WO2013179066A1

WO2013179066A1 - Nanopore for analysing biopolymers

Info

Publication number: WO2013179066A1
Application number: PCT/GB2013/051469
Authority: WO
Inventors: Jekaterina KULESHOVA
Original assignee: Base4 Innovation Ltd
Priority date: 2012-06-01
Filing date: 2013-06-03
Publication date: 2013-12-05
Also published as: GB201209856D0; EP2856151A1

Abstract

The present invention provides a device suitable for an analysing a biopolymer comprising a substrate perforated with at least one nanopore characterised in that the substrate is a composite comprised of stacked layers of at least two different solid inorganic compounds and wherein the inorganic compounds used in adjacent layers have different points of zero charge.

Description

NANOPORE FOR ANALYSING BIOPOLYMERS

The present invention relates to a nanopore device which is adapted to be an integral element of an apparatus for analysing a biopolymer or to be an easily replaceable component thereof. It is especially useful in sequencers designed to determine the sequence of nucleotides in polynucleotides such as DNA and RNA or amino acids in proteins and the like.

Next generation sequencing of genetic material is already making a significant impact on the biological sciences in general and medicine in particular as the unit cost of sequencing falls in line with the coming to market of faster and faster sequencing machines. For example, our co-pending application WO 2009/030953 discloses a new fast sequencer in which inter alia the sequence of nucleotides (bases or base pairs) in a single or double stranded polynucleotide sample (e.g. naturally occurring RNA or DNA) is read by translocating the same through a nano-perforated substrate provided with plasmonic nanostructures juxtaposed within or adjacent the outlet of the nanopores. In this device, the plasmonic nanostructures define detection windows (essentially an electromagnetic field) within which each nucleotide (optionally labelled) is in turn induced to fluoresce or Raman scatter photons in characteristic way by interaction with incident light. The photons so generated are then detected remotely, multiplexed and converted into a data stream whose information content is characteristic of the nucleotide sequence associated with the polynucleotide. This sequence can then be recovered from the data stream using computational algorithms embodied in corresponding software programmed into a microprocessor integral therewith or in a computing device attached thereto.

Another apparatus for fast sequencing polynucleotides is described for example in US 6627067, US 6267872 and US 6746594. In its simplest form, this device employs electrodes, instead of plasmonic nanostructures, to define the detection window across the substrate or in or around the outlet of the nanopore. A potential difference is then applied across the electrodes and changes in an electrical property of the ionic medium flowing therebetween, as a consequence of the electrophoretic translocation of the polynucleotide and associated electrolyte therethrough, is measured as a function of time. In this device, as the various individual nucleotides pass through the detection window they continuously block and unblock it causing 'blocking events' which give rise to characteristic fluctuations in current flow or resistivity. These fluctuations are then used to generate a suitable data stream for analysis as described above.

Whilst the nanopores employed in these apparatuses can be organic and suitably derived from biological material, it is preferred to create them in an inorganic, insulating substrate such as silicon nitride. In one embodiment of this approach, a device, akin to a computer chip, is fabricated by nano-perforating a thin wafer of the substrate to produce a membrane type structure. This can

3294976v1 then be located in a receiving assembly in the sequencer between analyte providing and receiving reservoirs which assembly is further adapted if required to provide the necessary electrical power to the device. In use, the analyte solution, being the polynucleotide analyte and an electrolyte carrier, is caused to translocate through the nanopores forming the basis for the measurement methodologies described above. After use the device can be removed and replaced by another thereby minimising the risk of sample contamination.

One problem encountered when using a device based on a simple silicon nitride membrane is that translocation of the analyte through the nanopore is very fast making it difficult to distinguish, the case of a polynucleotide, between adjacent nucleotides in the sequence. It would therefore be desirable to generate a 'smart' device whereby the translocation speed of the analyte could be optimised for a given situation.

A number of approaches to solving this problem have been discussed in the prior ait. Kejian et al (Applied Physics Letters 94 2009 014101) teach that the movement of an electrolyte through a solid state nanopore is a function of the zeta potentials between the bulk of the electrolyte and the stationary phase adhering to its walls. They thus predict that that a change in charge density on the nanopore will modify the analyte translocation rate and have modelled the effect in certain systems. Firnkes et al (Nanoletters 10 2010 2162-2167) have also examined the competitive actions of diffusion, electrophoresis and electroosmosis in the translocation of the model protein avidin through silicon nitride nanopores.

Siwy et al (JACS Communications 14^lh August 2004 126 10850) have investigated the effect that a gold nanotube with fixed surface charge and radius comparable to the thickness of the electrical double layer has on the rectification of the ion current through the pore.

Yen et al (Review of Scientific Instruments 83 2012 034301) teach that a twenty fold increase in DNA translocation through a silicon nitride nanopore can be achieved by applying a gate voltage thereto. However this results in a reduction in the number of nanopore blocking events in line with theoretical predictions.

The use of organic materials as nanopore modifiers to control surface charge has also been taught. Thus, Wananau et al (Nano Letters 7(6) 2007 1580-1585) teach various methods for chemically modifying 5-20nm nanopores fabricated in a silicon nitride substrate by coating the same with various organic polymers.

Yameen et al (JACS web published 21^st January 2009) demonstrate that the electrical properties of conical nanochannels in a nano-perforated membrane can be fine tuned by coating the inside with zwitterionic poly(methacryoyl- L-Lysine) 'brushes' . These brushes confer surface charge density characteristics which are pH dependent.

3294976v1 Vlassiouk and Siwy (Nano Letters 7(3) 552-557) have disclosed a nanofluidic diode comprising a conical nanopore fabricated in a poly(ethylene terephthalate) substrate which has been modified with an organic coupling agent comprising l-ethyl-3-[3- di(methylamino)propyl]carbodiimide hydrochloride.

The use of alternative substrates has been considered. Thus, Bashir et al (Advanced

Materials 21 2009 2771-2776 and Advanced Functional Materials 20 2010 1266-1275) have disclosed solid-state nanopore sensor employing only an alumina substrate. DNA translocation can be reduced by an order of magnitude due to high positive surface charge densities and the nucleation of charged nano-crystalline domains. It also describes the use of electron-beam radiation to modify the surface of the alumina membrane in terms of the various alumina phases present enabling the electrical properties of the fluid nanopore interface to be tuned. Finally, it discloses a manufacturing method whereby layers of alumina and silicon nitride are built up by sequential deposition on a silicon substrate and then the top and bottom silicon and silicon nitride layer are etched away to leave completely exposed alumina into which is drilled the nanopore using a focused electron beam. However it is our experience that the mechanical strength of these substrates can under certain circumstance be insufficient for the duty required of them.

Bashir et al (Biomed. Microdevices 13 2011 671-682) disclose alumina nanopore sensors which have been coated with a lipid biolayer. These devices exhibit improved superior biological functionality relative to coated silicon oxide or titanium oxide substrates by virtue of the lipids exhibiting higher diffusivities in the layer itself.

Chen et al (Nanopore Letters 4(7) 2004 1333-1337) have shown that atomic layer deposition of alumina on a silicon nitride nano-perforated membranes can neutralise surface charge and improve capture of DNA translocating therethrough from an analyte sample. The effect this has on ion-current flow and base pair blocking events is also discussed.

Wanunu et al (Nature Nanotechnology 24^th October 2010) show that thinning silicon nitride nanopore wafers to less than lOnm still allows the creation of nano-perforated membranes which are robust and improves signal amplitude from biomolecules. It is said that substrate thicknesses in the 3-1 Onm range allow discrimination between small polynucleotides.

Finally, Wanunu et al (Nature Nanotechnology 20^th December 2009) show that polynucleotide capture rates increase as their lengths increase from 800 to 8000bp and that capture rates increase when an ionic gradient is established across the nanopore. In particular, a 20 fold salt concentration gradient across the nanopore enables picomolar levels of DNA to be detected in a sample.

US 2012/0037919 Al discloses a nanopore electrical sensor having a layered structure. The structure includes, from bottom to top, a substrate, a first insulating layer, a symmetrical

3294976V1 electrode and a second insulating layer. The substrate may be the same material as the first insulating layer.

US 2005/0241933 Al discloses a method for molecular analysis. Sidewalls are formed extending through a structure between two structure surfaces to define an aperture. A layer of material is deposited on the aperture sidewalls and the two structure surfaces. The aperture with the deposited material layer is then configured in a liquid solution with a gradient in a chemical potential, between the two structure surfaces defining the aperture, that is sufficient to cause molecular translocation through the aperture.

US 2006/0154399 Al discloses a semiconductor device, or an arrangement of insulating and metal layers, having at least one detecting region which can include a recess or opening therein for detecting a charge representative of a component of a polymer proximate to the detecting region.

US 2010/0327255 Al discloses a field effect transistor comprising a reservoir bifurcated by a membrane of three layers. The membrane comprises two electrically insulating layers and an electrically conductive gate between the two insulating layers.

US 2011/0223652 Al discloses an apparatus using a piezoelectric material for controlling a polymer through a nanopore. A membrane having a nanopore therethrough comprises electrical conductive layers, piezoelectric layers, and insulating layers. The piezoelectric layers are operative to control a size of the nanopore for clamping/releasing a polymer when a voltage is applied to the piezoelectric layers. The insulating layers comprise the outermost layers of the membrane.

WO 2009/149125 A2 discloses a plasmonic nanostructure for enhanced light excitation. The nanostructure comprises a substrate, an adhesion layer disposed on top of the substrate, a surface plasmon resonance layer, and a cavity that extends into the surface plasmon resonance layer. The surface plasmon resonance layer is configured to concentrate an applied plasmon field to a bottom portion of the cavity.

We have now invented an improved a composite device in which nanopores are created in a substrate comprising stacked layers of different materials having differing points of zero charge. Not only does this device in certain circumstances allow the translocation speed of the analyte through the nanopore to be controlled or varied for a given analyte sample, but it can also in certain circumstances enhance the local concentration of the analyte within or around the inlet of the nanopore by electrostatic effects thereby improving the efficiency of the device itself.

Thus, according to the present invention there is provided a device suitable for analysing a biopolymer comprising a substrate perforated with at least one nanopore characterised in that the substrate is a composite comprised of stacked layers of at least two different solid inorganic

3294976V1 compounds and wherein the inorganic compounds used in adjacent layers have different points of zero charge.

In one embodiment of the present invention the device may further comprise electrodes juxtaposed either side of the substrate or within or around the outlet of the nanopore, hi another, the invention may further comprise plasmonic nanostructures juxtaposed within the nanopore or around its outlet. Finally, both electrodes and plasmonic structures can be employed if so desired. When electrodes are present, the flow of current therebetween (for example across the substrate or across the outlet of the nanopore) and the analyte blocking events which arise can be detected and measured. When plasmonic nanostructures are employed, these will typically be comprised of nano-sized elements made from a noble metal such as silver or gold whose exact sizes are chosen to generate maximum plasmon resonance upon stimulation by a coherent source of electromagnetic radiation such as a laser. This resonance in turn can stimulate an analyte fluorescing or Raman- scattering detection event in the corresponding electromagnetic detection window which these structures create between themselves. It is not intended herein to limit in anyway the morphological form or arrangements that these elements can take although studies have shown that annular, semi- annular or 'bow-tie' type arrangements work well. Further morphological forms are discussed in our WO 2009/030953 the contents of which are incorporated by reference. Typically these elements are milled out of a noble metal layer deposited on the topmost inorganic compound layer by for example fast cation bombardment although other methods, such as the direct deposition of noble metal nanospheres onto complementary receiving sites around the outlet of the nanopore are also envisaged.

The substrate employed in the device of the invention is a composite suitably comprised of at least two layers of at least two different inorganic compounds which are insulators; for example metal oxides, nitrides or carbides. Thus, in one embodiment the at least two different inorganic compounds are insulators. For example, the substrate may comprise an even number of layers for example 2, 4, 6, 8 or 10 comprised of strictly alternating layers of different materials. However this is merely illustrative and in principle any number of layers and inorganic compounds can be used providing that, for a given layer, the layers adjacent it are comprised of inorganic compounds which are different. Thus, for at least two adjacent layers of the substrate, a first layer of the two adjacent layers includes a first solid inorganic compound and a second layer of the two adjacent layers includes a second solid inorganic compound, wherein the first and second solid inorganic compounds are different, have different points of zero charge and are both insulators. This allows a rate of translocation of a biopolymer through a nanopore to be controlled, as will be explained in more detail below. The substrate typically consists of the at least two layers of at least two different inorganic compounds, i.e. no other components are present in or on the substrate. In one

3294976V1 embodiment, the substrate does not include an electrode within its structure. The substrate does not have a coating of one or more additional materials, because the alternating layers of the substrate need to be exposed in the walls of the nanopore to have the effect of allowing the rate of translocation of a biopolymer through the nanopore to be controlled, as described herein.

Typically, each layer is at least 2nm thick preferably at least 5nm thick, most preferably in the range 5 to 50nm. Suitably the whole stack of layers is from 5 to 200nm preferably 10 to lOOnm thick. Whilst in principle any number of layers can be used, it will be understood that, from a practical perspective, it is desirable that the total number is ten or less.

As regards the inorganic compounds themselves, it is preferred that they are chosen so that their characteristic point of zero charge is such that, in the operating range of the device, at least one layer is negatively charged and the other neutral; or at least one is positively charged and the other neutral; or at least one is positively charged and the other negatively charged. It is most preferred that this criterion is met in the pH range 4 to 9, most preferably 6 to 8. The following table provides examples of suitable inorganic compounds and the pH at which their associated points of zero charge occur.

Suitable binary combinations of these compounds include A1₂0₃ with NiO or with MgO (pH 8); Si₃N₄ with NiO or with MgO (pH 8); Zr0₂ with one of ZnO, NiO or MgO (pH 7) and Ti0₂ with one of A1203, ZnO, NiO or MgO (pH 6). Other combinations include Al- 20₃ with one of Si0₂, Ta₂0₅₎ SiC or Sn0₂; ZnO with one of Si0₂, Ta₂0₅, SiC or Sn0₂; NiO with one of Si0₂₎ Ta₂0₅, SiC or Sn0₂ and MgO with one of Si0₂, Ta₂0₅, SiC or Sn0₂. Ternary and higher combinations are also within the scope of the invention. Also, for example a silicon nitride layer can be employed simply to confer mechanical strength on the substrate and two or more layers of other inorganic compounds, for example alumina and a second (i.e. different) metal oxide selected from those disclosed above, can be stacked thereon.

3294976V1 In a preferred embodiment of the invention the top layer of the stack of inorganic compound is one which is positively charged at the pH of the electrolyte carrier.

Suitably the mean diameter of the nanopores in the device is from 2 to 15nm.

In use, the rate of translocation of the analyte biopolymer through the nanopore can be varied by fine tuning the pH of the electrolyte carrier of the biopolymer. Varying the pH affects the charge characteristics of each layer of inorganic compound causing them to attract or repel, to a greater or lesser extent, the charged biopolymer molecule as it translocates under electrophoresis caused by the application of a potential difference across the substrate using electrodes. For example, in the case of negatively charge DNA or RNA, a negatively charged layer of inorganic compound can either slow translocation down significantly or cause it to stop altogether. Translocation can then be restored by changing the pH to reduce the layer's negative charge density. This opens up the possibility that the device can be programmed to have an analyte gating function.

In one embodiment, the present invention therefore provides the use of a device comprising a substrate perforated with at least one nanopore characterised in that the substrate is a composite comprised of stacked layers of at least two different solid inorganic compounds and wherein the inorganic compounds used in adjacent layers have different points of zero charge, for controlling the rate of translocation of a biopolymer through a nanopore.

The present invention also provides a method for controlling the rate of translocation of a biopolymer through a nanopore, comprising:

(i) providing a device comprising a substrate perforated with at least one nanopore characterised in that the substrate is a composite comprised of stacked layers of at least two different solid inorganic compounds and wherein the inorganic compounds used in adjacent layers have different points of zero charge;

(ii) translocating a biopolymer present in an electrolyte through said nanopore; and

(iii) adjusting the pH of the electrolyte to control the rate of translocation of the biopolymer through the nanopore.

Typically, the at least two different solid inorganic compounds in the substrate are selected to produce the desired rate of translocation of the biopolymer. Typically, the electrolyte is

3294976V1 an aqueous electrolyte. The biopolymer is typically DNA, RNA or a polypeptide or protein.

Preferred features of these aspects of the invention are as described herein in relation to the device itself. In one embodiment, the at least two different inorganic compounds are insulators.

In an embodiment of the invention the device is used in association with an apparatus for sequencing or barcoding a biopolymer, preferably a polynucleotide, contained in an analyte solution. By the term 'barcoding' is meant the detection of detectable moieties characteristic of higher order structures within the biopolymer (for example particular short oligionucleotide fragments in a longer polynucleotide analyte as opposed to the individual nucleotides themselves). This apparatus comprises analyte providing and receiving chambers, means to receive a device according to the present invention and means for receiving a signal from the device. Optionally, the apparatus further comprises a means for processing the signal to reveal the sequence of the constituent parts of the biopolymer. Preferably the biopolymer is a polynucleotide such as DNA, RNA or a protein.

The device itself may comprise a single nanopore or a multiple array thereof. The device may be disposed on a supporting frame which makes it easy to locate reliably in a corresponding cavity located in the apparatus between the analyte providing and receiving chambers. Given that it is principally intended that device will be used as a replaceable component for the said apparatus, it is envisaged that the device may be sold separately and individually packaged in air-tight containers so that a plurality of them can be purchased at any one time and stored for later use without risk of contamination or degradation.

The device itself may further comprise a means for electrically connecting to a corresponding power source in the apparatus itself and/or a signal providing means.

The device of the present invention is now illustrated by the following Examples. Example 1

Onto one side of a 200 micron thick wafer of pure silicon is deposited a 30nm thick support layer of silicon nitride by chemical vapour deposition. Thereafter a lOnm layer of alumina is grown by atomic layer deposition (ALD) using a trimethylaluminium precursor.

3294976v1 Finally a lOnm layer of zinc oxide is also grown using ALD, in this case with a diethyl- zinc precursor..

The product so obtained is selectively etched on its silicon face with potassium hydroxide in the presence of a mask to expose square areas of exposed silicon nitride and the stacked metal oxide layers approximately 50nm thick in total and to create a precursor membrane-like material. Next, the zinc oxide face is coated with gold (30nm thick) by sputtering, and then nanopores of mean diameter 12nm are drilled through exposed areas from the gold side and bow-tie shaped nano-sized elements (dimensions in the range 1 to lOOnm) adjacent each nanopore are milled from the gold layer using gallium ion fast ion bombardment.

The product of the above-mentioned manufacturing process is fitted into a sequencing apparatus of the type described in WO 2009/030953 and used to sequence a fluorescently labelled polynucleotide 300 nucleotides long contained in an aqueous electrolyte (e.g. potassium chloride) by translocating the electrolyte and polynucleotide through the nanopores and the detection windows created by the plasmonic stractures adjacent their outlet using electrophoresis. Within the detection window the nucleotides undergo plasmon resonance induced fluorescence by interaction with the strong electromagnetic field these stracture create and the fluorescent photons so emitted are detected and transformed into a signal of characteristic of the nucleotide sequence itself using a photodetector and associated computer hardware and software. It is found that by fine tuning the pH of the electrolyte around 8 the rate of translocation can be changed to optimise the signal to noise ratio and information content of the signal detected by the photodetector.

Example 2

Example 1 is repeated except that nickel oxide is deposited onto the alumina by sputtering instead of zinc oxide.

Example 3

Example 1 is repeated except that (a) the first layer on top of the silicon nitride is titanium (IV) oxide produced by alternating pulses of tetramethyltitanium and water in an ALD process and (b) the second layer is zinc oxide produced in accordance with the method of Example 1. In use the pH of the electrolyte is fine tuned around 6.

Example 4

Example 1 is repeated except that the first layer deposited on the silicon nitride is silica (by sputtering) and the second is alumina (produced in accordance with Example 1).

3294976V1 Additionally, two further layers of silica and alumina are stacked on top of the alumina by repeating the cycle. In use the pH is tuned in the range 6 to 8.

Examples 5 to 8

The corresponding manufacturing methods of Examples 1 to 4 are followed but in each case, instead of plasmonic structures, electrodes are provided either side of the substrate along with a means to connect each of them to a power source and a current measuring device. The products so produced are located in an apparatus of the type described in for example US 6627067, US 6267872 and US 6746594 (the relevant parts of which are incorporated herein by reference) designed to monitor and measure current flow through the nanopores and to detect blocking events as a polynucleotide 300 nucleotides long is caused to translocate therethrough by electrophoresis. In these examples the pH is fine tuned as described in the corresponding earlier example.

3294976V1

Claims

Claims:

1. A device suitable for an analysing a biopolymer comprising a substrate perforated with at least one nanopore characterised in that the substrate is a composite comprised of stacked layers of at least two different solid inorganic compounds and wherein the inorganic compounds used in adjacent layers have different points of zero charge.

2. A device as claimed in claim 1 characterised in that it further comprises electrodes juxtaposed either side of the substrate or within or around the outlet of each nanopore for measuring the flow of current therebetween.

3. A device as claimed in claim 1 characterised in that it further comprises plasmonic nano structures juxtaposed within or around the outlet of each nanopore for measuring events stimulated by plasmon resonance in a detection window formed between the said structures.

4. A device as claimed in claim 3 characterised in that the detection event is either fluorescence or Raman scattering.

5. A device as claimed in any of the preceding claims characterised in that the mean diameter of the nanopores is from 2 to 15nm.

6. A device as claimed in any of the preceding claims characterised in that thickness of each layer of inorganic compound is in the range 5 to 50nm.

7. A device as claimed in any of the preceding claims characterised in that the stack of layers is from 5 to 200nm thick.

8. A device as claimed in any of the preceding claims characterised in that it comprises a stack of alternating layers of alumina and a second metal oxide on a silicon nitride support layer.

9. A device as claimed is in any of the preceding claims characterised in that it is adapted to fit into an apparatus for sequencing or barcoding a biopolymer contained in an analyte solution which apparatus comprises analyte providing and receiving chambers, means to receive the device and means for receiving a signal emitted by the device.

10. An apparatus for sequencing or barcoding a biopolymer contained in an analyte solution characterised in that it comprises analyte providing and receiving

3294976V1 chambers, means adapted to receive a device of the type claimed in any one of the preceding claims and means for receiving a signal emitted by the device

11. A process for producing the device of any one of claims 1 to 9 characterised in that it includes the step of depositing a layer of a first inorganic compound onto a support and thereafter depositing a layer of a second, different inorganic compound onto the first layer.

12. A process as claimed in claim 11 characterised in that it further comprises either attaching at least one plasmonic nanostructure within or around an outlet of each nanopore or arranging at least one pair of electrodes either side of the substrate or within or around an outlet of each nanopore.

13. A process as claimed in claim 12 characterised in that the plasmonic nano structures are produced by depositing a layer of noble metal atoms on the topmost layer and milling nano-sized elements from it using fast cation bombardment.

14. A process as claimed in claim 13 characterised in the nano-sized elements are milled so that the corresponding plasmonic nanostructure has a bow tie type morphology.

15. A process as claimed in claim 13 or 14 characterised in that noble metal is gold.

3294976v1