the fluid composition to pass through a nanopore between two liquid compartments in a plurality of translocation events, while the two liquid compartments are held at different voltages causing an analog current signal Ia(t) to flow from one liquid compartment to the other, where the analog current signal is measured as a function of time;
converting the analog current signal into a digital signal I(t) and recording I(t) in data files;
transforming the I(t) data to detect the translocation events and to provide intra-event data;
further transforming the measured I(t) values by computing ΔI(t) for at least a representative sample of translocation events by subtracting I(t) during the translocation events from a baseline I(t) signal;
finding the maximum ΔI value of the ΔI(t) from the translocation events measured in the preceding step;
calculating a distribution P(ΔI) of the maximum ΔI values of the measured events;
if P(ΔI) is normal:
calculating the volume A according to equation (1),
Δ
I
=
-
Λ
V
A
γ
ρ
(
L
P
+
0.8
D
P
)
2
S
(
d
M
D
P
)
(
1
)
where ΔI in equation (1) is the maximum of the normal distribution, γ is a shape factor, V A is the applied voltage, L P is the length of the nanopore, D P is the diameter of the nanopore, and ρ is the resistivity of the electrolyte, and S(d M /D p ) is a correction factor set equal to unity;
if P(ΔI) is not normal:
fitting P(ΔI) with a model that takes into account the distribution of electrical shape factors, equation (1), and the measurement errors of the technique to estimate ΔI min and ΔI max ;
substituting the estimated values of ΔI min and ΔI max into equations (S14) and (S15); and
Λ
(
m
)
=
{
Λ
(
γ
⊥
(
m
)
,
Δ
I
min
)
Λ
(
γ
∥
(
m
)
,
Δ
I
max
)
if
m
<
1
(
S14
)
Λ
(
m
)
=
{
Λ
(
γ
∥
(
m
)
,
Δ
I
min
)
Λ
(
γ
⊥
(
m
)
,
Δ
I
max
)
if
m
>
1
(
S15
)
solving the system for the excluded volume of the protein, Λ and the value of in;
converting intra-event ΔI(t) signals to angle of the particle θ(t); and
solving for rotational diffusion coefficient, D R , and dipole moment, μ of the macromolecule.
11. The method according to claim 10 , wherein the nanopore is lined with a fluid wall.
12. The method according to claim 10 , wherein the macromolecule is a protein.
13. The method according to claim 11 , further comprising processing the digital signal I(t) with a low-pass filter.
14. The method according to claim 11 , comprising recognizing the start of an event when the measured transient decrease in I(t) is above a threshold of five times the standard deviation of the I(t) signal.
15. The method according to claim 10 , comprising computing ΔI(t) values for all of the events.
16. A method of identifying one or more target macromolecules in a test composition containing a plurality of different macromolecules by measuring and comparing physical parameters of the different macromolecules in solution, the method comprising
(a) combining the plurality of different macromolecules in a test composition, or providing a composition comprising the plurality of different macromolecules;
(b) measuring electrical current I or equivalent electrical parameter as a function of time between two liquid compartments separated by and fluidically coupled through a synthetic nanopore upon translocation of particles comprising the plurality of different macromolecules in a plurality of translocation events from one liquid compartment through the synthetic nanopore to the other liquid compartment;
(c) detecting translocation events by recognizing a change in current ΔI relative to a baseline current,
(d) collecting values of intra-event values ΔI(t);
(e) deriving a volume Λ and/or a length to diameter ratio m from the intra-event values ΔI(t); and/or
(f) deriving one or more of a rotational diffusion coefficient D R and a dipole moment μ from the volume Λ, the length to diameter ratio m, and the intra-event values ΔI(t); and
(g) identifying the presence of the target macromolecule by matching the derived volume, length to diameter ratio, rotational diffusion coefficient, and/or dipole moment with the known values of the target macromolecule.
17. The method according to claim 16 , further comprising quantifying the relative number of the different macromolecules in the solution through the steps of
(h) carrying out step (g) for all translocation events or for a representative sample of translocation events;
(i) classifying each translocation event as translocation of one of the target macromolecules in the solution; on the basis of the parameter or parameters determined in steps (e) and/or (f); and
(j) counting the data in step (i) to quantify the target macromolecule.
18. The method according to claim 17 , wherein the solution comprises a first macromolecule, a second macromolecule, and a complex comprising both the first and second macromolecule, and where the method further comprises deriving an affinity constant for binding of the first macromolecule to the second macromolecule from the data of step (j).
19. The method according to claim 18 , wherein the target macromolecules are proteins.
20. The method according to claim 18 , wherein the first macromolecule is a protein and the second macromolecule is an antibody that binds the protein.