NL2007328C2

NL2007328C2 - A nanopore sensor and method for selective detection of analytes in a sample.

Info

Publication number: NL2007328C2
Application number: NL2007328A
Authority: NL
Inventors: Iwijn Vlaminck; Calin Plesa; Cees Dekker
Original assignee: Univ Delft Tech
Priority date: 2011-09-01
Filing date: 2011-09-01
Publication date: 2013-03-04

Description

A nanopore sensor and method for selective detection of analytes in a sample

The invention relates to a nanopore sensor assembly 5 and method for the selective detection of an analyte or analytes in a sample.

A nanopore sensor essentially comprises a small hole (a nanopore) in a layer of a material e.g. in a membrane, with electrodes provided on either side thereof. By applying 10 a voltage across the membrane and monitoring a current passing through the nanopore, passage of analytes through the nanopore can be detected. A disadvantage of simple nanopore sensors is their lack of selectivity; this has led to the development of nanopore sensors functionalised with analyte 15 binding moieties at the surface of the nanopore. Examples of such sensors are recited in United States patent application US 2005/0074778 and United States patent 7,439,055.

US 2005/0074778 relates to nanometer to micron scale functionalized apertures constructed on a substrate made of 20 glass, carbon, semiconductors or polymeric materials. The method of US 2005/0074778 is exemplified with reference to DNA sequencing and identification. The concept of DNA sequencing and identification with a functionalised aperture relies on forcing an unknown single DNA strand through a 25 molecular sized aperture that has been functionalised with a probe DNA strand. As the unknown strand of DNA passes through the aperture, it will bind with the DNA probe, if the DNA sequences are complementary, and then be released. The interaction time between two complementary DNA strands is 30 longer than between mismatched DNA strands. This interaction time is measured by continuously measuring the ionic current, the number of ions moving through the aperture with unit time, from top of the aperture through to the bottom of the aperture. When the strand to be analyzed enters the aperture, 35 the ionic flow is obstructed and the current changes. The duration and the amplitude of the current change can be correlated with the affinity between the probe DNA strand anchored in the aperture and the unknown DNA strand moving through the aperture.

40 US 7,439,055 discloses a device for detecting and 2 measuring the concentration of biomolecules in solution. In the device, nanoporous films are applied to a conducting electrode; pores of the nanoporous film are functionalised at their surface such that they can bind target molecules. When 5 the target molecules are captured, the nanopores are totally or partially blocked. As a result, the diffusion of indicator molecules in pore channels is affected and the redox current measured on the electrode is changed.

Disadvantages of the nanopore sensors of the prior 10 art are that concentration ranges over which they function are limited, e.g. they cannot be used with complex samples such as blood and other biological fluids; if samples being analysed contain particulates, these can block the pores and interfere with measurements; they cannot easily be adapted 15 such that a number of different analytes can be analysed with the same nanopores; their sensitivity is compromised if they are used to analyse concentrated or complex samples; it can be difficult to distinguish between clogging and binding of an analyte since the effect would be very similar; and 20 wherein the method relies on diffusion of the analyte, the sensor is inherently slow.

It is an object of the invention to provide a nanopore sensor assembly according to the appended claims that overcomes one or more of the problems/disadvantages of 25 the prior art.

In a first aspect, the invention relates to a nanopore sensor assembly for the selective detection of at least a first analyte wherein the nanopore sensor assembly comprises at least a first analyte binding surface adjacent 30 to a nanopore sensor i.e. the nanopore sensor assembly comprises at least two functionally distinct and spatially separate regions: the analyte binding surface and the nanopore sensor. The nanopore sensor may comprise either a single nanopore or a plurality of nanopores. Each nanopore prefera-35 bly has a diameter of less than 1 micron, more preferably

less than 100 nm. A smaller pore leads to a stronger blockade event. The pore needs to be larger than the size of the analytes to prevent clogging or obstruction of the pore during a detection phase . I

40 In a preferred embodiment the nanopore sensor assem- 3 bly comprises a plurality of analyte binding surfaces. Preferably each analyte binding surface is specific for a particular analyte. Wherein the nanopore sensor assembly comprises a plurality of analyte binding surfaces the nanopore 5 sensor assembly can advantageously be used for multiplexed detection of analytes.

The functional and spatial separation of the analyte binding surface or surfaces on the one hand and the nanopore sensor on the other hand, provides particular advantages: the 10 nanopore sensor and analyte binding surface or surfaces can be optimized independently for their specific tasks, for example, the functionalised surface or surfaces can be made large to allow for a large amount of analyte or analytes to be captured whilst the nanopore sensor can be made small to 15 allow for detection of translocation events with a high sig-nal-to-noise ratio. Furthermore, in use, it offers a possibility of temporally separating analyte binding and analyte detection. For example, a washing step could be performed to quickly and efficiently remove non-specifically bound com-20 pounds, thus overcoming a problem of the prior art of distinguishing analyte detection and non-specific clogging. 1

It is preferred that the analyte binding surface or surfaces are close to the nanopore sensor; a smaller separation leads to faster detection of the analyte after release.

25 During detection, charged analytes are driven towards the nanopore by an electric field generated by applying a voltage as described above. Active driving of analytes to the nanopore sensor using electric fields reduces the time required for detection compared to methods that rely on diffu-30 sion of analytes.

In an alternative embodiment, instead of fixedly integrating the nanopore sensor and analyte binding surface into the nanopore sensor assembly, the analyte binding surface is a surface of a bead with a diameter in the range of 35 several nanometres (nm) nm to several microns (pm), said bead being reversibly positioned adjacent to the nanopore sensor.

As an example, the bead can be reversibly positioned by providing the nanopore sensor in a well. Use of the nanopore sensor assembly comprises a step wherein the bead is provided 40 such that it is reversibly positioned adjacent to the 4 nanopore sensor. The bead is functionalised aforehand and separately such that its outer surface is the analyte binding surface. Functionalisation of the bead can thus occur outside of the electronic device, removing any restrictions that 5 might be imposed on the functionalisation steps (for example high temperature steps or use of corrosive chemicals). A generic nanopore sensor provided in a well can then advantageously be 'loaded' with any active surface.

In a preferred example, at least the first analyte 10 binding surface comprises an analyte binder. The analyte binder is any species that is able to specifically bind an analyte. Suitable examples of analyte binders include (i) a protein such as an antibody or a zinc finger; (ii) a component such as of a binding system such as biotin-streptavidin, 15 His-tag, FLAG tag, HS tag, MYC tag, GST tag, MBP tag and snap tag systems;(iii) a nucleic acid such as a deoxyribonucleic acid (DNA) strand or a ribonucleic acid (RNA) strand; (iv) a lipid; (v) a chemical compound such as an amine, a thiol, an alcohol, a carboxylic acid; an amine reactive compound such 20 as an active ester, carboxylic acid, epoxy group or aldehyde; a thiol reactive compound such as a maleimide, a pyridyl disulfide or a vinyl sulfone; a carboxylic acid reactive compound such as an amine; an alcohol reactive compound such as an epoxy group; (vi) a polymer such as a modified polymer 25 membrane such as a polypropylene membrane modified with polyaniline; (vii) a hydrogel on metal such as a hydrogel on gold; and (viii) combinations thereof including for example DNA-encoded antibodies.

Examples of analytes include proteins, DNA and RNA, 30 but can in principle be any species that can be specifically bound to an analyte binder. The term analyte is taken to include an analyte that has been modified in a processing step such that it includes a recognition element; suitable means to achieve this are known to a person skilled in the art and 35 include for example a process leading to an analyte being functionalised with a tag, such as for example biotinylation. In an alternative embodiment the processing step involves generation of a fusion protein.

The analyte binder is more preferably an antibody, 40 an RNA strand or a DNA strand and is most preferably an anti- 5 body. Providing an analyte binder constitutes a simple means for selectively binding analytes to an analyte binding surface. Since many analyte binders are known, the nanopore sensor assembly of the present invention can readily be applied 5 to a wide variety of applications. Antibodies, RNA strands and DNA strands are particularly advantageous analyte binders since they have inherent specificity for particular analytes such as an antigen, a complementary RNA strand or a complementary DNA strand respectively.

10 Each analyte binding surface may comprise only a single analyte binder or it may comprise a number of different analyte binders.

In a further preferred example, the analyte binder is attached to the analyte binding surface by a linker. The 15 linker is most preferably cleavable; suitable examples of both linkers and cleavable linkers are known to a person skilled in the art. Particularly interesting examples are DNA-based linkers; such linkers can be designed to include specific enzymatic cleavage sites. A linker serves to dis-20 tance an analyte binder from the analyte binding surface; this is often beneficial to ensure efficient analyte binding, for example when a substrate has particular surface properties such as a surface charge that would otherwise hinder binding. Cleavable linkers constitute a simple, practical and 25 controllable means for releasing an analyte.

In an example, the nanopore sensor is a solid-state nanopore sensor.

Nanopore sensors in general and in particular solid-state nanopore sensors are some of the simplest electrical 30 sensors imaginable comprising essentially only a hole e.g. in a membrane, and a pair of electrodes. The nanopore sensor assembly of the invention is manufacturable at low cost, in large quantities and using many different methods. Furthermore the combination of the nanopore sensor and the analyte 35 binding surface or surfaces provide ultra high selectivity (e.g. for single analytes) and ultrahigh sensitivity (e.g. single molecule detection). Nanopore sensors further permit additional information about a sample to be determined such as concentration and size of the analyte being detected. The 40 amplitude and duration of the decrease in ionic current 6 provide a measure of the size of analytes passing through the nanopore and the concentration of analyte in a sample can be determined by monitoring the number of events per unit time.

In a second aspect, the invention relates to a proc-5 ess for the selective detection of at least a first analyte in a sample using the nanopore sensor assembly wherein the process comprises the steps: (i) providing a sample on at least a first analyte binding surface of the nanopore sensor assembly, (ii) allowing a period of time for binding of an 10 analyte optionally to a corresponding analyte binder or analyte binding surface, (iii) removing non-bound compounds from the analyte binding surface, such as by rinsing, (iv) releasing the analyte from the analyte binding surface, (v) providing a voltage across a nanopore of the nanopore sensor and 15 detecting.

In step (i) the sample can be any fluid or solution that potentially contains an analyte. Suitable examples include biological fluids such as blood, urine or serum. The analyte is preferably a biomarker such as for cancer. In an 20 alternative embodiment an additional step is performed wherein an analyte is functionalised with a recognition element .

In step (ii) the period of time is dependent in particular on the nature of the analyte binder and/or analyte 25 binding surface and/or the nature of the interaction between a particular analyte binder and analyte; suitable time ranges are known to a person skilled in the art.

In step (iii) compounds that are not bound to an analyte binding surface and/or an analyte binder are removed; 30 typically this may be done by rinsing.

In a preferred example, step (iv), releasing the analyte from the analyte binding surface is by one of: electrical, optical, thermal, chemical, biological or mechanical means. Specific examples are given in table 1 wherein the 35 analyte binder is an antibody. These examples are not limiting of the invention.

In step (v) a voltage is applied across a nanopore or nanopores of the nanopore sensor; the resulting electrical field serves to drive analytes towards the nanopore(s). De-40 tection involves monitoring the decrease in ionic current as 7 analytes pass through the nanopore(s) (wherein the nanopore sensor comprises multiple nanopores, each nanopore is effectively a separate detector i.e. the drop in current is measured across each nanopore or the nanopore sensor individu-5 ally). The duration and amplitude of the decrease in ionic current give a measure of the size of the analyte. The number of events per unit time give a measure of the concentration of the sample.

In a third aspect, the invention relates to a device 10 for use in diagnostic applications comprising at least the nanopore sensor assembly of the first embodiment of the invention, but more preferably an array comprising a plurality of nanopore sensor assemblies wherein each nanopore sensor assembly is specific for a particular analyte, thus permit-15 ting multiplexed detection of analytes. In an example, the plurality of nanopore sensor assemblies are concurrently sub-jectable to a single sample for the performance of multiple analysis on said sample. Multiplexed detection is important for the accuracy of many diagnostic assays. The device is 20 preferably a "stand-alone" device incorporating a computer or other means for processing and presenting data collected by the device. In an alternative embodiment, the device is preferably connectable to a computer. The device is useable for example in a hospital, however it also envisageable that such 25 a device is useable at home, for example where periodic monitoring of a condition is required or for self-diagnosis e.g. along the lines of modern home pregnancy tests, but in an electronic version that is applicable to a wide range of biomarkers and that is able to detect multiple biomarkers simul-30 taneously.

Particular advantages of the device are that it permits rapid, quantitative and multiplexed detection of analytes even in complex media such as whole blood. It is de-sireable to be able to analyse blood since it contains the 35 largest representation of the human proteome. Incorporation of the nanopore sensor assembly provides high selectivity and single-molecule sensitivity.

In a fourth aspect, the invention relates to a kit comprising the nanopore sensor assembly of the first embodi-40 ment of the invention incorporated into a device for use in 8 diagnostic applications and further comprises materials necessary to perform the method of the second embodiment of the invention.

The process of the invention can advantageously be 5 used as an inexpensive, user friendly alternative to tech- nigues such as ELISA, optical detection methods, lateral flow tests, magnetic immunoassays, radioimmunoassay and radiobinding assays.

The invention is hereinafter clarified with refer-10 ence to the drawing of Figures 1-3 showing a preferred example of the invention which is not limiting of the appended claims .

In the drawing:

Figure 1 shows a preferred embodiment of the 15 nanopore sensor assembly and process of the invention;

Figure 2 shows a schematic of a nanopore sensor detecting an analyte;

Figure 3 shows a schematic of the output from a nanopore sensor detecting analytes wherein the analyte is 20 present at a low and a high concentration.

Whenever in the Figures the same reference numerals | are used, these numerals refer to the same parts.

With reference to Figure 1 showing the nanopore sensor assembly (1) of the invention, comprising an analyte 25 binding surface (2) with analyte binders (2') and a nanopore sensor (3) comprising a nanopore (3'). A representation of a sample is also shown comprising an analyte of interest (A), in this case a protein, and other sample constituents (B, C and D). The analyte binding surface (2) and nanopore sensor 30 (3) are adjacent, but spatially separated; thus the analyte binding domain (2) and the nanopore sensor (3) can be optimised separately and a compromise does not need to be drawn ! that would inevitably effect the performance of the nanopore sensor assembly (1), for example decreasing its sensitivity.

35 In this preferred embodiment, the analyte binder (2') is an antibody. The analyte binder is optionally linked to the analyte binding surface (2) by a linker (L). The nanopore sensor (3) is shown as comprising only a single nanopore (3') however it should be appreciated that it would equally be com- ! 40 pletely in keeping with the spirit of the invention for the 9 nanopore assembly (3) to comprise a plurality of nanopores (3') .

Panel (I) of Figure 1 shows a sample being provided; the sample is provided at least on the analyte binding sur-5 face (2). Panel (II) shows an analyte in the sample binding to the analyte binder (2'); the analyte binder is specific for a single analyte. In an alternative embodiment, the nanopore sensor assembly (1) may comprise a plurality of analyte binding surfaces (2), each analyte binding surface (2) 10 being specific for a single analyte; preferably no two analyte binding surfaces bind the same analyte. Wherein the nanopore sensor assembly (1) comprises a plurality of analyte binding surfaces (2) each analyte binding surface (2) is adjacent to, but spatially separated from the nanopore sensor 15 (3). In a further alternative embodiment, the analyte binding surface (2) contains a multiple analyte binders (2'), preferably such that each analyte binder is selectively cleavable optionally by use of multiple linkers. Sufficient time is allowed for the analyte to bind to the analyte binder (2'), in 20 this case an antibody. Wherein the analyte binder is an antibody, the period of time is for example of the order of sec onds to minutes. Panel (III) shows the removal of constituents of the sample that are not bound to an analyte binder (2) this is for example by rinsing with a buffer. Panel (IV) 25 shows an analyte, in this case a protein, being released from the analyte binding surface. The analyte may be released either in the form in which it was bound, or as a composition comprising the analyte and at least a portion of the analyte binder and/or a linker. Panel (V) shows a detection step 30 wherein a voltage is applied across the nanopore (3') such that analytes are drawn through the nanopore (3'). As an analyte passes through the nanopore (3') the ionic current is ! monitored.

With reference to Figures 2 and 3, a nanopore sensor 35 (3) is shown comprising a nanopore (3')^ electrodes (4A and 4B) positioned on either side of the nanopore (3') such that a voltage can be applied across the nanopore (3'), a power source (5) and an amperemeter (6). In the plot, time is the abscissa and ionic current the ordinate. Application of a 40 voltage across the nanopore (3') causes analyte (A) to trans- 10 locate through the nanopore (3'). As the nanopore becomes partially blocked by the analyte, the resistance across the nanopore increases and thus a decrease in ionic current is recorded on an amperemeter (6). The duration and amplitude of 5 the decrease in ionic current are a measure of the size of the analyte. The plots of Figure 3 are equivalent to the plot of Figure 2, but demonstrate that information about concentration of an analyte is similarly obtainable using a nanopore sensor such as the nanopore sensor (3) of the 10 nanopore sensor assembly (1).

The combination of an analyte binding surface (2) functionalised with analyte binders (2') allows for very sensitive (single molecule ) and selective detection. Spatially separating the analyte binding surface (2) and the nanopore 15 sensor (3) allows optimising the functionality of both components. The multiphase detection method offers the possibility of temporal separation of the analyte binding and detection steps. This is important because it provides a means for a cleaning or a purification step, allowing even complex fluids 20 such as blood, serum and urine to be analysed.

11

Table 1

Type__Method__Examples/Comments_ !

Electrical Electrochemically Application of an electric current to the area I

reversible where the antibodies are bound breaks the __chemistry__bond between the antibody and the surface.

electrophoretic force Applied field generates a force strong enough to remove the target molecule from ___the antibody._

Optical photo-activated A pulse of light is used to cleave the linkage __linkers__between the antibody and the surface._ photo-induced Light is focused on the area containing the heating antibodies and causes local heating of the buffer and substrate, which in turn causes the _______protein-antibody complex to dissociate._

Thermal integrated heater An integrated resistive element heats the local area when an current is passed __through._ _______heating entire system The entire system is heated externally._ exothermic reaction Heat is released into the system through a ____chemical reaction._

Chemical salt concentration Alteration of the buffer salt concentration causes dissociation of the target-antibody ________complex._ pH of buffer Alteration of the buffer pH causes ________dissociation of the target-antibody complex.

addition of chemical Addition of a chemical agent to the buffer agent which specifically targets the antibody- _________surface or target molecule-antibody binding.

Biological restriction enzymes Target molecule bound to DNA which is ___cleaved by restriction enzyme._ proteases Target molecule binds to a polypeptide or ___protein which is then cleaved by a protease.

Mechanical AFM, stylus/probe Target molecule-antibody complex is physically removed from surface by __ mechanical shearing._ 5

Claims

A nano-hole sensor assembly (1) for selective detection of at least a first analyte (A), characterized in that the nano-hole sensor assembly (1) comprises at least a first analyte binding surface (2) adjacent to a nano-hole sensor (3).

A nanogat sensor assembly (1) according to claim 1, characterized in that at least the first analyte binding surface (2) is a surface of a bead, which bead is reversibly positioned adjacent to the nanogat sensor (3).

The nano-hole sensor assembly (1) according to claim 2, characterized in that the nano-hole sensor (3) is provided in a well.

A nanogat sensor assembly (1) according to at least one of the preceding claims, characterized in that at least the first analyte binding surface (2) comprises an analyte binder.

A nanogat sensor assembly (1) according to claim 4, characterized in that the analyte binder (2 ') is one or more of: (i) a protein such as an antibody or zinc finger; (ii) a component such as from a connecting system such as bi-otin-streptavidin, His tag, FLAG tag, HS tag, GST tag, MBP tag and snap tag systems; (iii) a nucleic acid such as a strand of deoxyribonucleneic acid (DNA) or a strand of ribonucleic acid (RNA); (iv) a lipid; (v) a chemical compound such as an amine, a thiol, an alcohol, a carboxylic acid; an amine reactive compound such as an active ester, carboxylic acid, epoxy group or aldehyde; a thiol reactive compound such as a maleimide, a pyridyl disulfide or a vinyl sulfone; a carboxylic acid reactive compound such as amine; an alcohol-reactive compound such as an epoxy group. (vi) a polymer such as a modified polymer membrane, such as a polypropylene membrane modified with polyaniline; (vii) a hydrogel on metal such as a hydrogel on gold; and | - 13 - (viii) combinations thereof,

A nanogat sensor assembly (1) according to at least one of claims 4 and 5, characterized in that the analyte binder (2 ') is selected from a group comprising: an antibody, a strand of RNA and a strand of DNA .

A nanogat sensor assembly (1) according to at least one of claims 4-6, characterized in that the analyte binder is adhered to the analyte binding surface by a linker (L).

A nanogat sensor assembly (1) according to claim 7, characterized in that the linker (L) is a cleavable linker.

The nano-hole sensor assembly (1) according to at least one of the preceding claims, characterized in that the nano-hole sensor (3) is a semiconductor nano-hole sensor.

A process for the selective detection of at least a first analyte (A) in a sample by using a nano-hole sensor assembly (1) of one or more of the preceding claims, characterized in that the process comprises the following steps: (i ) applying a sample to at least a first analyte binding surface (1), (ii) waiting a period of time for the analyte (A) to bind to optionally an associated analyte binder (2 ') and / or an analyte binding surface (2), (iii) removing unbound compounds (2) such as by rinsing, (iv) releasing analyte (A) from the analyte binding surface (2), (v) applying a voltage across the nano hole of the nano hole sensor (3) and detection.

11. Process according to claim 10, characterized in that step (iv) is by electrical, optical, thermal, chemical, biological or mechanical means.

12. Process according to at least one of claims 10 and 11, characterized in that the analyte-binding surface (2) is provided on a bead, and that the process further comprises the step of placing the bead such that it reverses is placed adjacent to the nano-hole sensor.

Device for clinical diagnostic applications, characterized in that the device consists of a nanogat sensor assembly or a plurality of nanogat sensor assemblies according to at least one of claims 1-10. - 14 -

Device according to claim 13, characterized in that the multi-nano-hole sensor assemblies can be used simultaneously on a single sample for performing multiple analyzes of the sample.

Diagnosis kit, characterized in that the kit comprises the device of claim 13 or 14.