WO2025131976A1 - System for polypeptide identification using a pore field-effect transistor - Google Patents

Abstract

A system for polypeptide identification including pore field-effect transistors (FET) and electrical readout modules. The pore FET has a gate, source, drain, and channel region, plus a pore with distinct openings, parts, and a sensing region. The pore, less than 10 µm in effective diameter, has a smaller aperture part with an effective cross-section diameter, dA, less than 10 nm. During operation, the gate is controlled by the sensing region of the pore. The readout module records electrical signals from electroactive labels on different amino acid types when a polypeptide translocates through the pore. The system also incorporates a processor to match recorded signals to these fingerprints, and a translocation unit for moving the delivered polypeptides through the pore.

Description

System for Polypeptide Identification Using a Pore Field-Effect Transistor

Field of the Invention

The invention relates to the field of proteomics, and more specifically to systems for polypeptide identification using pore technology.

Background of the Invention

High-throughput next-generation DNA sequencing has transformed genomics, impacting diverse fields from healthcare to archaeology. However, while our understanding of the genome has significantly increased, our understanding of the proteome— the complete set of proteins expressed by a cell, tissue, or organism at a particular time-lags behind. Current proteomics techniques are largely restricted to specialized institutes and involve large, complex equipment operated by highly trained personnel. This results in long queue times and extended run-times, limiting their application mainly to research purposes.

The dynamic range of protein concentrations in plasma can vary significantly, often exceeding a factor of 10¹², with biomarker proteins of interest often present at the lowest abundance levels. Current mass spectrometry (MS) techniques, with a dynamic range of 10³-10⁵, are inadequate for detecting these low- abundance proteins (see Fig. 1). As a workaround, plasma protein depletion is often applied to measure less abundant species. However, this approach also removes proteins of interest that are bound to the depleted plasma proteins.

Further, single-cell proteomics— a crucial area of research— remains largely unrealized due to the limitations of MS. For instance, the typical MS limit of detection is greater than 0.1 fmole, or 6xl0⁷ copies, which is inadequate for detecting the less than 1000 copies of low abundance proteins in a cell. Moreover, the so-called "dark proteome," consisting of approximately 3,000 human proteins that have never been directly identified, remains a challenge for current MS-based techniques.

Another key challenge in mass spectrometry-based proteomics is the capture of all individual variants of proteins, known as proteoforms. While there are approximately 20,000 human protein-coding genes, transcription with splice variants increases the number of proteins to around 70,000. Post-translational modifications further create hundreds of thousands of protein variants, many of which play crucial roles in diseases such as cancer. The current MS-based toolset struggles to capture all these proteoforms.

Silicon CMOS-based nanotechnology has the potential to revolutionize proteomics with high- throughput, high-accuracy, and low-cost read-out. The key challenges for the next generation of proteomics tools include enhancing the measurable protein concentration dynamic range, lowering the limit of detection, making the tools widely available through microchip technology, and mapping of proteoforms. Addressing these challenges would transform proteomic research and clinical analysis, enabling the measurement of low-abundance protein species in clinical samples, high-throughput biomarker discovery, single-cell proteomics, uncovering of the dark proteome, mapping of proteoforms, and the wide clinical application of proteomics. Such a tool could have a significant impact on cancer research and other areas of healthcare, potentially paving the way for true personalized medicine and precision health.

There is therefore a need in the art for proteomics tools that meet at least partially one or more of the above challenges.

Summary of the Invention

It is an object of the present invention to provide good tools for polypeptide identification and analysis.

The above objective is accomplished by a system for polypeptide identification using a pore field-effect transistor according to the present invention, as well as by a method of operating the same.

In a first aspect, the present invention relates to a system for polypeptide identification comprising: al) One or more pore field-effect transistors, each comprising: i. A field-effect transistor having a gate, a source, a drain, and a channel region between the source and the drain, and ii. a pore comprising: at least a first and a second opening; two contiguous parts, a lumen part and an aperture part, such that the lumen part is fluidically connected to the first opening and that the aperture part is fluidically connected to the second opening; a sensing region located in the lumen part, the aperture part, or both parts of the pore such that a polypeptide travelling first opening to the second opening will pass through the sensing region; wherein the effective diameter of a transverse cross-section, at all points within the pore, is less than 10 pm, preferably less than 5 pm, more preferably less than 1 pm; wherein the aperture part inside the pore has all its effective transverse cross-section diameters, dA, being less than 10 nm; wherein an area of a transverse cross section in the aperture part, AA, is smaller than an area of a transverse cross section in the lumen part, AL, such that AL>2AA, wherein the gate is controlled by the sensing region of the pore when the system is in operation, a2) one or more an electrical readout modules, each being connected to the drain and/or source of one or more of said field-effect ransistors, configured to record an electrical signal indicating the presence of electroactive labels on at least two different amino acid types of a linearized polypeptide when the linearized polypeptide translocates through the pore; b) A processing unit configured to identify a polypeptide from the recorded electrical signal, either by matching it to a polypeptide fingerprint in the database or by applying learned patterns from the neural network to the signal, c) A translocation unit for translocating the linearized polypeptide from the first opening to the second opening of the pore.

In a second aspect, the present invention relates to a method for polypeptide identification comprising the steps of: translocating the linearized polypeptide from the first opening to the second opening of the pore, recording an electrical signal as said linearized polypeptide translocates through said pore, said electrical signal indicating the presence of the electroactive labels on the amino acids of the linearized polypeptides, identify a polypeptide from the recorded electrical signal, either by matching it to a polypeptide fingerprint in a database or by running a neural network on the signal.

In a third aspect, the present invention relates to a computer program comprising instructions to cause the system of any embodiment of the first aspect to execute the steps of the method of the second aspect.

In a fourth aspect, the present invention relates to a computer-readable medium having stored thereon the computer program of the third aspect.

The embodiments of the present invention boast numerous advantages. They utilize Pore Field Effect Transistor (FET) sensor technology, providing high-throughput, high-accuracy, and low-cost read-outs. This technology increases accessibility for various applications and users. The invention can be leveraged in a handheld form factor, further enhancing the availability and user-friendliness of proteomics tools, potentially revolutionizing both research and clinical diagnostics. Furthermore, this novel technology could usher in a wide clinical application of proteomics, significantly impacting fields such as cancer research, personalized medicine, and precision health. Additionally, it introduces a new high-bandwidth approach to protein fingerprinting, greatly expediting the process of identifying proteins at the single molecule level. For background on protein fingerprinting, see Y. Yao et al., Phys. Biol. 12 (2015) 055003. It is also an advantage of embodiments of the present invention that they offer the potential for a high current signal, thereby improving scalability and reducing circuit size.

It is also an advantage of embodiments of the present invention that they can provide excellent measurable protein concentration dynamic range. In particular, they are able to enhance the dynamic range versus mass spectrometry by multiple orders of magnitude, allowing for more accurate and comprehensive measurements.

Embodiments of the present invention carry significant advantages in their enhanced detection capabilities. These include enabling a low limit of detection that facilitates comprehensive mapping of proteoforms, the revealing of the dark proteome, and the empowering single-cell proteomics. Furthermore, they have to potential to allow for the detection and measurement of low abundance proteins in clinical samples, often key in proteomics research and clinical diagnostics. The technology also holds the potential to detect currently undetectable proteins in the human proteome, a breakthrough that could significantly advance our understanding of biology and disease. Collectively, these features could revolutionize single-cell proteomics, making it not only feasible and practical but also opening up a myriad of new opportunities for research and clinical applications.

Particular and preferred aspects of the invention are set out in the accompanying independent and dependent claims. Features from the dependent claims may be combined with features of the independent claims and with features of other dependent claims as appropriate and not merely as explicitly set out in the claims.

Although there has been constant improvement, change, and evolution of devices in this field, the present concepts are believed to represent substantial new and novel improvements, including departures from prior practices, resulting in the provision of more efficient, stable, and reliable devices of this nature.

The above and other characteristics, features, and advantages of the present invention will become apparent from the following detailed description, taken in conjunction with the accompanying drawings, which illustrate, by way of example, the principles of the invention. This description is given for the sake of example only, without limiting the scope of the invention. The reference fig.s quoted below refer to the attached drawings.

Brief description of the drawings

Fig. 1 illustrates the detection limitations of mass spectroscopy.

Fig. 2 a) shows the specification of a modelled pore usable in embodiments of the present invention; Fig. 2 b) shows the signal in V expected for different aperture sizes as a function of the position of a labelled molecule with respect to the pore exit for the pore modelized as in Fig. 2 a). Fig. 3 shows graphs of the signal and various sources of noise for the pore simulated in Fig. 2 when the molecule labelled with a 6-fold charged label.

Fig. 4 is a vertical cross-section through a schematic representation of a pore usable in embodiments of the present invention. The pore is depicted with a labelled and linearized polypeptide passing through it.

Fig. 5 is a schematic representation of an embodiment of a method according to an embodiment of the present invention.

Fig. 6 shows two examples of electrical readout modules usable in embodiments of the present invention. Electrical readout module b) enables a larger bandwidth than the electrical readout module a).

Fig. 7 shows a vertical cross-section (left) and a transverse cross-section (right) of a pore-FET wherein the pore is within the channel region of the field-effect transistor.

Fig. 8 shows a vertical cross-section (left) and a transverse cross-section (right) of a pore-FET wherein the pore is physically separate from the channel region of the field-effect transistor.

Fig. 9 is a schematic representation showing various parameters used to define a pore as used in embodiments of the present invention. These parameters are indicated on a vertical cross-section through a pore according to embodiments of the present invention.

Fig. 10 is a perspective view of a cross-section through a pore as used in embodiments of the present invention.

Fig. 11 is a perspective view of a cross-section through a pore as used in other embodiments of the present invention where the lumen perpendicular to the aperture.

Fig. 12 illustrates an example of a lateral device design.

Fig. 13 illustrates a further example of a lateral design.

Fig. 14 illustrates a further example of a lateral design comprising multiple sub-lumens.

Fig. 15 illustrates the correlation between the resistances Rc/Rp and the optimal signal modulation.

Figs. 16A-D illustrate alternative designs of the present devices, where the location of the main electrode or conductive layer acting as channel may be varied.

Figs 17A-B illustrate alternative designs of the present devices, where several electrodes or conductive layers acting as channel may be used, and that the location of these may vary.

Fig. 18 shows schematically two embodiments of a system according to the present invention. Fig. 19 illustrates the pore bandwidth (BW) and SNR trade-off. fig. 13 (left) shows the impact of the design measures on the SNR, while fig. 13 (right) illustrates the impact of the design measures on the BW.

In the different figures, the same reference signs refer to the same or analogous elements.

Description of illustrative embodiments

The present invention will be described with respect to particular embodiments and with reference to certain drawings but the invention is not limited thereto but only by the claims. The drawings described are only schematic and are non-limiting. In the drawings, the size of some of the elements may be exaggerated and not drawn on scale for illustrative purposes. The dimensions and the relative dimensions do not correspond to actual reductions to practice of the invention.

Furthermore, the terms first, second, third, and the like in the description and in the claims, are used for distinguishing between similar elements and not necessarily for describing a sequence, either temporally, spatially, in ranking, or in any other manner. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments of the invention described herein are capable of operation in other sequences than described or illustrated herein.

It is to be noticed that the term "comprising", used in the claims, should not be interpreted as being restricted to the means listed thereafter; it does not exclude other elements or steps. It is thus to be interpreted as specifying the presence of the stated features, integers, steps, or components as referred to, but does not preclude the presence or addition of one or more other features, integers, steps or components, or groups thereof. The term "comprising" therefore covers the situation where only the stated features are present (and can therefore always be replaced by "consisting of" in order to restrict the scope to said stated features) and the situation where these features and one or more other features are present. The word "comprising" according to the invention therefore also includes as one embodiment that no further components are present. Thus, the scope of the expression "a device comprising means A and B" should not be interpreted as being limited to devices consisting only of components A and B. It means that with respect to the present invention, the only relevant components of the device are A and B.

Reference throughout this specification to "one embodiment" or "an embodiment" means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrases "in one embodiment" or "in an embodiment" in various places throughout this specification are not necessarily all referring to the same embodiment, but may. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner, as would be apparent to one of ordinary skill in the art from this disclosure, in one or more embodiments.

Similarly, it should be appreciated that in the description of exemplary embodiments of the invention, various features of the invention are sometimes grouped together in a single embodiment, fig., or description thereof for the purpose of streamlining the disclosure and aiding in the understanding of one or more of the various inventive aspects. This method of disclosure, however, is not to be interpreted as reflecting an intention that the claimed invention requires more features than are expressly recited in each claim. Rather, as the following claims reflect, inventive aspects lie in less than all features of a single foregoing disclosed embodiment. Thus, the claims following the detailed description are hereby expressly incorporated into this detailed description, with each claim standing on its own as a separate embodiment of this invention.

Furthermore, while some embodiments described herein include some but not other features included in other embodiments, combinations of features of different embodiments are meant to be within the scope of the invention, and form different embodiments, as would be understood by those in the art. For example, in the following claims, any of the claimed embodiments can be used in any combination.

Furthermore, some of the embodiments are described herein as a method or combination of elements of a method that can be implemented by a processor of a computer system or by other means of carrying out the function. Thus, a processor with the necessary instructions for carrying out such a method or element of a method forms a means for carrying out the method or element of a method. Furthermore, an element described herein of an apparatus embodiment is an example of a means for carrying out the function performed by the element for the purpose of carrying out the invention.

In the description provided herein, numerous specific details are set forth. However, it is understood that embodiments of the invention may be practiced without these specific details. In other instances, well-known methods, structures, and techniques have not been shown in detail in order not to obscure an understanding of this description.

We now refer to Fig. 18. In a first aspect, the present invention relates to a system for polypeptide identification comprising: al) One or more pore field-effect transistors, each comprising: i. A field-effect transistor (3) having a gate, a source (S), a drain (D), and a channel region between the source (S) and the drain (D), and ii. a pore (4) comprising: at least a first (5) and a second (6) opening; a sensing region (11) located in the pore (4) such that a polypeptide travelling from the first opening (5) to the second opening (6) will pass through the sensing region (11); wherein the effective diameter of a transverse cross-section of the pore, at all points within the pore (4), is less than 1 pm; wherein the narrowest part (8) of the pore (4), also called aperture part (8), has an effective transverse cross-section diameter, dA, less than 10 nm; wherein the gate is controlled by the sensing region (11) of the pore (4) when the system is in operation, a2) one or more electrical readout modules (14), each being connected to the drain (D) and/or source (S) of one or more of said field-effect transistors (3), configured to record an electrical signal indicating the presence of electroactive labels (12) on at least two different amino acid types of a linearized polypeptide (13) when the linearized polypeptide (13) translocates through the pore (4); b) A processing unit (10) configured to identify a polypeptide from the recorded electrical signal, either by matching it to a polypeptide fingerprint in a database or by running a neural network on the recorded electrical signal, c) A translocation unit (15) for translocating the linearized polypeptide (13) from the first opening (5) to the second opening (6) of the pore (4).

In preferred embodiments of the first aspect, the present invention relates to a system for polypeptide identification comprising: al) One or more pore field-effect transistors, each comprising: i. A field-effect transistor (3) having a gate, a source (S), a drain (D), and a channel region between the source (S) and the drain (D), and ii. a pore (4) comprising: at least a first (5) and a second (6) opening; two contiguous parts, a lumen part (7) and an aperture part (8), such that the lumen part (7) is fluidically connected to the first opening (5) and that the aperture part (8) is fluidically connected to the second opening (6); a sensing region (11) located in the lumen part (7), the aperture part (8), or both parts of the pore (4) such that a polypeptide travelling from the first opening (5) to the second opening (6) will pass through the sensing region (11); wherein the effective diameter of a transverse cross-section of the pore, at all points within the pore (4), is less than 1 pm; wherein the aperture part (8) inside the pore (4) has an effective transverse cross-section diameter, dA, less than 10 nm; wherein an area of a transverse cross section in the aperture part (8), AA, is smaller than an area of a transverse cross section in the lumen part (7), AL, such that AL>2AA, wherein the gate is controlled by the sensing region (11) of the pore (4) when the system is in operation, a2) one or more electrical readout modules (14), each being connected to the drain (D) and/or source (S) of one or more of said field-effect transistors (3), configured to record an electrical signal indicating the presence of electroactive labels (12) on at least two different amino acid types of a linearized polypeptide (13) when the linearized polypeptide (13) translocates through the pore (4); b) A processing unit (10) configured to identify a polypeptide from the recorded electrical signal, either by matching it to a polypeptide fingerprint in a database or by running a neural network on the recorded electrical signal, c) A translocation unit (15) for translocating the linearized polypeptide (13) from the first opening (5) to the second opening (6) of the pore (4).

These configurations are advantageous as it allows for the accurate and efficient identification of polypeptides, and preferably entire proteins. The present invention is better at identifying entire proteins than polypeptides or protein segments.

The system comprises one or more pore field-effect transistors. The system may comprise multiple pore field-effect transistors. These pore field-effect transistors could, for instance, be in the range of from 1 pore transistor to 10 million pore transistors. Preferably, an array of pore transistors is used. In embodiments, the one or more pore transistors may be a thousand or more, preferably a million or more. This scalability potentially enables high-throughput polypeptide analysis, beneficial for applications like proteomics.

Each pore transistor has an electrical readout module connected to its drain (D) and/or source (S). Both, the pore field-effect transistor and the electrical readout module are typically present on the same chip. Each pore transistor can have its own electrical readout module or a plurality of pore transistors can share an electrical readout module.

The pore field-effect transistor includes a field-effect transistor (FET) with a gate, a source (S), a drain (D), and a channel region between the source (S) and the drain (D). It also has a pore with a maximum effective diameter of less than 10 pm, preferably less than 5 pm, more preferably less than 1 pm. As used herein, the term "pore field-effect transistor" should be construed as the combination of an FET and a pore, wherein the pore is used for modulating a gate voltage of the FET. We now refer to Figs. 7, 8, and 18. The pore (4) is either configured to extend through the channel region of the field-effect transistor (3) between the source (S) and the drain (D) (see Figs. 7 and 18 (left)) or the pore (4) is physically separate from the channel region of the field-effect transistor (3) (see Figs. 8 and 18 (right)). Hence, in embodiments, the pore may be configured to extend through the channel region of the FET between the source (S) and the drain (D) or may be physically separate from the channel region of the FET. In embodiments, the pore may be arranged in relation to the channel region, such that the pore may control the gate voltage directly, whereby the gate material is represented by the fluid inside the pore. For instance, the pore may be configured to extend through the channel region. Thus, in one embodiment, an FET gated by an electrolyte-filled pore running through the channel of the FET is provided, typically a silicon gate. In alternative embodiments, the pore may be separately arranged from the channel region of the FET. For instance, the pore could be provided with a pore electrode which is directly connected to a gate electrode of the FET, such that a voltage of the gate electrode could be controlled by the pore electrode. In these alternative embodiments where the pore is physically separate from the channel region of the FET, the pore electrode may be wrapped around the pore or located in the pore, this electrode is connected to a remote FET. Thus this FET has an extended gate. An extended gate is advantageous because it permits the use of foundry-based CMOS rather than full-custom CMOS.

A pore FET is advantageous versus a conventional pore setup, i.e., a setup wherein each pore is provided with a galvanically isolated electrode. In particular, it permits to increase the density of pores compared to the conventional pore setup. Also, a pore FET is typically capable of delivering higher currents, e.g., in the order of microamps, thereby permitting an easier detection of the signal, a higher bandwidth and a simpler associated electronic circuit.

In embodiments, the density of pores may be up to 1 pore per pm², e.g., from 1 pore per 100 pm² to 1 pore per pm², or from 1 pore per 10 pm² to 1 pore per pm², for embodiments where the pore is configured to extend through the channel region.

In embodiments, the density of pores may be up to 1 pore per 100 pm², e.g., from 1 pore per 1000 pm² to 1 pore per 100 pm², for embodiments where the FET has an extended gate.

The pore comprises a first opening, a lumen part, an aperture part, a second opening, and a sensing region.

The pore of the pore FET device comprises two main contiguous parts, a lumen part and an aperture part or aperture, wherein each part contains one opening of the pore. One opening of the aperture pore part is fluidically connected to an aperture reservoir. The pore further comprises a plurality of openings. In an embodiment, the pore comprises two openings, a first and a second opening, such that the lumen part is connected to the first opening and the aperture part is connected to the second opening. In other embodiments, the lumen part comprises several sub-lumen parts, each in direct fluidic connection with different openings of the pore. Thus, the lumen part may comprise multiple sub-lumen parts each fluidically connecting the other end of the aperture part (the end not connected to the aperture reservoir) to a different reservoir (excluding the aperture reservoir). In an embodiment, the lumen part comprises two sub-parts, a first sub-lumen and a second sub-lumen, wherein the first sub-lumen is directly fluidically connected to the first opening and the second sub-lumen is connected to a third opening. The openings in the pore leads to a plurality of reservoirs, such as one reservoir per opening, wherein the inside of the pore is fluidically connected to the reservoirs. With multiple reservoirs, one reservoir is connected to the aperture parts, and the other to different sub-lumen parts. In one embodiment, the device comprises a first and a second reservoir, the pore being fluidically connected to said first and second reservoir, wherein the first reservoir is in direct fluidic connection with the lumen part of the pore via the first opening, and the second reservoir is in connection with the aperture part of the pore via the second opening. In another embodiment the device comprises a third reservoir and the lumen part comprises two sub-parts, a first sub-lumen and a second sub-lumen directly fluidically connected to the first and third openings, respectively, wherein the third reservoir is in direct fluidic connection with the second sub-lumen of the pore via the third opening, and wherein the pore is fluidically connected to the first, second and third reservoir.

The device further comprises a pore central pathway running from the first opening to the other openings of the pore, wherein the one or more lumen part and the aperture part of the pore are oriented along the pore central pathway. It should be noted that the pore central pathway is not necessarily linear, but may be bent, and also it may be split if the pore comprises more than two openings, such that the pore central pathway may head in two different directions towards different openings. Thus, the pore central pathway may be seen as a curve running through the center of the pore tubular structure. The center is the center of mass of the cross-section, or 2D cross-sectional shape (assuming uniform mass density) in a plane orthogonal to the pore central pathway. Target polypeptides may translocate through the pore along the pore central pathway, e.g. from a first reservoir into a second (or further) reservoir. In some embodiments, the target polypeptides enter the pore from a first reservoir via a first opening in the lumen part and exit the pore via the second opening in the aperture part, where the lumen part and aperture are oriented along the pore central pathway. In other words, the device includes a pore central pathway that extends from the first opening to the second opening of the pore. This pathway serves as a reference line for the orientation of the one or more lumen parts and the aperture part of the pore. The central pathway through the pore need not be straight and may even divide into branches. In embodiments, the central pathway may comprise a straight axis or a plurality of straight axis connected to one another at an angle or via a curved section. When the pore has more than two openings, multiple straight axes may be defined, each extending toward different openings. These axes can be understood as straight lines that pass through the center of mass of the pore's cross- sectional shape, assuming uniform mass density, in a plane that is perpendicular to each straight axis. Target polypeptides may move along pathways that are generally aligned with these axes, for example, transitioning from a first reservoir to a second or subsequent reservoir.

In the current invention, the pores may have a maximum effective diameter of up to 10 micron, preferably less than 5 pm, more preferably less than 1 pm, i.e., nanopores.

Pores can be categorized into solid-state pores (or inorganic pores), biopores (or organic pores), or hybrid pores. Solid-state pores are fabricated holes in a thin membrane made from robust materials such as silicon, silicon nitride, graphene, hafnium oxide, or other suitable materials. They can be manufactured with high precision, allowing for controlled properties such as diameter, thickness, and surface charge density. Notably, they display robust stability, enabling them to withstand a broad range of temperatures, pH levels, and chemical environments, and are resistant to protein denaturing agents.

Additionally, solid-state pores can be fabricated in arrays, enabling high-throughput applications. In contrast, creating arrays of biopores is more challenging. Solid-state pores also offer reusability, as they can often be cleaned and reused, whereas biopores are typically single-use. Finally, solid-state pores can be readily integrated into electronic devices and circuits, offering a key advantage for applications such as detecting and quantifying molecules in solution.

On the other hand, biopores are pores composed of biological materials, typically proteins. They occur naturally in the cell membranes of organisms, functioning as gateways for the transport of ions and molecules in and out of cells.

Hybrid pores combine the features of both solid-state pores and biopores, integrating a biopore into a solid-state membrane. This approach seeks to leverage the durability of solid-state materials and scalability of solid-state based CMOS process technology, along with the precise size control and specific recognition capabilities of biopores.

Given their various advantages, solid-state pores are preferred.

In embodiments, the lumen part of the pore may have inner walls that have a higher surface charge density than the inner walls of the aperture part. This could help in better signal transduction and measurement of polypeptides. A lumen part with a high surface charge density is preferred for optimal performance. This can be promoted by the deposition of a high surface charge density layer on its surfaces. In embodiments, the lumen part of the pore may have inner walls made of a high-k dielectric (e.g., HfCh). Atomic Layer Deposition (ALD) of Hafnium Dioxide (HfCh) can be employed to form such a layer. Hafnium dioxide (HfC>2) indeed has a high dielectric constant and can thus accumulate a high surface charge density when in contact with an electrolyte.

Alternative approaches can be utilized, where organic coatings such as Self-Assembled Monolayers functionalized with charged groups such as carboxy or amine groups are applied to the lumen part. Conversely, the aperture part of the pore device is preferably treated to limit its surface charge density, thereby creating a more pronounced bottleneck. This can be achieved through selective functionalization, e.g., with electrically neutral chains, preferably non-polar chain, or by providing the inner walls of the aperture part with a material which has a lower k value, i.e., a lower dielectric constant than the material of the inner walls of the lumen part. The k value of the material of the inner walls of the aperture part may, for instance, be lower by at least 0.1, preferably at least 1, more preferably at least 4 with respect to the k value of the material of the inner walls of the lumen part. The pore can be designed in a multitude of shapes, including but not limited to a stepped cylindrical shape (see Figs. 2, 7, and 8), a conical shape (see Fig. 4), a pyramidal shape (see Fig. 5), a L-shape (see Fig. 11) or a combination thereof (see Fig. 9). The shape can also be irregular. Transverse cross-sections of the pore, i.e., cross-sections taken perpendicularly along the pore central pathway, can have any shapes such as circular, hexagonal, square, or irregular. In embodiments, the pore may have a minimum effective diameter of less than 9 nm, preferably less than 8 nm, more preferably less than 7 nm, yet more preferably less than 6 nm and most preferably less than 5 nm. This makes it possible to detect smaller polypeptides and offers higher resolution. In embodiments, the pore may have a minimum effective diameter of at least 1 nm, such as at least 2 nm. This enable the polypeptides to pass through. The minimum effective diameter of the pore is in the aperture part thereof.

Pores in solid state materials are fabricated by various means including ion beam sculpting, focused ion beam fabrication, electron beam fabrication, track-etching, dielectric break down, laser-assisted dielectric breakdown, laser-assisted etching, wet etching, and atomic layer etching. These techniques make it possible to create pores with varying shapes and surface chemistries in a range of materials including silicon, silicon nitride, silicon dioxide, hafnium oxide, aluminum oxide, graphene, glass and polymer films. A convenient way to fabricate the pore is by exposing a silicon substrate having the same thickness as wished for the aperture diameter to tetramethylammonium. For instance, to obtain a 5 nm aperture diameter, a mask can be created on a silicon substrate having a thickness of 5 nm. The mask can have an opening of 12 nm diameter exposing the silicon. This opening could be done using a technique like electron beam lithography or focused ion beam milling, both of which can create features at the nanometer scale. The silicon substrate is then exposed to the tetramethylammonium etchant, which removes material where the substrate is exposed.

After the etching process, the mask is removed, leaving behind a pore of the desired size.

We now refer to Fig. 9 where parameters of a pore are defined. The first opening of the pore is an entry point of the pore. It is directly connected to the lumen part of the pore, serving as the initial entry point for polypeptides. The effective diameter of a transverse cross-section at this opening is always less than 10 pm, preferably less than 5 pm, more preferably less than 1 pm, allowing only tiny particles to pass through. The specific shape of this two-dimensional opening can be circular, hexagonal, square, or irregular, reflecting the diversity of the possible pore designs. The lumen part of the pore is directly fluidically connected to the first opening and provides a passage for the traveling polypeptides. The lumen part's alignment is parallel to the pore central axis, corresponding here to the pore central pathway, thereby maintaining a direct route for the polypeptides.

The device may further comprise an electrical center (9) and at least one sensor region. The at least one sensor region is such that a target molecule travelling along the pore central pathway will pass through the sensor region (sensitive region). The sensor region may also be referred to as an electrode region when the FET has an extended gate. The sensor region of the pore comprises a sensitive gate surface exposed to the electrolyte of the pore. When the pore is embedded in the FET, such that the FET is directly gated by an electrolyte-filled pore running through the channel region of the FET, the sensor region is in between a source (S) and drain (D) region (source drain axis). The sensor region then comprises the semiconductor channel of the FET with the pore running through or beside the semiconductor channel. The channel has an electrical contact to both the source (S) region and the drain (D) region. When the FET is instead remote, there are no source (S) and drain (D) regions near the sensor region but the sensor region comprises a conducting (e.g. metal) pore electrode with the pore running through or beside it, and one contact to the pore electrode can suffice. This contact is in electrical connection to the gate of a remote FET.

When the FET has a remote gate, the electrical center and the sensor region overlaps. Thus, in this embodiment, the device comprises an electrical center comprising a sensor region of the device. In embodiments, the sensor region may overlap with at least one of the lumen part and the aperture part of the pore. This sensor region may be mostly in the lumen region to allow for a sufficiently large electrode size to enable low noise. The pore electrode area may be exposed for at least three quarters to the lumen part of the pore. In embodiments, the sensor region may be located mostly in the lumen region. This is advantageous because, since the lumen is larger than the aperture, it allows for a larger sensitive gate surface area then would be possible in the aperture region. The larger sensitive gate surface area allows low noise while still being sufficiently small to attain high bandwidths. The pore electrode is made of an electrically conducting material, such as but not limited to TiN, Ru, Pt, and doped silicon. A thin solid dielectric material (0.5-10nm), such as but not limited to AI2O3, TiOz, HfOz, SiOz, may be present between the pore electrode and the space inside the pore. The pore electrode is typically located on the wall of the pore. The sensor region comprise either the semiconductor of a metal-oxide- semiconductor field effect transistor (MOSFET) channel or it comprises a pore electrode electrically connected to the gate of a metal-oxide-semiconductor field effect transistor (MOSFET). In the first case, a dielectric may be covering the inner walls of the pore, thereby serving as a gate dielectric. Additional pore electrodes may be present apart from the pore electrode. Thus, in some embodiments the device comprises a main pore electrode and one or more additional pore electrodes. The additional electrodes may be located in the aperture part or the lumen part of the pore. In embodiments of the extended gate embodiments, the sensor region design of electrical center in the lumen may be "decoupled" from the aperture. The aperture determines the translocation signal. The decoupling enables an aperture with a small diameter and low height (<50nm) for a strong molecular signal, and a lumen, containing the sensor region, with larger diameter and height for low noise. The maintenance of the small scale (<10pm) for the lumen allows for maintaining high bandwidth.

In embodiments, less than 25% of the pore electrode area of the electrical center is in the aperture part. The pore electrode may be situated entirely in the lumen part, and there may be a spacer in the lumen part separating the pore electrode from the aperture part.

In embodiments, the lumen part may house the sensing region, allowing for the detection of the polypeptides as they pass through. Additionally, the area of a transverse cross-section in the lumen part (AL) is typically greater than the aperture part's transverse cross-section area (AA), with AL preferably being more than double that of AA. This feature helps to enhance signal resolution while simultaneously reduce pore clogging, thereby promoting system reliability, preferably, the area of every transverse cross-section in the lumen part (AL) is typically greater than the area of every transverse cross-section in the aperture part (AA), with AL preferably being more than double that of AA. In embodiments, at least 3/4 of the transverse cross sections of the lumen part of the pore may have an area which is at least four times larger than the area of any aperture part transverse cross section, such that they fulfil the requirement AL>4AA. This configuration facilitates improved signal resolution and reduces the likelihood of pore clogging, thereby enhancing system reliability.

The aperture part of the pore is an integral piece of the pore, connected to the pore's second opening. Its alignment, like the lumen part, is typically oriented along the pore central pathway, providing a clear path for polypeptides. Its minimum effective diameter, dA, is less than 10 nm, preferably less than 8 nm, yet more preferably less than 6 nm, and most preferably less than 5 nm, ensuring that only very small entities can pass through. The minimum effective diameter of the aperture part is preferably at least 1 nm, such as at leat 2 nm to ensure that the linearized protein can pass through it. In embodiments, the height of the aperture part of the pore may measure at most 50 nm, preferably at most 15 nm.

By adjusting the size of the lumen part and the aperture part of the pore in the device, such that the lumen is longer and wider than the aperture, both high bandwidth and low noise may be attained. The asymmetric design allows to decouple the FET design in the lumen from the aperture, where the aperture determines the translocation signal. In this way, in the case of the FET with remote gate, the FET can be made larger to control the short channel effects, while still maintaining an optimal translocation signal, because the aperture can be made very small without affecting FET properties. The FET active area determines the bandwidth but can still be kept sufficiently small to realize bandwidths > 1-lOMHz for which SNR > 1. Hence, these embodiments of the present disclosure allow a high-quality FET with suppressed short channel effects, an optimal signal magnitude and resolution pore detector and high bandwidths (>l-10MHz bandwidth with SNR>1.)

In embodiment, the aperture part may also contain the sensing region, facilitating the detection and analysis of the passing polypeptides. The second opening of the pore acts as the terminal point of the journey for the polypeptides through the pore. It is directly linked to the aperture part of the pore.

An embodiment of the invention is illustrated in fig. 9, showing the lumen part (7) of the nanopore as the wide and long part of the pore, while the aperture (8) is narrow and short. Cross-section may be made at a point on the pore axis, here corresponding to the pore central pathway (1); 2D shape formed by the cross-section of the pore tubular structure and a plane orthogonal to the pore central pathway running through the point on the pore central pathway.

Thus, the effective cross-section diameter (or radius) may be an average diameter (or radius) at a certain location if the pore is not circular in shape. Further, in embodiments, the aperture part inside the pore has an effective cross-section diameter, dA, less than 10 nm, preferably smaller than 8 nm, for all its transverse cross-sections. Any cross section (2) of the lumen part is at least 8 nm (e.g., when all crosssections of the aperture part are smaller than 8 nm), and preferably at least 10 nm. In embodiments, the area of at least 90% of the cross sections of the lumen part, e.g., all of them, is larger than twice the area of the largest aperture cross section. This may also be defined as that the largest cross sectional area of the aperture part is smaller than twice the area of the smallest cross sectional area taken across 90% of the rest of the pore (90% of the lumen part). Typically, the difference in size is even larger, such that the area of the majority, such as 3/4 (i.e. >75%), of the lumen cross sectional areas are at least 4 times larger than minimum pore cross section of the aperture part (AL>4AA), or even 8 or 16 times larger or more than minimum pore cross section of the aperture part (Ai>8AA Or AL>16AA). When having multiple sublumens, each sub-lumen of the lumen part may have larger dimensions (effective cross-section diameter, along length) than the aperture part, as defined above. For instance, where more than three quarters of the cross-section areas are at least 4x larger than the minimum cross section area of the aperture parts, preferably 8x larger, even more preferably 16x larger.

The area of the cross-section of the first reservoir at the connection with the lumen part is at least 1pm. This distinguishes the reservoir from the lumen. Typically, the cross-section of the first reservoir is at least 1 pm along its whole length.

The area of the cross-section of the first reservoir at the connection with the aperture part is at least 10 nm. This distinguishes the reservoir from the aperture. Typically, the cross-section of the first reservoir is at least 10 nm along its whole length.

Further, in embodiments, the pore may be such that at the ends of the pore, the cross-section area shows an abrupt enlargement, larger than a factor of four. Thus, the cross section area at each opening of the pore, Ao, may show an enlargement of the pore compared to an area inside the pore, Ai, such that at least Ao>4Ai.

In fig. 9, the double arrow on the right points to two regions of the pore having approximately the same resistance.

An example of a pore shape is illustrated in fig. 10, a schematic cross-section of a pore having a narrow aperture at the top, and a wide lumen part below, with a pore electrode area surrounding the top part of the lumen facing the aperture. For a maximal signal, the resistance of the aperture should approximately equal the resistance of the lumen, where the resistive divider criterion is thus defined as RA=RL. The reason to have an asymmetry between the aperture and the lumen is that a small aperture give rise to a high translocation signal and high resolution, while a large lumen gives no translocation signal and can fit a large pore electrode. Larger pore electrodes mean less noise, and large pore electrode capacitance makes a device less sensitive to parasitic interconnect capacitance, providing better coupling to a remote FET, but it should not be too large to maintain bandwidth.

In fig. 10, the pore has an aperture at the top, and a lumen part below, with a pore electrode area surrounding the top part of the lumen facing the aperture. In that embodiment, the layer thickness determines the electrode size, and thus the electrode size cannot be varied on a same wafer. The pore electrode size is fixed by the lumen effective diameter, and to keep the lumen resistance equal to the aperture resistance, the lumen depth is set by the effective lumen diameter. Varying diameter on the same wafer is hence difficult, as one cannot change thicknesses easily. The maximum pore electrode size is limited by the achievable vertical etch aspect ratio. By rotating the lumen in fig. 1090”, one attains a pore as illustrated in fig. 11, where the lumen and the aperture are perpendicular instead of parallel, thus attaining a lateral design. In this design, one may vary the pore electrode size. Full pore electrode sizing freedom is attained since sizing is determined by layout, and in this case it is also be more easy to integrate and vary materials. This design also provides lumen geometric freedom in layout, as the lumen size can be decoupled from the pore electrode size. However, one needs to mind parasitic capacitance. In this design no aspect ratio limitations due to the process (as geometry is mainly defined by mask) is present, nor are there any pore electrode size limitations due to the process. There is more freedom to shape the lumen, such as by mask design rather than by etch, and it is possible to obtain lots of different shapes on the same wafer.

A further example of a lateral device design is illustrated in fig. 12, showing the aperture (8), the lumen (7), a planar pore electrode (11), an insulator (diagonal pattern), a FET (3), a base of foundry Si (dotted pattern), a fluidics wall (dark rectangle at the top), a cis reservoir directly fluidically connected to the lumen, and trans reservoir directly fluidically connected to the aperture. The cis reservoir is the reservoir in which the polypeptides are provided before that pass through the pore for analysis. The trans reservoir is the reservoir in which the polypeptides end up after they passed through the pore. fig. 13 illustrates a further example of a lateral design, in a zoomed out view of the cross section. The device comprising a foundry wafer (dotted pattern), a trans reservoir in direct fluidic connection with an aperture (8), a sensing pore electrode (11), a bias electrode (further on the right from the sensing pore electrode), and a large channel (>30pm) to a cis reservoir, wherein the cis reservoir and the trans reservoir are separated by bonded glass or Si (dark upper part), fig. 14 illustrates a further example of a lateral design similar to fig. 13, but comprising multiple sub-lumens (7a, 7b).

The device typically comprises a wall/walls having a width/thickness surrounding the pore. In embodiments, the area and the thicknesses of the insulating walls surrounding the pore may be such that the total summed capacitances between the electrolyte inside the pore and the outside electrical conductors, such as the first and second reservoirs and including the pore electrode, is smaller than 50fF, preferably smaller than lOfF even more preferably smaller than If F.

In some embodiments, the geometry of the pore may be such that 1) if the pore and reservoirs are uniformly filled (in simulation) with a single isotropic and uniformly conducting test material or test liquid and 2) if it is made sure (in experiment or theory) that the main pore electrode makes electrical contact with this test material or liquid with negligible contact resistance (by removing any thin dielectric on the pore electrode) and negligible pore electrode resistance, then the (test) resistance between the aperture reservoir as electrically contacted by that reservoir's electrode and the main pore electrode (Rl) approximately equals the (test) resistance between the lumen reservoir as electrically contacted by that reservoir's electrode and the main pore electrode (R2). Approximately equal resistance here refers to equal within a factor of 1/6 to 6 or more preferably within a factor of 1/3 to 3. In other words, R1/R2 may be from 1/6 to 6 or from 1/3 to 3.

In other embodiments, the geometry of the pore is such that if the pore and reservoirs are uniformly filled (in experiment or theory) with a single isotropic and uniformly conducting test material or test liquid and 2) if it is made sure in experiment or theory that the main pore electrode makes electrical contact with this test material or liquid with negligible contact resistance (by removing any thin dielectric on the pore electrode) and negligible pore electrode resistance, then the lumen test resistance (RL) approximately equals theta (0) times the aperture test resistance (RA), i.e. 0* RA . Approximately here refers to equal within a factor of 1/6 to 6 or more preferably within a factor of 1/3 to 3. Theta (0) is the resistivity of the electrolyte of the aperture reservoir/second reservoir (the pore device is meant to be used with) Rel, divided by the resistivity of the electrolyte of the lumen reservoir (the pore device is meant to be used with) Re2, i.e. 0 = — . [Equation 1]

For use with symmetric electrolytes theta equals one. In some embodiments, the resistance of the lumen part, RL, and the resistance of the aperture part, RA, is approximately the same when no molecule is translocating through the pore, such that RA=RL. RL may be defined [Equation 2],

and RA may be defined Equation s .

Thus, the correlation between the resistances may be defined as:

i.e. the resistance condition of the geometry independent of electrolyte.

Alternatively, the resistance may be defined as: Equation 5 ,

where p is the resistivity.

A(l) is the area of the cross section at position along pore central pathway /, and top, bottom, pore electrode bottom and pore electrode top correspond to positions along the pore central pathway, where pore electrode refers to the main pore electrode, rtop and rbottom is the effective cross-section radius at the ends of the pore.

Further, the pore above and pore below the main pore electrode will have approximately equal resistance, where the pore above and below the electrical center may correspond to the lumen and aperture part, or may be partially overlapping as the main pore electrode may be partially situated in the different parts as discussed above, thus the pore above and below the main pore electrode is not necessarily the same as pore lumen part and pore aperture part, in which the resistance of pore below the pore electrode may be referred to as Rp (which may be identical to RA) and the resistance of the pore above the pore electrode may be referred to as Rc (which may be identical to RL). Typically, the largest part (>75%) of the area of the main pore electrode is exposed to the lumen part of the pore (the wide part of the pore). A pore where the pore part above and pore part below the (main) pore electrode have approximately equal resistance may be realized in different ways. It can for example be translated in purely geometric criterium independent of electrolyte. The pore electrode satisfying this equal resistance criterium most closely is main pore electrode, others are optional auxiliary pore electrodes. It should be noted that a pore electrode does not need to entirely wrap around the pore. The electrode is typically made of a conductive material, which may be selected from (but not limited to) the of following materials; titanium nitride, TiN, ruthenium, Ru or platinum, Pt, or doped silicon, doped-Si. The pore electrode can for example be covered with a thin dielectric. That the resistance is "approximately" equal could for example be defined as meaning that x40 leeway in Rc/Rp possible, this would imply a reduction by at most a factor of 10 versus the optimal signal modulation. In another example, xl/2 of optimal signal modulation means x6 leeway in Rc/Rp, or in a third example, x3/4 of signal optimal modulation means x3 leeway in Rc/Rp. Approximately equal resistance here may refer to equal within a factor of 1/6 to 6 or more preferably within a factor of 1/3 to 3. This is illustrated in fig. 15, where the relation between the ratio of resistances and the optimal signal modulation ( which is related to the desired signal to noise ratio, SNR), such that xl/2 of optimal signal modulation corresponds to x6 leeway in Rc/Rp, and similarly that x3/4 of optimal signal modulation corresponds to x3 leeway in Rc/Rp.

In some embodiments, the lumen part is so large, the height of the lumen part of the pore can be larger than 1 micron (i.e. the length), such as tenths of microns, and the cross sections large, but typically smaller than 10 micron in effective diameter, and preferably smaller than 1 micron in effective diameter (i.e., a nanoscale pore or nanopore), that when a target molecule passes through, no significant modulation of the resistance ( RL) occurs, while the aperture part is so narrow, the height being 50 nm or smaller and the effective diameter being 10 nm or smaller, that when a target molecule passes through, a significant modulation of the resistance (RA) occurs. The lumen part may have a uniform shape (typically of equal width) or may be tapered, becoming smaller and smaller when approaching the aperture, to relax requirements on the top part. Thus, it should be realized that the lumen part of the pore need not necessarily have a uniform cross-section, but the pore may have a shape of a truncated cone, such that the pore provides a minimum cross-section at one end of the pore and a maximum crosssection at an opposite end of the pore.

In fig. 7 is illustrated a pore-FET device according to an embodiment, wherein the pore is embedded in the FET, such that the FET is gated by an electrolyte-filled pore running through the channel region of the FET, where fig. 7 (left) illustrates a side view of the device/pore, and fig. 7 (right) illustrates a top view. The device comprises a wall having a width/thickness w surrounding the pore, where the wall thickness w is 10 nm or less, such as 5nm or less, which wall thickness is illustrated as Si wall thickness in fig. 7 (right). Wall width/thickness is the width of the pore electrode/channel between lumen and opposing channel edge. For example, the Si wall thickness is the width of the silicon channel formed at the narrowest constriction between the pore and sidewall passivation. As illustrated in fig. 7 (right), the wall comprises at least three different layers comprising at least two different materials, the layers being arranged on top of each other such that a dielectric material is arranged at the top and bottom of the pore, the top dielectric layer having a height box top and the bottom dielectric layer having a height /)ox_bottom, and a conductive/semi-conductive material is arranged in the middle of the pore and having a height hsi, the semiconductive material typically being silicon (Si) and having a height defined as hsi of 5- lOOnm, or more. It may thus be seen that in this example, the height of the lumen part of the pore, HL, is defined by h = box to_P+ hsi, wherein hi is in the range of 50 nm-1 pm, and the height of the aperture part of the pore, HA, is defined by /)A= box bottom, wherein /)A is typically less than 50 nm. The cross-section of the pore orthogonal to the pore axis has an Area which defines the effective cross-section radius r=(A/n)^1/2 and an effective cross-section diameter defined by d=2r. The effective diameters of the lumen part and aperture part (i.e. the measurement of the pore parallel to the cross sections shown in fig. 9, where the effective radius, r, of the cross sections relate to the effective diameter, d, such that 2r=d, and the effective diameter is perpendicular to the height of the lumen and aperture parts) relate such that the area of any cross section of the lumen part is at least twice the area of the smallest area of any cross section of the aperture part, as defined above. The effective diameter of the lumen part, di is in the range of 50-300 nm, and the effective diameter of the aperture part, C/A is less than 10 nm. The wall width and the FET width from a top view of the device/pore are illustrated in fig. 7 (left).

It is preferred for the pore FET to realize a high signal readout with low noise. To achieve that, getting a negligible signal from the lumen part of the pore is advantageous. This can be attained by the majority of the lumen part of the pore being wider, and by the area of cross sections of the lumen part being larger than the aperture part, such as typically 4 or even 8 times larger, as described above. A second advantageous feature, to attain the high signal and high resolution, is that the aperture part of the pore should preferably be smaller, such as being shorter (a low height FIA) and narrower (a small effective diameter dA and thus a small area AA of a cross section of the aperture part) than the lumen part. The signal strength may be independent of the resolution as defined by

[Equation 6] where hA is the height (length) of the aperture part, and AA is the area of an aperture cross section. Thus, it may be seen that the resolution corresponds to the heigh/length, i.e. resolution = hA. Thus, the size of the aperture may be adapted based on the application of the pore FET device, such that desired resolution and process time limits are met, where the size of the aperture opening, i.e. the effective diameter of the aperture, is typically sub 10 nm. A third preferred feature of the device is the resistive divider criterion, to obtain the highest signal for the overall pore, such that the resistance of the lumen part is approximately the same as the aperture part.

The advantageous features above are structural features, wider pore in lumen part (or part above electrical center) and narrower and shorter pore in the aperture part (or part below electrical center), which are independent of electrolyte. Assuming a uniform electrolyte, and as we aim for equal resistance RL=RA or Rp ^r=Rc, ' which corresp i-onds ap i-pi-roximately 7 to

,

_J (Equation 4) , resistivity drops out of the equation, resulting in a purely

geometric definition. The criterion would be the same if the empty space is filled with any material of uniform conductivity. Here, the surface charge is not accounted for. To account for surface charge, the 11 definition can be altered, where a parameter can be introduced which depends on electrolyte conditions.

In the case of asymmetric electrolyte, the resistivity p of cis and trans reservoir electrolyte is different, which may be characterized by an asymmetry factor theta 9 = — [Equation 7].

P2

The demands on wide pore in lumen part (or part above electrical center) and narrow and short pore in the aperture part (or part below electrical center), are independent of electrolyte still, but for the RL=RA or Rp=Rc, the resistivity does not drop out of the equation, and we thus introduce the asymmetry factor theta;

[Equation 8],

where the criterion would be the same if empty space is filled with any material with uniform conductivity, adjusting criterion to 0R1=R2.

Thus, in embodiments, the pore of the FET-based pore device may comprise two parts, an aperture with a small effective diameter and low height as well as a lumen with larger effective diameter and height. The aperture with smaller effective diameter may be defined in a dielectric layer below the silicon semiconductor layer. The thickness of the aperture dielectric layer may be reduced in order to increase the resolution with which features can be resolved on a translocating molecule. For optimal signal magnitude the thickness of the bottom insulator is preferred to be of a similar thickness (or somewhat smaller) as the molecular feature to be detected. The aperture effective diameter is preferred as small as possible. The lumen with larger effective diameter is defined in the silicon layer and an insulator layer on top of the silicon. The silicon layer is preferably between 5 and lOOnm thick. The top oxide thickness may be chosen to obtain an optimal signal magnitude, which is obtained for an optimal resistive divider condition, where the electrolyte in the top insulator part or lumen part (including access and spreading resistances) should have equal resistance as the electrolyte in the aperture part (including access and spreading resistances). In embodiments of the pore FET of the present disclosure, the effective diameter of the lumen is chosen larger than the effective diameter of the aperture resulting in an "asymmetric device" (large lumen, small aperture), in order to still allow for an optimal signal magnitude while allowing a thicker silicon or pore electrode layer (5-100nm). The lumen part of the pore may have a uniform shape (substantially equal effective diameter throughout) or a tapered shape. The relation between the sizes of the lumen and aperture parts are advantageous, as this enables the larger bandwidth and low noise. The present design allows to decouple the FET design in the lumen from the aperture which determines the translocation signal. In this way the FET can be made larger to control the short channel effects, while still maintaining an optimal translocation signal, because the aperture can be made very small without affecting FET properties. The FET active area determines the bandwidth but can still be kept sufficiently small to realize bandwidths > l-10MHz. Hence, in embodiments, the present pore FETs allows a high-quality FET with suppressed short channel effects, an optimal signal magnitude and resolution pore detector and high bandwidths (>l-10MHz.). Thus, the devices of the present invention preferably operated with bandwidths over 100kHz, even more preferably with bandwidths higher than 1MHz due to nanoscale cross-sections of fluidic passage.

In embodiments, to reduce short channel effects, different designs of the device may be beneficial. For example, the pore effective diameter in the lumen part (e.g., silicone region) should not be made too small, such that it may be beneficial to maintain a pore effective diameter larger than lOnm, preferably larger than 20nm. Typically increasing pore effective diameter decreases signal strength. This is avoided here by providing the narrower 'aperture' pore in the bottom insulator. This aperture will determine signal strength. Thus, by combining a larger lumen which can house a larger main pore electrode with a small aperture, the drawbacks of the prior art may be mitigated. Further, it may be beneficial to reduce the (silicon) wall thickness. For example, the wall thickness is the width of the silicon channel formed at the narrowest constriction between pore and sidewall passivation. This wall thickness could be reduced below lOnm, preferably below 5nm. In addition, thinning down the silicon of the FET further suppresses short channel effects. Further, it should be considered that short channel effects depend on the channel length of the FET device (the distance between source (S) and drain (D) junctions) and the effects become worse for decreasing gate length. Hence, the distance of the source (S) and drain (D) junctions may be adjusted to regulate the short channel effects.

As described above in relation to fig.s 10 and 11, in an embodiment the FET may be embedded in the pore, and the aperture may have a height corresponding to the height of the bottom layer. In view of this, different designs may be possible. One variation allows for a thicker dielectric on the bottom of the silicon to prevent shunting currents running in the bottom of the silicon. Another variation makes use of a 2D material (e.g., boron nitride, graphene, dichalcogenide) as the aperture material to realize the thinnest possible aperture membrane for very high molecular resolution. A fourth layer may be arranged below the bottom dielectric material and having a heigh fu, wherein hi_= box to_P+ h_Si + /j_Ox_bottom and /)A= / . Introduction of a dielectric multi-layer consisting of multiple different dielectric materials allows for a thicker dielectric on the bottom of the (silicon) wall while still having a thin aperture height (haperture, FIA). This may be advantageous for suppressing shunt currents on the bottom of the Si, which grow larger with thinner oxides on the bottom of the silicon. The thin aperture height allows a higher resolution for resolving closely spaced features on target polypeptides. Introducing a 2D material (as discussed above) below the bottom oxide allows for an atomically thin aperture layer (low f ) for which a very high resolution to resolve closely spaced features on molecules is attained.

In an alternative embodiment of the invention, an extended gate variation of the present device is presented, where the silicon in the pore is replaced with a pore electrode material, wherein the device comprises a remote, extended gate FET, where a pore electrode wrapped around the pore is connected to a remote FET. The pore electrode material is typically a metal, such as titanium nitride, TiN, ruthenium, Ru or platinum, Pt, which is coupled to the gate of a silicon transistor remote from the pore. The design allows a sufficiently large metal-electrolyte surface area of the pore electrode material in the lumen and hence a larger capacitance of the pore electrode. This allows to limit the signal reduction due to the coupling to a readout transistor, which may be implemented in relatively standard CMOS technology. A standard CMOS FET typically has a larger capacitance than aggressively scaled device. Having an increased pore electrode capacitance leads to a reduced signal decrease due to the capacitive divider which determines signal transfer between pore electrode and remote FET. A larger pore electrode with larger capacitance also leads to less signal reduction due to the interconnect capacitance due to the pore electrode-to-FET interconnect. More standard CMOS technology may be preferred due to cost reasons, and also because analogue design prefers larger FETs to suppress noise and variability.

Figure 8 illustrates an example of the remote gate sensor embodiment, fig. 8 (left) illustrating a side view of the device/pore, and 8 (right) illustrating a top view. In the schematic cross-sections of the device design for an extended gate NPFET, the metal pore electrode is connected to the gate of a nearby FET implemented in standard CMOS technology. Compared to fig. 7, the middle layer, his, in this embodiment is made from a metal, and the metal wall thickness is illustrated in fig. 8 (right).

The two main embodiments, the embedded FET (e.g., silicon pore-FET) and the remote FET (extended gate pore-FET) have several main characteristics in common. By increasing the lumen size and the active area, the noise is decreased. The larger lumen does not affect the signal (which is caused by the polypeptide in the aperture) as long as lumen resistance approximately equals aperture resistance. The resistance of the lumen and the aperture region, or the region above and below the electrical center should be approximately the same. The aperture effective diameter and height are kept low to enhance signal magnitude and resolution, respectively. Hence, noise reduction with maintained signal means the Signal-to-noise ratio (SNR) is improved when enlarging the lumen. Further, the capacitance associated with the active area, determines the bandwidth limit due to the electrolyte. When enlarging the lumen to enhance SNR, the lumen can still be kept sufficiently small to allow the SNR gains but also realize bandwidths above l-10MHz.

Also, for the embedded FET, due to the design (respective size difference between the lumen and aperture) the FET can be made larger to control the short channel effects of the silicon pore-FET. The larger pore FET also entails lower FET noise, and hence higher SNR.

For the extended gate pore-FET embodiment, due to the design one can have a large metal-electrolyte surface area of the pore electrode material in the lumen and this means a larger capacitance of the pore electrode. The larger capacitance allows to strongly limit the signal reduction due to the capacitive coupling to the readout FET. Moreover, this also allows to choose a larger readout transistor as typically preferred in analogue technology, and for cost reasons.

Figure 16 illustrates some possible alternative designs of the present devices, fig. 16A illustrates the general concept having a first part of the pore, a lumen part, and a second part of the pore, an aperture part, where a conductive material, acting as channel or having an pore electrode connected thereto, is present in the lower part of the lumen part. In fig. 16B a first variation of the concept is illustrated, where parts of the pore are the same, but the main pore electrode is located both in the lumen (first) part and aperture (second) part of the pore. In fig. 16C, a second variation of the main concept is illustrated, where the conductive material, acting as channel or having an pore electrode connected thereto, is present in the lower part of the lumen part, but with a spacer between the main pore electrode and the aperture part of the pore. In fig. 16D, a third variation of the main concept is illustrated, where a slanted/sloped main pore electrode is used.

Besides the main pore electrode, a plurality of additional pore electrodes may be used, such as one or two additional pore electrodes besides the main pore electrode, thus the apparatus may have multiple sensor regions. As illustrated in fig. 17A, depicting a lumen part, and an aperture part, where a conductive layer, acting as channel or having a pore electrode connected thereto, is present in the lower part of the lumen part, and in addition, a second pore electrode in present in the aperture part. Thus, it is possible to measure the change in resistance in different parts directly. As an alternative, as illustrated in fig. 17B, both the main pore electrode and the additional pore electrode is located in the lumen part, thus enabling direct charge sensing.

In embodiments, the system may comprise a first and second reservoir, optionally comprising an electrically conducting fluid, such as an electrolyte, and a pore connecting the two reservoirs. In embodiments, the target polypeptides to be analyzed may be present in the first reservoir (cis), and may during operation of the device exit the first reservoir (trans), enter the lumen of the pore of the device, translocate through the pore along the pore central pathway and exit through the aperture into the second reservoir, wherein the lumen/ electrolyte in the lumen has a resistance RL and the aperture/electrolyte in the aperture has a resistance RA, and the aperture has a size such that a significant modulation of the resistance RA occurs when a target molecule translocates through the aperture, while the lumen has a size such that no significant modulation of the resistance RL occurs when a target molecule translocates through the lumen.

The pore sensor may comprise a first and second electrolyte reservoir, respectively, being separated by a barrier comprising a pore. The sensor/system may further comprise electrodes for translocating molecules through the pore from the first electrolyte reservoir to the second electrolyte reservoir, wherein at least one of the first and second electrolyte reservoirs comprises the target molecule. The pore may have an aperture through which the molecule is translocated. As pores are not necessarily circular, the term "effective diameter" of the pore at a location refers to the average diameter of the pore at that location.

According to an embodiment, the resistance of the pore parts may be tuned. This may be performed by adjusting the size of said parts. Alternatively, the carrier concentration of the electrically conductive fluid (e.g. a salt concentration of an electrolyte) may be adjusted. Alternatively, the surface charge may be adjusted, by controlling the pH, as discussed in more detail below. It was noted that a significant conductivity increase can be obtained by having a low surface charge shallow pore (aperture part) combined with a high surface charge high pore (lumen part).

In the present devices, it is possible to provide surface charges on a surface of the pore along the extension from the first side to the second side, wherein the surface charges are of an opposite sign to a charge of the molecule to be detected. Having surface charges on the surface of the pore implies that the fluid in the pore may exhibit charge carriers of an opposite sign. Hence, when the molecule to be detected is moved through the pore, there may be a local depletion of charges of the same sign in the fluid. The local depletion is induced by the molecule to be detected being arranged in the pore. This implies that the introduction of the molecule to be detected in the pore may change the fluid current flow through the pore to a large extent so as to increase effective difference in the gate voltage when the molecule to be detected is moved through the pore. According to an embodiment, a concentration of the surface charges is in a range of lxlO¹² - lxlO¹⁵ cm'².

Surface charges may be formed on the surface of the pore by (de)-ionization of OH-groups or other ionizable groups at the surface of the thin dielectric layer. This may be caused by exposing the pore FET to the electrolyte fluid. For instance, if silicon dioxide (SiCh) is used as the thin dielectric layer, depending on pH, silanol groups ionize when the silicon dioxide is immersed in an aqueous solution (which may be used as the electrolyte fluid), and ionization of silanol molecules at the surface of the thin dielectric layer would form negative surface charges on the surface of the pore for a neutral (pH = 7) solution. Charge density may have a dependence of pH value of the electrolyte fluid, such that control of pH value may provide a control of concentration and sign of the surface charges.

If the surface charge concentration is very high, the surface charges could cause a high ion concentration in the fluid in the pore, such that an impact of the charge of the particle to be detected on the effective gate voltage may be reduced and, hence, blockage of electrical potential distribution in the pore by the particle to be detected may be reduced. Therefore, the surface charge concentration should preferably not be too high.

The sensing region of the pore is where the detection of the polypeptides occurs. It can be located in the lumen part, the aperture part, or both. In embodiments, the lumen part of the pore may have inner walls that have a higher surface charge density than the inner walls of the aperture part. This helps in better signal transduction and measurement of polypeptides. When a molecule passes through the sensing region, it disrupts the ionic current flowing through the pore, which can then be detected and analyzed.

The gate is controlled by the sensing region of the pore when the system is in operation.

In embodiments, typically in operation, the pore may be filled by an electrically conducting fluid.

The system comprises one or more electrical readout modules connected to the drain (D) and/or source (S) of one or more of said field-effect transistors, configured to record an electrical signal indicating the presence of electroactive labels on at least two different amino acid types of a linearized polypeptide when the linearized polypeptide translocates through the pore. In embodiments, the one or more electroactive labels may be a first electroactive label applied to one specific type of amino acid and a second distinct electroactive label applied to another specific type of amino acid of the polypeptide. This feature allows identifying a sequence of two amino acids and the corresponding interdistances. This is sufficient to identify the large majority of proteins. In embodiments, the electroactive labels may be charged labels, zwitterionic labels, and/or labels with a hydrodynamic radius measuring from 5% to 49% of the minimum effective diameter of the aperture. This variety allows for a wider range of detectable labels, broadening the types of polypeptides that can be analyzed.

In embodiments, each electrical readout module may operate at a frequency of at least 1 kHz, preferably at least 1 MHz, and potentially up to 100 MHz. For instance, it may operate at a frequency with the range of from 1 kHz and 1 Ghz. This is advantageous because it makes it capable of quickly and accurately capturing the electrical signal when a polypeptide translocates through the pore. This fast operation speed enhances the throughput of polypeptide identification.

In embodiments, each electrical readout module may include a pre-amplifier, typically an on-chip preamplifier. The pre-amplifier may comprise a source-follower. This is advantageous as it allows to accommodate a larger bandwidth and in particular higher-frequency signals.

In embodiments, each electrical readout module may comprise a pre-amplification system comprising:

(a) A source-follower configured to bias the Field Effect Transistor (FET) of the pore field-effect transistor (e.g., the drain (D) of the pore field-effect transistor is directly electrically connected to the gate of the source-follower); and

(b) A further FET designed to operate in at least one frequency within a bandwidth of from 100 MHz to 1GHz and configured to be biased by the source-follower (e.g., the gate of the further FET is directly electrically connected to the source (S) of the source-follower).

Typically, each electrical readout module further comprises an analog-to-digital convertor configured to receive an analog output from the further FET and to transmit a digital output to the processing unit. The system may comprise a memory (18) storing a database of polypeptide fingerprints or storing the parameters of a neural network configured to output a polypeptide when receiving a recorded electrical signal as input. The neural network may have been trained on electrical signals indicating the presence of electroactive labels on (e.g., at least two) different amino acid types of a linearized polypeptide when the linearized polypeptide translocates through the pore.

The memory can be part of the system or the system can comprise a port for connecting to the memory. The capacity of the memory could range from gigabytes to terabytes or even petabytes, depending on the size and complexity of the polypeptide fingerprint database or neural network. This memory could be in the form of RAM, flash memory, or even cloud-based storage in which case the system is adapted to access said cloud-based storage. The database might include fingerprints for known polypeptides, including variations caused by post-translational modifications. Each fingerprint is a unique representation that characterizes a specific polypeptide. Identifying fingerprints instead of complete sequences can provide significant advantages in terms of efficiency and cost-effectiveness. Fingerprinting techniques focus on identifying unique, characteristic regions or patterns in a molecule, which is typically faster than sequencing the entire molecule when the aim is a mere identification of the molecule. This focused approach also contributes to the cost-effectiveness of fingerprinting, as high- resolution sequencing can be quite expensive and time-consuming. This fingerprint is derived from the sequence of amino acids that make up the polypeptide. This fingerprint typically comprises a sequence of at least two different types of amino acids present in the polypeptide and may also incorporate their interdistances. This fingerprint typically comprises in a sequence of less than 10, preferably at most 4, more preferably at most 3 different amino acids present in the polypeptide. Most preferably, the fingerprint consists in a sequence of only 2 different amino acids as it is sufficient to identify a large majority of all existing polypeptides. Utilizing fingerprints comprised of only two amino acids provides certain advantages such as simplicity, speed, and resource efficiency. By focusing on just two types of amino acids, the complexity of the system is greatly reduced, potentially decreasing the likelihood of errors during identification. The limited possibilities to consider could also expedite the identification process, making it quicker than methods involving larger sets of amino acids. This narrowed focus additionally simplifies the electroactive labeling process, as fewer types of labels are needed, thereby saving on resources. Furthermore, a two-amino-acid fingerprint system could streamline database management. With fewer variations to account for, the complexity and size of the fingerprint database are diminished, leading to more efficient data handling and search processes.

The memory might store the parameter of a neural network trained on a myriad of electrical signals, each signal indicating the presence of the electroactive labels on at least two different types of amino acids found in a distinct linearized polypeptide when it translocates through the pore. In this scenario, rather than maintaining a database of known fingerprints, the system leverages the capability of the neural network to recognize patterns from the learned data. Each training signal is a unique representation of a specific polypeptide that allows the neural network to learn how to characterize and identify various polypeptides based on their respective signals.

Training neural networks on these signals can provide distinct advantages in terms of speed and adaptability. Neural networks excel at identifying patterns in large data sets, which makes them well- suited for complex tasks such as characterizing and identifying unique electrical signals corresponding to different polypeptides. Neural networks can learn to identify these patterns much faster than traditional computational methods, leading to more efficient polypeptide identification.

Training the neural network on a plurality of electrical signals reduces the complexity of the polypeptide identification system. The network does not need to consider the entirety of the polypeptide sequence or account for all possible variations; rather, it learns to recognize and distinguish between unique patterns in the electrical signals. This focus on pattern recognition over sequence analysis greatly simplifies the system and reduces the likelihood of errors during polypeptide identification.

A neural network-based system also lends itself to continuous learning and adaptation. Unlike a static database, a trained neural network can easily be retrained or fine-tuned as new data becomes available, allowing for continuous improvement in the identification accuracy.

Moreover, a neural network model storing the learned patterns is typically able to better detect the fingerprints in noisy environments.

Therefore, using a neural network for polypeptide identification provides various benefits, including speed, adaptability, reduced complexity, and efficient resource usage, making it a robust alternative to traditional fingerprinting techniques.

In embodiments, all cysteine and lysine amino acids present within the polypeptide are labelled. In embodiments, this is performed by maleimide and succinimide chemistries.

In embodiments, the labels used are selected from polymer chains, preferably hydrophilic polymer chains such as polyethylene glycol chains; dendrimers, preferably hydrophilic dendrimers such as PEG- dendrimers; zwitterionic moieties; and charged moieties such as polysulfonate, polyphosphonate, polyglutamic acid, and polylysine. If a charged label is used, it preferably comprises at least two charges and more preferably at least 3 charges. For instance, one of two labels could bear three negative charges and the other of the two labels could bear six negative charges.

The system further includes a processing unit: The processing unit is configured to identify a polypeptide from the recorded electrical signal, either by matching it to a polypeptide fingerprint in the database (17) or by applying learned patterns from the neural network to the signal. This processing unit may consist of a single-core, multi-core, or a cluster of processors. It might also utilize a variety of algorithms for this purpose, including machine learning techniques like neural networks, or more traditional pattern matching methods. The processing unit may also be supported by GPU or FPGA hardware to accelerate these computational tasks. In some embodiments, the processing unit may directly apply a neural network trained to recognize specific polypeptide signals. In others, it may execute an algorithm that produces a sequence of labels and interlabel distances to be matched against a polypeptide fingerprint in the database.

The system may comprise a polypeptide Delivery Unit. The polypeptide delivery unit delivers a linearized polypeptide bearing electroactive labeled amino acids to a location that enables it to reach the first side of the pore. This unit could use a variety of methods for delivery, such as capillary action, microfluidics, or electrophoresis. The orientation and position of this delivery unit can be tailored to the system design for efficient delivery of the polypeptide.

The system comprises a translocation Unit. The translocation unit moves the linearized polypeptide from the first side to the second side of the pore. It may comprise a first electrode (named cis electrode) for setting a first voltage at the first side of the pore, and a second electrode (trans electrode) for setting a second voltage at the second side of the pore. These voltages can be carefully controlled to ensure proper translocation speed of the polypeptide. Alternatively, the translocation unit may be adapted to set a pressure difference between both side of the pores.

In embodiments, the voltage range may be between 3 mV to 3V. These electrodes do not need to be galvanically isolated. They are preferably not galvanically isolated.

In embodiments, a same cis-electrode and a same trans-electrode can be used with a plurality of pores, e.g., with all pore of the system.

The translocation unit can be made of conductive materials like platinum, gold, or other suitable materials.

In embodiments, the system may also comprise at least a first and a second reservoir, with the pore being fluidically connected to said first and second reservoir. This setup can facilitate fluid handling and increase the efficiency of the system. In embodiments, one of the reservoir may contain a electroactive labelled polypeptide as defined in any embodiments.

In embodiments, a same first reservoir and a same second reservoir can be used with a plurality of pores.

In embodiments, the first reservoir may contain the cis-electrode, and the second reservoir may contain the trans-electrode, wherein the cis and the trans-electrodes may be configured so that a potential can be created between them, thereby driving the polypeptide through the pore by electrophoresis.

In embodiments, the reservoir may be elongated and part of a lateral microfluidic conduit. This design could contribute to a more streamlined and compact system architecture. In a second aspect, the present invention relates to a method for polypeptide identification comprising the steps of: translocating the linearized polypeptide from the first opening to the second opening of the pore, recording an electrical signal as said linearized polypeptide translocates through said pore, said electrical signal indicating the presence of the electroactive labels on the amino acids of the linearized polypeptides, identify a polypeptide from the recorded electrical signal, either by matching it to a polypeptide fingerprint in a database or by running a neural network on the signal.

In an embodiment of the second aspect, the present invention relates to method of operating a system according to any embodiment of the first aspect for polypeptide identification comprising the steps of: delivering a linearized polypeptide bearing electroactive labeled amino acids at a location that enables it to reach the first side of the pore, translocating the linearized polypeptide from the first opening to the second opening of the pore, recording an electrical signal as said linearized polypeptide translocates through said pore, said electrical signal indicating the presence of the electroactive labels on the amino acids of the linearized polypeptides, operating the processing unit so as to identify a polypeptide from the recorded electrical signal, either by matching it to a polypeptide fingerprint in the database or by applying learned patterns from the neural network to the signal.

Any feature of the second aspect may be as correspondingly described in any embodiment of the first aspect.

In a third aspect, the present invention relates to a computer program comprising instructions to cause the system of any embodiment of the first aspect to execute the steps of the method of the second aspect.

In a fourth aspect, the present invention relates to a computer-readable medium having stored thereon the computer program of the third aspect.

Example 1: simulation of protein detection based on pore dimensions

We now refer to Fig. 2 (left). The pore electrolyte conductivity has been modeled as a chain of resistors. In this model, each resistor symbolized a thin horizontal section of the pore. The chain included the access resistances, a set of resistances representing the wider lumen part of the pore, and a few resistors symbolizing the narrow aperture part of the pore. The effective diameter of the lumen part was set at 12 nm, the height of the lumen part was set at 5 nm, the effective diameter of the aperture part was varied between 2 and 12 nm, and the height of the aperture part was set at 1.2 nm (equal to the gate dielectric thickness).

The FET was gated by the lumen part of the pore, and we approximated the effective gate voltage as the average voltage across the lumen part. Factors determining the resistance of the elements include the bulk conductivity of the electrolyte, the ions in the double layers due to surface charge density, the ions shielding the translocating molecule's charge, and the ions displaced by the molecule's volume.

The salt concentration in the electrolyte here was set at 150 mM and the surface charge density was set at lxlO¹³ charges, cm'².

A simulation was conducted on a molecule designed to mimic the behavior of a section of a linear protein with charged labels at cysteines and lysines, which are typically separated by an average of 3 nm.

The simulation results revealed that detection becomes possible when the effective diameter of the aperture part falls below 10 nm (see Fig. 2, right). Signal strength exhibited considerable variation with effective aperture part diameter, reaching 8.8 mV for a 5 nm aperture part and about 20 mV for a 2 nm aperture part. This highlights the importance of a narrow aperture part in signal discrimination.

We now refer to Fig. 3 where the simulated signal (d) for a 6-fold charged 1.2 nm long model molecule is depicted together with various expected sources of noises: FET noise (a), thermal noise (c) at 100MHz and Schottky shot noise (b) at 100 MHz. For the FET, a noise level (a) of around 1 millivolt is anticipated due to the flicker and thermal noise of the FET itself at 100 MHz. However, the majority of the noise is from the pore, with Schottky's shot noise (b), or current-related noise, being the most significant source.

Yet, we also need to consider that the noise could be substantially reduced by the anti-correlation of ions, a factor not covered in Schottky's model. In other words, variations in the density of carriers might lead to an accumulation of charge. These accumulations will be balanced out by Coulomb forces, making the shot noise (b) depicted in Fig. 3 likely an overestimation. So, for this specific label, a signal-to-noise ratio of at least 1 is predicted for an effective aperture part diameter of 7 nm or less. A more bulky label and a correction of the Schottky shot noise overestimation is predicted to lead to a signal-to-noise ratio of at least 1 already at an effective aperture part diameter of 9 nm or less.

Example 2: Fabrication process of a pore field effect transistor

We now refer to Fig. 4. A FET is fabricated with an Si channel layer having a thickness of 5 nm. A pore is wet-etched anisotropically in the middle of that Si channel by using an anisotropic diluted TMAH (Tetramethylammonium hydroxide) solution on the Si channel masked by mask having a 12 nm effective diameter opening. This process creates a self-aligned, tapered pore in the silicon that is 12 nm wide at the top and narrows to 5nm at the bottom.

TMAH is an anisotropic silicon etchant that etches the (111) planes of silicon at a much slower rate than other surfaces. These planes are angled at 54.7 degrees to the wafer surface, resulting in the tapered etch. To achieve the preferred dimensions, the silicon channel is thinned down to 5 nm before starting the etching process.

Example 3, signal-to-noise ratio (SNR) of different designs

As discussed above, a smaller lumen allows higher bandwidth, and a certain bandwidth target determines a ceiling for lumen radius and silicon or pore electrode thickness. However, SNR favors increasing lumen size.

To test the implications of the properties on the bandwidth and the SNR, a device was constructed in which the lumen radius was 40nm and the Si thickness lOOnm. It was observed upon use that the bandwidth is still above 100MHz. It was thus concluded that the lumen can still be considerably large while still allowing high bandwidth. For the processing, smaller lumen reduces the height-to-effective diameter aspect ratio of the lumen - AR becomes smaller for smaller radius, and higher aspect ratios are more difficult to realize/process.

Noise decreases with increased lumen radius. This dependence is mainly due to FET 1/f noise and much less due to the noise of the electrolyte in the pore. This means that if FET 1/f noise is low vs. other noise sources (e.g. for foundry quality FETs) lumen radius will have limited impact on noise. For low 1/f noise, there is still a considerable SNR advantage when scaling the lumen radius up to ~20nm since signal is boosted with increasing lumen radius considerably for radii up to ~20nm. For good quality FETs (with medium noise), noise is reduced considerably when increasing lumen effective diameter up to ~20-40nm (which is determined by the relevance of FET 1/f noise vs. electrolyte noise). Beyond 20-40nm limited reductions in noise are obtained.

The SNR grows with the square root of the Si (or pore electrode) thickness. This is due to a decrease in noise. The dependence of signal on Si thickness is weak. There is no SNR advantage when making silicon thin. A pore design, where the lumen radius is large vs. the aperture radius, results in high resolution because of the thin aperture. There is no strong lumen geometry dependence of the resolution for said design, as the resolution depends on the aperture.

To determine the impact of the size of the lumen part radius (top radius) and the Si thickness in the devices of the present disclosure, different designs where tested and the results summarized in fig. 19, illustrating the pore bandwidth (BW) and SNR trade-off. fig. 19 (left) shows the impact of the design measures on the SNR, while fig. 19 (right) illustrates the impact of the design measures on the BW. As shown, nanoscaling is driven by bandwidth, with SNR gains for larger devices becoming limited. Processing considerations also favor smaller effective diameter, and lumen aspect ratio becomes difficult for larger radius.

The main pore electrode area is key to obtain the desired SNR and bandwidth properties. A simulation of the bandwidth obtained for which SNR > 1 as a function of pore electrode side Seiectrode and the square root of FET gate area,

was performed The pore electrode is assumed square for this simulation with area Seiectrode². Based on the results it became clear that preferably the pore electrode area is larger than lOOxlOOnm (lOOOOnm²). Around that size and smaller sizes SNR drops below 1 and hence negligible bandwidth with SNR>1 is obtained. Bandwidths of 10MHz are obtained with pore electrode side ~< 1 micron illustrating the bandwidth benefit of the pore.

Example 4 : Surface charge density Manipulation of the pore

A lumen part with a high surface charge density is preferred for optimal performance. This can be promoted by the deposition of a high surface charge density layer on its surfaces. Atomic Layer Deposition (ALD) of Hafnium Dioxide (HfCh) can be employed to form such a layer. Hafnium dioxide ( HfCh) indeed has a high dielectric constant and can thus accumulate a high surface charge density when in contact with an electrolyte.

Alternative approaches can be utilized, where organic coatings such as Self-Assembled Monolayers functionalized with carboxy or amine groups are applied to the lumen part.

Conversely, the aperture part of the pore device is preferably treated to limit its conductivity, thereby creating a more pronounced bottleneck. This implies a lower surface charge density, which is achieved through selective functionalization, e.g., with non-polar chain, or material contrast.

For instance, the pore is first thoroughly cleaned with acetone, isopropyl alcohol, and deionized water in an ultrasonic bath to remove any potential surface contamination. This is followed by a rinse in deionized water and drying under a stream of nitrogen.

A high-quality layer of Hafnium Dioxide ( Hf O2 ) is then deposited on the inner surface of the lumen part using an Atomic Layer Deposition (ALD) system.

Once the process for enhancing the lumen part's surface charge density is completed, the device is prepared for the next process: lowering the surface charge density of the aperture part. This can be done, for instance, through the use of selective functionalization to create a low surface charge density. For this purpose, a controlled deposition of Self-Assembled Monolayers (SAMs) functionalized with a hydrophilic polymer, such as polyethylene glycol, is carried out on the aperture part. For instance, a silane comprising a polyethylene glycol moiety can be used to form that SAM. Through this protocol, a high-performance pore device with a high surface charge density lumen part and a low surface charge density aperture part is fabricated, exhibiting a differential surface charge density that enhances the device's overall performance.

Example 5: Design and Implementation of a High-Bandwidth On-Chip Pre-Amplification System for the Pore Field-Effect Transistors

A conventional electrical readout module as depicted in Fig. 6a could be used but it has some limitations. The limited drive current of the pore field-effect transistor, paired with large external capacitances greater than 1 pF due to on-chip interconnects, off-chip Printed Circuit Board (PCB) or cable routing, and amplifier input capacitance, inhibits its ability to achieve up to 1 GHz bandwidth. Furthermore, the bandwidth of an off-chip amplifier is constrained by parasitic input capacitance. Consequently, it is preferred to use an electrical readout module comprising an on-chip preamplification circuit, as depicted in Fig. 6b, to bypass these limitations.

The improved design (see Fig. 6b) is characterized by a pre-amplifying, here a source-follower, which biases a broad, quasi-RF FET (M3) with dimensions of, for instance, L = 250 nm, W = 7.5 pm. Fluctuations in gate bias, the signals from the molecule, within the pore field effect transistor, are transposed onto this wide quasi-RF FET by the intermediary of the source-follower, resulting in a boost in bandwidth. Preferably, all transistors are designed to be either pMOS or nMOS in order to simplify the processing steps. Preferably, nMOS are use as it allows a higher bandwidth due to its superior mobility.

The capacitance driven by the pore field-effect transistor is significantly reduced by the pre-amplifier. The quasi-RF FET is set to drive a reduced external capacitance (~1 pF) due to the on-chip interconnect and is designed to drive a 50 ohm transmission line connected to a high bandwidth external readout.

In this manner, a bandwidth of approximately 1 GHz can be achieved, while the large quasi-RF FET contributes only around 4% of the noise of the pore field-effect transistor (based on the square root of the "area" ratio).

Example 6: linearization of proteins

Denaturing agents, including Sodium Dodecyl Sulfate (SDS) or its variants, urea, guanidium chloride, and organic solvents such as ethanol, are being utilized for the denaturation of proteins. SDS is often favoured due to its consistent impartation of negative charge on proteins, enabling a smooth, unidirectional translocation facilitated by uniform electrophoresis. This also aids in avoiding potential obstructions or oscillatory movement. However, it should be noted that proteins coated with SDS may not all translocate linearly. The design of the Pore Field-Effect Transistor (NPFET) is being optimized to enhance protein translocation linearity. This is achieved by the adoption of an approach known as entropic constriction, which necessitates the selection of an appropriately narrow aperture. Pull tags, such as DNA, polyglutamic acids, or polysulfonates, are being attached to the protein head to ensure a headfirst entrance into the orifice.

In spite of being denatured, proteins often do not adopt a linear formation but instead tend to coil. This can disrupt the orderly, single-file translocation of proteins into the NPFET. To counteract this issue, a strategy of pre-linearisation using a nanochannel is being implemented. Denatured proteins are electrophoretically stretched and aligned within this channel, resulting in the pore being fed with pre-linearised molecules.

The importance of maintaining a low-resistance path from the cis and trans electrodes to the NPFET cis and trans openings for the sake of bandwidth preservation is recognized. This can be facilitated by the construction of wider fluidic channels or the inclusion of on-chip electrodes.

A charged pull-tag, such as DNA or polyglutamic acid, is being placed on the N-terminal (protein head) for the pre-linearisation strategy. Electrophoretic bias application and counterflowing electroosmotic flow serve to straighten the protein tail. Surface charge density modification of the channel or pore can induce this. The naturally negative charge of SiO2 or HfO2 walls at neutral pH are found to straightforwardly induce such a flow. Although this method could be supplemented with a charged tag on the C-terminal (protein tail), this approach is less practical for labelling in proteomics experiments on a full scale, so the use of a pull tag alone is favoured.

Example 7: labelling of proteins

In our experimental design, we primarily focus on the labeling of cysteine and lysine amino acids present within proteins. We accomplish this using well-established maleimide and succinimide chemistries. The labels used include PEG-chain and PEG-dendrimer labels, zwitterionic volume labels, as well as various charged labels such as polysulfonate, polyphosphonate, polyglutamic acid, and polylysine. Through the application of maleimide and succinimide chemistries, these labels are individually attached to cysteine and lysine amino acids within the proteins, respectively. Following the completion of each reaction, the successful attachment of labels to the proteins is verified via analytical techniques such as mass spectrometry or NMR spectroscopy. The proteins, now labeled, are stored properly for future use.

Example 8: Signal Interpretation and Polypeptide Identification

We now refer to Fig. 5. A electroactive labelled polypeptide is translocated through the pore by applying a potential difference between a cis and a trans electrode situated on either part of the pore, thereby generating an electrical signal. The electrical readout module picks up this signal and is channeling its output to a processing unit, which is designed to interpret the recorded electrical signal by using a stored model or reference data in a memory. Depending on the approach used, the processing unit could either match the electrical signal to a polypeptide fingerprint from a stored database or apply a trained neural network model for the identification of the corresponding polypeptide.

A memory is provided, storing a database of polypeptide fingerprints for a traditional pattern-matching approach, or holding the model of a trained neural network for a machine learning approach.

For a system that uses a neural network for interpretation, the memory stores the weights, biases, and structure of the neural network, which are used to interpret the signals from the electrical readout module and identify the corresponding polypeptide. This "model" essentially represents the knowledge that the neural network has learned from its training data, which may be a collection of electrical signals associated with specific electroactive labeled polypeptides.

Suitable neural networks are, for instance, convolutional neural networks and recurrent neural networks.

In contrast, for a system that uses a traditional pattern-matching algorithm, the memory stores a database of polypeptide fingerprints. Each fingerprint in the database is associated with a specific polypeptide, and the algorithm identifies the polypeptide by finding the best match between the electrical readout module output and the fingerprints in the database.

It's also worth noting that a hybrid approach could be possible, where both a neural network model and a database of polypeptide fingerprints are stored and used in tandem. This could potentially leverage the strengths of both techniques.

It is to be understood that although preferred embodiments, specific constructions and configurations, as well as materials, have been discussed herein for devices according to the present invention, various changes or modifications in form and detail may be made without departing from the scope of this invention. Steps may be added or deleted to methods described within the scope of the present invention.

Claims

Claims

1. A system for polypeptide identification comprising: al) One or more pore field-effect transistors, each comprising: i. A field-effect transistor (3) having a gate, a source (S), a drain (D), and a channel region between the source (S) and the drain (D), and ii. a pore (4) comprising: at least a first (5) and a second (6) opening; two contiguous parts, a lumen part (7) and an aperture part (8), such that the lumen part (7) is flu id ica I ly connected to the first opening (5) and that the aperture part (8) is flu id ical ly connected to the second opening (6); a sensing region (11) located in the lumen part (7), the aperture part (8), or both parts of the pore (4) such that a polypeptide travelling from the first opening (5) to the second opening (6) will pass through the sensing region (ii); wherein the effective diameter of a transverse cross-section of the pore, at all points within the pore (4), is less than 1 pm; wherein the aperture part (8) inside the pore (4) has all its effective transverse cross-section diameters, dA, being less than 10 nm; wherein an area of a transverse cross section in the aperture part (8), AA, is smaller than an area of a transverse cross section in the lumen part (7), AL, such that AL>2AA, wherein the gate is controlled by the sensing region (11) of the pore (4) when the system is in operation, a2) one or more electrical readout modules (14), each being connected to the drain (D) and/or source (S) of one or more of said field-effect transistors (3), configured to record an electrical signal indicating the presence of electroactive labels (12) on at least two different amino acid types of a linearized polypeptide (13) when the linearized polypeptide (13) translocates through the pore (4); b) A processing unit (10) configured to identify a polypeptide from the recorded electrical signal, either by matching it to a polypeptide fingerprint in a database or by running a neural network on the recorded electrical signal, c) A translocation unit (15) for translocating the linearized polypeptide (13) from the first opening (5) to the second opening (6) of the pore (4).

2. The system according to claim 1, wherein at least 3/4 of the transverse cross sections of the lumen part (7) has an area which is at least four times larger than the area of any aperture part (8) transverse cross section, such that they fulfil the requirement AL>4AA.

3. The system according to claim 1 or claim 2, wherein the one or more electroactive labels (12) are a first electroactive label applied to one specific type of amino acid and a second distinct electroactive label applied to another specific type of amino acid of the polypeptide (13).

4. The system according to any one of the preceding claims, wherein the one or more pore transistors are a thousand or more, preferably a million or more.

5. The system according to any one of the preceding claims, wherein the pore (4) is configured to extend through the channel region of the field-effect transistor (3) between the source (S) and the drain (D) or wherein the pore (4) is physically separate from the channel region of the field-effect transistor (3).

6. The system according to any one of the preceding claims, wherein the pore (4) has a minimum effective diameter of less than 5 nanometers.

7. The system according to any one of the preceding claims, wherein the electroactive labels (12) are charged labels, zwitterionic labels, and/or labels having a hydrodynamic radius measuring from 5% to 49% of the minimum effective diameter of the aperture (4).

8. The system according to any one of the preceding claims, wherein the pore (4) is a solid-state pore.

9. The system according to any one of the preceding claims, wherein each electrical readout module (14) operates at a frequency of at least 1 kHz.

10. The system according to any one of the preceding claims, wherein each electrical readout module (14) comprises an analog-to-digital converter.

11. The system according to any one of the preceding claims wherein the lumen part (7) has inner walls that have a higher surface charge density than the inner walls of the aperture part (8).

12. The system according to any one of the preceding claims, wherein the height of the aperture part (8) measures at most 15 nm.

13. A method for polypeptide identification comprising the steps of:

• translocating the linearized polypeptide (13) from the first opening (5) to the second opening (6) of a pore (4),

• recording an electrical signal as said linearized polypeptide (13) translocates through said pore (4), said electrical signal indicating the presence of the electroactive labels (12) on the amino acids of the linearized polypeptides (13),

• identify a polypeptide from the recorded electrical signal, either by matching it to a polypeptide fingerprint in a database or by running a neural network on the signal

14. A computer program comprising instructions to cause the system of any one of claims 1 to 12 to execute the steps of the method of claim 13.

15. A computer-readable medium having stored thereon the computer program of claim 14.