Method to determine the throughput speed of a pore

Abstract

A method to determine the throughput speed v of a pore, comprising the steps of feeding, by means of a driving force F, a filiform calibration element through the pore, the calibration element having a plurality of markers spaced apart by known distances and configured to produce an interaction event that transmits a signal away from the pore upon interaction with the pore, detecting a plurality of interaction events, and determining a time interval Δt between successive interaction events, and/or a frequency ω of interaction events.

Claims

1. A method to determine the throughput speed v of a pore, comprising the steps of: feeding, by means of a driving force F, a filiform calibration element through the pore, the calibration element having a plurality of markers spaced apart by known distances and configured to produce an interaction event that transmits a signal away from the pore upon interaction with the pore; detecting a plurality of interaction events; determining a time interval Δt between successive interaction events, and/or a frequency ω of interaction events; determining, from the known distances and from the time interval Δt and/or the frequency ω, the speed with which the filiform calibration element passes through the pore as the throughput speed v; and further comprising: determining a throughput resistance R of the pore as a ratio of the speed v per unit of the driving force F.

2. A method to determine the throughput speed v of a pore, comprising the steps of: feeding, by means of a driving force F, a filiform calibration element through the pore, the calibration element having a plurality of markers spaced apart by known distances and configured to produce an interaction event that transmits a signal away from the pore upon interaction with the pore; detecting a plurality of interaction events; determining a time interval At between successive interaction events, and/or a frequency ω of interaction events; and detecting at least one increase in the mechanical resistance that the filiform calibration element experiences when moving through the pore as an interaction event.

3. A method to determine the throughput speed v of a pore, comprising the steps of: feeding, by means of a driving force F, a filiform calibration element through the pore, the calibration element having a plurality of markers spaced apart by known distances and configured to produce an interaction event that transmits a signal away from the pore upon interaction with the pore; detecting a plurality of interaction events; determining a time interval Δt between successive interaction events, and/or a frequency ω of interaction events; and measuring an intensity of light that is emitted from a marker with a detector outside the pore, and detecting at least one change of the intensity as an interaction event.

4. The method of claim 3, wherein the change of the intensity is an increase of the intensity that results from an amplification of the light by the inner wall of the pore, or an extinction of the light produced by the inner wall of the pore.

5. A method to determine the throughput speed v of a pore, comprising the steps of: feeding, by means of a driving force F, a filiform calibration element through the pore, the calibration element having a plurality of markers spaced apart by known distances and configured to produce an interaction event that transmits a signal away from the pore upon interaction with the pore; detecting a plurality of interaction events; determining a time interval At between successive interaction events, and/or a frequency ω of interaction events; and wherein the feeding a filiform calibration element specifically comprises: contacting the pore with a liquid solution comprising a plurality of filiform calibration elements.

6. A method to determine the throughput speed v of a pore, comprising the steps of: feeding, by means of a driving force F, a filiform calibration element through the pore, the calibration element having a plurality of markers spaced apart by known distances and configured to produce an interaction event that transmits a signal away from the pore upon interaction with the pore; detecting a plurality of interaction events; determining a time interval At between successive interaction events, and/or a frequency ω of interaction events; and wherein the driving force F specifically comprises one or more of: a pressure gradient, a concentration gradient, and an electrostatic force.

7. A filiform calibration element, the filiform calibration element comprising: a strand; a plurality of light sources coupled to the strand, wherein the filiform calibration element is configured to pass through a pore of at most 5 μm diameter, wherein the plurality of light sources are configured to absorb excitation light of a first wavelength λ.sub.1 and to emit light of a second wavelength λ.sub.2 in response to the excitation light, as markers, wherein the light sources are quantum dots, and wherein the strand is a DNA strand with at least one sticky end, at least one quantum dot is coupled to a counterpart of the sticky end by means of a thiol group, and the counterpart is attached to the sticky end of the DNA strand.

8. A filiform calibration element, the filiform calibration element comprising: a strand; a plurality of light sources coupled to the strand, wherein the plurality of light sources are configured to absorb excitation light of a first wavelength λ.sub.1 and to emit light of a second wavelength λ.sub.2 in response to the excitation light, as markers, and wherein the filiform calibration element is configured to pass through a pore of at most 200 nm diameter.

9. A filiform calibration element, the filiform calibration element comprising: a strand; a plurality of light sources coupled to the strand, wherein the filiform calibration element is configured to pass through a pore of at most 5 μm diameter, wherein the plurality of light sources are configured to absorb excitation light of a first wavelength λ.sub.1 and to emit light of a second wavelength λ.sub.2 in response to the excitation light, as markers, and wherein the filiform calibration element is further functionalized to be electronegative, so as to facilitate the driving of the filiform calibration element through the pore by means of an electrostatic driving force F.

10. A liquid solution as a tool for determining the throughput speed v of at least one pore, comprising: a solvent and a plurality of filiform calibration elements comprising: a strand; a plurality of light sources coupled to the strand, wherein the filiform calibration element is configured to pass through a pore of at most 5 μm diameter, and wherein the plurality of light sources are configured to absorb excitation light of a first wavelength λ.sub.1 and to emit light of a second wavelength λ.sub.2 in response to the excitation light, as markers.

11. The liquid solution of claim 10, wherein the solvent comprises buffered KCl.

12. The liquid solution of claim 11, wherein the concentration of the buffered KCl is up to 1 M KCl and the concentration of filiform calibration elements is between a few fM to hundreds of nM.

Description

DESCRIPTION OF THE FIGURES

(1) In the following, the invention is illustrated using Figures without any intention to limit the scope of the invention. The Figures show:

(2) FIG. 1: Exemplary embodiment of the method 100 according to the invention;

(3) FIG. 2: Mechanical interaction 3 between markers 21 and the inlet of the pore 1;

(4) FIG. 3: Easing of an electronegative strand 22 into the pore 1 by means of an electric field E;

(5) FIG. 4: Detection of a single quantum dot 21 with a macroscopic detector 57.

(6) FIG. 1 illustrates an exemplary embodiment of the method 100 according to the invention. FIG. 1 is schematic and not drawn to scale. A glass base 55 and a cylindrical wall 54 define a space that is divided into a trans-space 51 and a cis-space 52 by a solid-state membrane 53. Seals between the glass base 55, the cylindrical wall 54 and the solid-state membrane 53 are formed by polymethyldisiloxane (PDMS) 56. The solid-state membrane 53 comprises a conical pore 1 with an inner wall 1a. Part of the inner wall 1a of the pore 1, and the surrounding area on the surface of the membrane 53, is covered by PDMS 56 as well as a result of the fabrication process.

(7) Both the trans-space 51 and the cis-space 52 are filled with a solution 20 that comprises a solvent 24 and filiform calibration elements 2. Only one filiform calibration element 2 is shown in FIG. 1 for clarity.

(8) The filiform calibration element 2 comprises a DNA-like strand 23 to which a plurality of quantum dots 21 as markers are attached, spaced apart by known distances 22 along the length of the strand 23. For reasons of legibility, only two quantum dots 21 are labelled with reference signs in FIG. 1, and the distance 22 is indicated only once as well.

(9) In step 110 of the method 100, the filiform calibration element is fed and pulled through the pore 1 in the membrane 53 by means of a driving force F. At the same time, the inside of the pore 1 is irradiated with light of wavelength λ.sub.1 by a laser 58. Every quantum dot 21 that happens to be irradiated with the light of wavelength λ.sub.1 will be excited to emit light of wavelength λ.sub.2 in return. When the quantum dot 21 reaches the bottom of the pore 1, a resonance condition is fulfilled, and the interaction with the inner wall 1a of the pore 1 results in a drastic amplification of the light of wavelength λ.sub.2 that is emitted from the quantum dot 21. This amplification is used as the interaction event 3 to calibrate the throughput speed v.

(10) This amplified light is detected by a detector 57 in step 122. As per step 120, a plurality of interaction events 3 is detected. In step 130, a time interval Δt between successive interaction events, and/or a frequency ω of interaction events 3, is determined. In step 140, the desired throughput speed v is evaluated from the time interval Δt, and/or from the frequency ω, in combination with the known distance 22 between any two quantum dots 21. Optionally, in step 150, the throughput resistance R may be determined from the throughput speed v in combination with the amount of the driving force F.

(11) FIG. 2 illustrates a different possible interaction 3 between markers 21 on a DNA-like strand 22 that may be used to calibrate the throughput velocity v of a pore 1 in a membrane 53. Akin to FIG. 1, the membrane 53 separates a trans-space 51 and a cis-space 52. The filiform calibration element 2 is pulled through the pore 1 from the trans-space 51 into the cis-space 52 by applying a driving force F, which is at the same time measured. Each time one of the markers 21 hits the entrance of the pore 1, it produces a mechanical resistance that sends a force signal through the strand 22 to the measurement instrument that is used to measure the driving force F. This is the interaction event 3 that is used to denote precise points in time where a marker 21 is exactly at the entrance of the pore 1, and that can be detected in step 121 of the method 100. Once the mechanical resistance has been overcome by the driving force F, the marker is flipped over and slides against the inner wall 1a of the pore 1 with only little resistance. After having passed the pore 1, the marker 21 is flipped back to its previous orientation perpendicular to the strand by means of a restoring force.

(12) FIG. 3 illustrates how a filiform calibration element 2 that is floating in a solvent 20 may be eased into a pore 1. Between the trans-space 51 and the cis-space 52, a voltage is applied from a voltage source 60 by means of electrode 59a in the trans-space 51 and electrode 59b in the cis-space 52. The field lines of the electric field E converge on the pore 1 and re-orient the electronegative strand 22 of the filiform calibration element 2 so that its end will be pulled into the pore 1 by the electric field E.

(13) FIG. 4 is a real measurement result acquired with a macroscopic detector 57 as illustrated in FIG. 1. The detector 57 is sensitive to the wavelength λ.sub.2 of the light that is emitted by the quantum dot 21. Its sensitivity is set so that the light intensity emitted from a quantum dot 21 is only noticeable if an interaction event 3 is occurring at the bottom of the pore 1 and the light emitted from the quantum dot 21 is amplified accordingly, as illustrated in FIG. 1. Therefore, it does not matter that the quantum dot 21 is much smaller than the wavelength λ.sub.2. The diffraction limit may dictate that two or more quantum dots 21 with comparable light intensities cannot be distinguished from one another, but this does not preclude the detection of one single sufficiently intense light source.

(14) Only three field lines of the electric field E are exemplarily drawn in FIG. 3 for clarity.

LIST OF REFERENCE SIGNS

(15) 1 pore 1a inner wall of pore 1 2 filiform calibration element 20 solution, comprising filiform calibration elements 2 21 markers of filiform calibration element 2 22 spacing between markers 21 23 strand of filiform calibration element 2 24 solvent 3 interaction event 51 trans-space 52 cis-space 53 membrane, comprising pore 1 54 cylindrical wall 55 glass base 56 polymethyldisiloxane (PDMS) 57 detector for light of wavelength λ2 58 laser for light of wavelength λ1 59a electrode in trans-space 51 59b electrode in cis-space 52 60 voltage source E electric field F driving force λ.sub.1 first wavelength, emitted by laser 58 to excite marker 21 λ.sub.2 second wavelength, emitted by marker 21 R throughput resistance of pore 1 Δt time interval between interaction events 3 v throughput speed of pore 1 ω frequency of interaction events 3 100 method 110 feeding filiform calibration element 2 through pore 1 120 detecting interaction events 3 121 detecting increase in mechanical resistance as interaction event 3 122 detecting change in light intensity as interaction event 3 130 determining time interval Δt and/or frequency ω 140 determining throughput speed v of pore 1 150 determining throughput resistance R of pore 1