Nanopore system using short recognition molecules

Abstract

The present disclosure relates to novel recognition molecules and systems thereof for use in nanopore sensing, the recognition molecules having a short skeleton comprising densely spaced modifications, as well as methods for detecting target molecules in a sample.

Description

Technical Field

[0001] This disclosure relates to molecular sensing using nanopores. More particularly, the proposed technology relates to novel recognition molecules for use in nanopore sensing, wherein the recognition molecules are short and have densely spaced backbone modifications. This disclosure also relates to a system for reading the novel recognition molecules, the system comprising a field effect transistor (FET) nanopore. Background Art

[0002] Nanopore sensing has been used for DNA and RNA detection and sequencing for some time, and research interest has also shifted to nanopore sensing of biomolecules such as proteins and peptides. The possibility of selectively screening a range of proteins at the single-molecule level in biofluids is a key feature for developing new therapeutic and diagnostic strategies. Nanopore sensing is attractive because it has the potential to simultaneously detect multiple different types of biomolecules, such as DNA, RNA, and proteins, in complex biological samples, and it can be integrated into small, portable devices.

[0003] One method for detecting the presence of an analyte in a sample is based on engineering DNA molecules with structural modifications, including dumbbells, coupling them to an antibody capable of binding to a target, and then incubating them with the target to form a complex with the target that can be transported through a solid-state nanopore (NAW Bell et al. Nature nanotechnology 11 (2016): 645-652). Thus, the engineered DNA molecules can act as "barcodes," where the structural modifications produce unique signal patterns when transported through a nanopore sensor. These barcodes can identify the complex and, therefore, the analyte.

[0004] However, engineered DNA molecules that act as barcodes have several drawbacks, such as folding and forming structures where some modifications are shielded from detection in the nanopore and therefore may not be read correctly. Therefore, enhanced methods for detecting and quantifying molecules using nanopore sensing are needed. Summary of the Invention

[0005] The goal of the present disclosure is to provide new recognition molecules, methods and systems that offer alternative and more efficient ways to detect and quantify molecules using nanopore sensing, with advantages such as improved accuracy, low cost and high throughput.

[0006] This object is achieved by providing a recognition molecule for use in nanopore sensing, wherein the recognition molecule is capable of generating a unique recognition signal when transported through the nanopore sensor, the recognition molecule comprising a backbone molecule having a plurality of backbone modifications, wherein each backbone modification causes a modulation of the signal amplitude when transported through the nanopore sensor, and wherein the distance between the backbone modifications of the recognition molecule is 10-40 nt, such as 30 nt, 20 nt or 15 nt, corresponding to 3.4-13.6 nm, such as 10.2 nm, 6.8 nm or 5.1 nm. The closely spaced modifications allow the use of shorter recognition molecules, which have the following advantages: compared to longer recognition molecules, due to greater rigidity, folding and secondary structure formation are reduced.

[0007] Thus, in embodiments, the length of the backbone molecule is 136 nm or less, corresponding to 400 nucleotides (nt) or less, such as 12-136 nm. In embodiments, the length is 102 nm, 68 nm, 34 nm, 27.2 nm, 20.4 nm, 13.6 nm or 6.8 nm, corresponding to 300 nt, 200 nt, 100 nt, 80 nt, 60 nt, 40 nt or 20 nt, wherein the length of the backbone is greater than the distance between the backbone modifications.

[0008] In the recognition molecules of the present invention, the backbone modifications are selected from a plurality of modification types, and when read in the nanopore sensor, each signal amplitude modulation obtained from the plurality of modifications is assigned an identification value, and the total number of identification values ​​corresponding to the modifications yields an array of identification values ​​corresponding to the unique recognition signal of the recognition molecule. The assignment of these identification values ​​is based on i) the distance between the modifications, or ii) the modification type.

[0009] In some embodiments, two modification types are used, and each signal amplitude modulation of these two modification types is assigned an identification value of "0" or "1", and all identification values ​​of these corresponding modifications produce a binary identification value array corresponding to the unique identification signal of the identification molecule.

[0010] In a preferred embodiment, the recognition molecule has a biopolymer backbone, such as a DNA molecule backbone, preferably a double-stranded DNA molecule. The backbone modification is selected from bulky DNA structures, such as DNA hairpin structures, cruciform DNA, DNA origami, and quadruplex DNA; DNA modifications, such as biotin, oligonucleotides, peptides, PNA, carbohydrates, or organic molecules; and differentiated DNA structures, such as ssDNA differentiated into ssDNA hybridized with oligonucleotides or polyethylene glycol moieties, or a combination thereof. Single-stranded DNA can also be used as the backbone, whereby oligonucleotides of complementary DNA that bind to the single-stranded DNA are used as the backbone modification.

[0011] In a preferred embodiment, the nanopore sensor has a field effect transistor (FET) embedded in the nanopore, such that the FET is controlled by an electrolyte-filled nanopore that passes through the channel region of the FET. In an alternative embodiment, the nanopore sensor comprises a remotely extended FET, wherein electrodes surrounding the nanopore are connected to a remote gate sensor.

[0012] Further provided is a system for target molecule sensing and sequencing using a nanopore sensor, the system comprising: a first electrolyte reservoir and a second electrolyte reservoir, the first and second reservoirs being separated by a barrier, the barrier comprising one or more FET-embedded nanopores, the nanopores comprising an pore having a predetermined height and a predetermined diameter; and optionally, an electrode for transporting molecules from the first electrolyte reservoir to the second electrolyte reservoir through the nanopore, wherein at least one of the first and second electrolyte reservoirs comprises a recognition molecule of the present disclosure, wherein the recognition molecules are capable of generating a unique recognition signal when transported through the nanopore. The recognition molecule can be in a complex or optionally separated from the target-recognition molecule or the target-assay-recognition molecule.

[0013] The pore size is the narrowest constriction in a nanopore device that reads the recognition molecule. The narrow diameter and low height of the pore generate a strong signal when the recognition molecule is transported through the nanopore. The pore size also determines the resolution of the nanopore device, which is the shortest distance between modifications on the backbone that the nanopore device can still distinguish.

[0014] In embodiments, the pore height h of the FET-embedded nanopore is predetermined based on the distance between the backbone modifications of the recognition molecule, such that a shorter distance between the backbone modifications determines a shorter pore height. The pore height can be in the range of 3,4-14 nm, preferably 7 nm or less. This corresponds to a spacing between modifications of approximately 10-40 nt, preferably 20 nt or less.

[0015] The pore diameter is in the range of 3-20 nm, preferably 10 nm or less. The nanopore FET preferably has a bandwidth of 1 MHz or higher.

[0016] The system can be used to perform a method for detecting one or more target molecules in a sample using a nanopore sensor, the method comprising: providing (S1) a sample containing one or more target molecules to be detected; providing (S2) at least one recognition molecule according to the present disclosure for the corresponding target molecule, wherein the recognition molecule is capable of generating a unique recognition signal when transported through the nanopore sensor, and the association between the unique recognition signal of the recognition molecule and the bound target molecule is known; optionally linking the at least one recognition molecule to an assay molecule (S3); incubating the at least one recognition molecule with a sample that may contain one or more target molecules (S4), thereby allowing the at least one recognition molecule to bind to the one or more target molecules to form at least one target-recognition molecule or target-assay-recognition molecule, and allowing the recognition molecule to be transported through the nanopore sensor (S5) to obtain at least one unique recognition signal, wherein the recognition molecule is in a complex or optionally separated from the target-recognition molecule or target-assay-recognition molecule; and detecting (S6) any presence of the one or more target molecules in the sample by associating the obtained at least one unique recognition signal with the one or more target molecules.

[0017] Other objects and advantages will become apparent to those skilled in the art from a reading of the ensuing detailed description and the appended claims, wherein the detailed description proceeds with reference to the following illustrative drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The above and additional objects, features and advantages of the present invention will be better understood through the following illustrative and non-limiting detailed description of various embodiments of the present invention with reference to the accompanying drawings, in which:

[0019] Figure 1A Nanopore readouts are shown using two different types of side-chain modifications to generate peaks that are assigned unique identification signals / barcodes. Figure 1B The molecules and their resulting signal readouts are presented.

[0020] Figure 2A The backbones with different types of side chain modifications are shown. Figure 2B The peak pattern generated when the backbone with side chain modification passes through the nanopore sensor is shown. Figure 2C Peak patterns generated by backbones with modifications are shown and can be converted to values ​​(these values ​​can be represented as 0 and 1).

[0021] Figure 3 shows the modified dsDNA backbone passing through the nanopore, where Figure 3A A general schematic diagram is shown. Figure 3B A nanopore FET is shown.

[0022] Figure 4AA barcode linked to a nucleic acid assay molecule is presented, which enables detection of a nucleic acid target molecule; Figure 4B Barcodes linked to antibody assay molecules are demonstrated, enabling the detection of protein antigens.

[0023] Figure 5A Demonstrates different side chain modifications on the backbone forming the recognition molecule, Figure 5B Shows Figure 5A The resulting readout of the identified molecule in

[0024] Figure 6 Different approaches to generate recognition molecules are demonstrated: modifications are made to the nucleotide oligomers (e.g. forming vertical DNA stretches, thus generating branched oligonucleotides) whereby different branches can have different lengths, thereby generating peaks of different sizes upon nanopore transport.

[0025] Figure 7 A method for synthesizing recognition molecules based on DNA ligation is demonstrated.

[0026] Figure 8 The recognition molecules of the present disclosure are presented.

[0027] Figure 9 Schematic cross-section of a nanopore FET device. DETAILED DESCRIPTION

[0028] Various aspects of the present disclosure will be described more fully below with reference to the accompanying drawings. However, the systems and methods disclosed herein can be implemented in many different forms and should not be construed as limited to the aspects set forth herein. Throughout the text, like reference numerals in the accompanying drawings refer to like elements. For the sake of clarity and simplicity, the method will be described in terms of "steps." It is emphasized that steps are not necessarily processes that are defined or separated by time, and more than one "step" can be performed simultaneously in a parallel manner.

[0029] The terms used herein are for the purpose of describing particular aspects of the present disclosure only and are not intended to limit the present disclosure. Unless the context clearly indicates otherwise, as used herein, the singular forms "a", "an", and "the" are intended to include the plural forms as well.

[0030] In some embodiments, the non-limiting term "molecule" is used as in "target molecule." This refers to any type of analyte that can be detected, identified, and quantified using the methods and systems of the present invention.

[0031] In some embodiments, the non-limiting term "biomolecule" is used. The term "biopolymer" may also be used interchangeably. The terms "target biomolecule" or "target biopolymer" may be used to refer to the biomolecule / biopolymer that the methods of the present invention attempt to detect. The target biopolymer or biomolecule herein can be any type of biopolymer, such as a polynucleotide, polypeptide, lipid, or polysaccharide, including DNA or RNA polymers, peptides, and proteins.

[0032] In some embodiments, the general term "recognition molecule" is used. When the recognition molecule includes a barcode, the term "barcode molecule" can be used interchangeably therewith. Recognition molecules or barcode molecules as used herein refer to engineered elongated molecules that, when transported (read out) on a nanopore sensor, produce recognition signals or barcode signals. Recognition molecules have a "skeleton", which in some embodiments may be referred to as a "skeleton molecule", "scaffold" or "scaffold skeleton", such as a biopolymer skeleton or a synthetic non-natural polymer skeleton, which includes one or more modifications / skeleton modifications, and the addition of these modifications is to obtain recognition molecules. The skeleton molecule is preferably a double-stranded (ds) DNA molecule biopolymer skeleton. The biopolymer skeleton can also be, for example, double-stranded (ds) RNA, peptides, carbohydrates and lipids, and the synthetic skeleton can be, for example, polyesters, polyamides, rigid rod polymers and / or modified polymers with carbon linking segments. The skeleton can also be an uncharged molecule, for example a polyether, such as polyethylene glycol (PEG) or a variant thereof.

[0033] The term "modification" or "backbone modification" refers to modifications on the backbone of the recognition molecule that produce a unique recognition signal or signal change, or signal alternation when read out in a nanopore sensor. In some aspects, modifications on the backbone (e.g., modifications to the backbone itself) are configured to produce a unique signal pattern, signal amplitude modulation (such as a current peak pattern) when the recognition molecule is transported through the nanopore. The characteristics of the signal pattern can be used as a barcode to identify the target molecule. For example, the change in the signal (signal amplitude modulation) can be a "rising" or "falling" signal, or a change in the characteristics of the signal, such as a change in the duration or amplitude of a "rising" or "falling" signal (peak). In some embodiments, the signal pattern of the overall change can be used as a barcode. In some embodiments, a series of changes can be assigned a value in the barcode. An example is shown in Figure 1. Figure 1A Nanopore readouts are shown using two different types of side-chain modifications to generate peaks that are assigned unique identification signals / barcodes. Figure 1B The molecules and their resulting signal readouts are presented.

[0034] In some embodiments, modification of the backbone can be achieved by adding protruding modifications to the backbone, in which case the modification / backbone modification may also be referred to as a "side chain modification" (e.g., a DNA hairpin), which generates a peak signal when the recognition molecule is transported through the nanopore. For example, a DNA structure extension (e.g., a hairpin) may be placed at a single nucleotide position and protrude from the backbone molecule. The modification protrudes from the backbone and can therefore be recorded by the nanopore sensor, such as changes in current during transport of the recognition molecule. The modifications may be of different types, referred to as "modification types," wherein the modification types may be, for example, different DNA hairpins, such as 8 base pairs (8bp) long DNA hairpins or 16bp DNA hairpins. When the recognition molecule is transported through the nanopore sensor, each modification generates a signal amplitude modulation (e.g., a peak). Different modification types may generate peaks of different amplitudes and / or peaks of different durations. In some embodiments, two modifications that are very close in distance may appear as a longer signal amplitude modulation and may not necessarily form two distinguishable peaks.

[0035] When the recognition molecule is transported through the nanopore, the signal amplitude modulation (e.g., peak) generated in the signal is, for example, caused by the current change during the transport of the recognition molecule. Therefore, this signal amplitude modulation or "peak" can be a "current peak", but it can also be a "voltage peak" (voltage threshold peak), in which case the nanopore acts as a voltage divider in the device.

[0036] Each signal amplitude modulation or peak can be assigned an "identification value" (also referred to as "value"), which may depend on the amplitude and / or duration of the signal amplitude modulation / peak (i.e., indirectly based on the modification type). These values ​​can have two or more "types" or "components", such as "0" and "1" being different components. The exact implementation may depend on multiple factors, such as the resolution of the hole. For DNA hairpins, significant differences in hairpin size will produce different signal amplitude modulation / peak sizes based on the length of nucleotides protruding from the backbone. For example, a hairpin of 8 nucleotides (nt) will produce a smaller peak than a 24nt hairpin. This size difference may be enough to mark all 8nt hairpins as, for example, "0" and all 24nt hairpins as "1". The peak size difference between an 8nt hairpin and a 9nt hairpin may be so small that they are classified as having the same size and, therefore, the same value. This also applies to other types of modifications. In one example, several signal amplitudes (such as 1-5) may be assigned the same identification value, while a signal amplitude of 6-10 may be assigned another value. In addition, the distance between different modifications can also be used to assign a specific identification value, where, for example, distance 1 is assigned a value of 0 and distance 2 is assigned an identification value of 1; or, for example, the presence or absence of a modification is assigned a specific identification value. In the case where the unique signal is a barcode signal, the "identification value" can be referred to as a "barcode value". In one embodiment, two modification types are used, and the barcode value is one of two components, namely "0" or "1". When more than two modification types are used, several modification types may produce the same value / component. In some aspects, the amplitude of the peak signal in the nanopore sensor is used to assign an identification value / barcode value, where, for example, "1" and "0" correspond to different signal amplitudes, while in other aspects, the spacing between peak signals can be used to assign an identification value / barcode value, where, for example, "1" is assigned when a peak is present and "0" is assigned when a peak is not present. All identification values / barcode values ​​of the recognition molecules together form a pattern, i.e., an array of identification values / barcode values, which is referred to as a unique "identification signal" or "barcode signal". In one embodiment, multiple modification types (eg, three or more) can be used, corresponding to multiple identification values / barcode values, thereby generating a more complex identification signal / barcode signal.

[0037] The backbone molecules of the present invention are generally shorter than conventional molecules, for example, with a length in the range of 6.8-136 nanometers (nm), such as 6.8-102nm, or preferably 6.8-68nm. Since the distance between two nucleotides in the DNA double helix is ​​0,34nm, this corresponds to a length of 20-400 nucleotides (nt), such as 20-300nt, or preferably 20-200nt. This can be achieved because the spacing of the backbone modifications is very dense, such as the distance between the backbone modifications of the recognition molecules is 10-40nt, such as 30nt, 20nt or 15nt. This dense spacing allows more modifications to be made per unit backbone length, thereby making the backbone of the recognition molecule shorter than conventional backbone. In order to accommodate multiple modifications in a recognition molecule, the length of the backbone is always greater than the distance between the backbone modifications.

[0038] The recognition molecules of the present invention may have a backbone encoded as a chain structure with "blocks" of different sizes and / or shapes, such as origami tubes, spheres, cubes or plates. These blocks may have a stable and semi-rigid structure and may be formed from one or more layers of nucleic acids, peptides or proteins. The origami structure may be used as a backbone to which extensions are added, which can be used to generate barcodes. For example, one could have a "tube-tube-tube-tube" structure where the "-" is a linker and whereby a different hairpin extension or another type of side chain modification is added to each "tube" block; alternatively, one could construct a structure such as a "tube-sphere-cube-plate-sphere-tube" where the "-" is a linker and each geometric structure or block would generate a different signal due to its size, i.e. a sphere would generate a different signal than a cube, and in this way a unique barcode could also be generated.

[0039] In some embodiments, an assay molecule is used to bind a recognition molecule to a target molecule. The term "assay molecule" refers to a molecule that is designed or inherently capable of binding to a target molecule in an assay. It can be any type of biomolecule or synthetic molecule that can be used in an affinity assay. Each assay molecule is capable of specifically binding to a target biomolecule in a sample. An assay molecule can be linked to one or more recognition molecules to form an assay-recognition molecule. When incubated with a sample containing a target biomolecule, the assay-recognition molecule can bind to the target biomolecule, thereby forming a target-assay-recognition molecule. The assay molecule can be selected from, for example, proteins, peptides, DNA, RNA, molecularly imprinted polymers, compounds, metal ions, lipids, polysaccharides, vesicles, whole cells, and polystyrene beads. In some embodiments, the assay molecule is an antibody or a binding fragment thereof, such as a single-chain variable region fragment, an aptamer, or an affimer. The target-assay-recognition molecule can be separated from the sample to be analyzed, such as by washing the assay-recognition molecule that is not bound to any target molecule in the sample, and then the intact target-assay-recognition molecule or only the separated recognition molecule can be transported through the nanopore to detect the target.

[0040] The nanopore sensor of the method of the present invention preferably comprises a solid-state nanopore. In a preferred embodiment, the nanopore sensor comprises a nanopore field effect transistor (FET). The nanopore sensor may comprise a first and a second electrolyte reservoir, respectively, both separated by a barrier comprising a nanopore. The sensor / system may further comprise an electrode for transporting molecules from the first electrolyte reservoir to the second electrolyte reservoir through the nanopore, wherein at least one of the first and second electrolyte reservoirs comprises an isolated recognition molecule for detecting the target molecule. The nanopore may have an pore size through which the recognition molecule is transported, wherein the pore diameter is related to the approximate diameter of the recognition molecule used. Depending on the resolution of the nanopore, side chain modifications (such as DNA hairpin structures) need to be spaced far enough apart to avoid peak overlap. Similarly, the resolution of the nanopore will determine the minimum length required for the hairpin DNA to extend to produce a peak. Nanopores with high resolution (such as nanopore-FETs) can still distinguish peaks even when using shorter DNA hairpins that are closer to each other. Therefore, in order to achieve the application of short recognition molecules with densely spaced modifications, the design of the nanopore FET needs to be adjusted.

[0041] The properties of the nanopore FET need to be suitable for reading the short recognition molecules in this article. For example, the nanopore FET will require a high bandwidth, such as above 1 MHz. The nanopore FET will need to be designed to have a high signal-to-noise ratio (SNR) for short recognition molecules at a high bandwidth. Therefore, the characteristics of the nanopore FET aperture can be based on the distance between the backbone modifications of the recognition molecules. The height h of the aperture can, for example, vary with the distance between the backbone modifications. The aperture should preferably be in the range of 3,4-14 nm, preferably less than 10 nm. The distance or spacing s between the modifications in base pairs (bp) can be divided by approximately 2,94 to obtain the aperture height h in nm, i.e. h=s / 2,94. Therefore, for the distance (spacing) between modifications of 10-40 bp, a height of 3,4 nm to 14 nm can be used (for a spacing of 10 bp, 3,4 nm is used; for a spacing of 20 bp, 7 nm is used; and for a spacing of 40 bp, 14 nm is used). Furthermore, the diameter d of the nanopore aperture needs to be precisely controlled, with the diameter being 20 nm or less, preferably 10 nm or less, such as 7 nm or 5 nm. The larger the height and diameter of the aperture, the lower the signal intensity and signal-to-noise ratio. It should be noted that these measurements are relative to the aperture diameter, not the entire pore. By adjusting the size of the nanopore to match the size of the recognition molecule, background signal is generally reduced and foreground signal is enhanced.

[0042] In some aspects, the present invention relates to a device or system comprising a nanopore FET, wherein the nanopore-containing device is configured to perform the methods defined above and below. In some aspects, the system or device of the present invention comprises multiple nanopores that can perform simultaneous sensing to achieve high throughput of the system.

[0043] In some aspects, a system comprising a microfluidic delivery system is provided, wherein the microfluidic delivery system delivers different molecules, such as a target sample, a recognition molecule, an assay molecule, and an assay-recognition molecule. The system may also include a detection unit, wherein the detection unit comprises two reservoirs, wherein the reservoirs are separated by a nanopore. The microfluidic delivery system can be connected to one of the two reservoirs of the detection unit, wherein one of the two reservoirs contains the recognition molecule. The recognition molecule can be in a complex or optionally separated from the target-recognition molecule or the target-assay-recognition molecule. Thus, the recognition molecule can be isolated / independent, i.e., not bound to any other molecules, as it may have been separated from the other molecules prior to transport through the pore; or it can be bound to, for example, the target / complex and thus contained in the entire target-assay-recognition molecule complex, which is transported through the pore. The detection unit can detect one or more unique recognition signals obtained when the recognition molecule or the entire target-assay-recognition molecule complex is transported through the nanopore, and can be configured to detect any presence of one or more target molecules in the sample by associating at least one unique recognition signal obtained with one or more target molecules. The system may further comprise a control unit that controls the delivery of molecules by the microfluidic delivery system and controls the detection unit and provides instructions to the system to perform the methods described above and below, such as incubating at least one assay-recognition molecule with the sample; removing assay-recognition molecules that are not bound to any target molecule from the sample; optionally separating at least one recognition molecule from at least one target-assay-recognition molecule; and transporting the target-assay-recognition molecule or the separated at least one recognition molecule through the nanopore sensor to obtain at least one unique recognition signal. The incubation step of incubating at least one assay-recognition molecule with the sample can be performed at different locations in the microfluidic system, such as pre-mixed in the microfluidic delivery system, as a separate unit, or in one of the reservoirs. The step of removing any unbound assay-recognition molecules can employ a washing or removal mechanism that is configured to wash / remove any assay-recognition molecules in the sample that are not bound to any target molecule to the microfluidic output channel. The step of separating the recognition molecule from the complex can employ a separation mechanism configured to separate or isolate at least one recognition molecule (the recognition molecule from the target-assay-recognition molecule) by photolysis, heating, pH change, chemical removal, or enzymatic removal, such as by delivering a chemical substance / enzyme through at least one microfluidic channel, or providing heating / pH adjustment through at least one electrode, or comprising an illumination source. For example, the recognition molecule or target-assay-recognition molecule can be transported between the two reservoirs through the nanopore by applying a voltage to at least one electrode in each reservoir. If the target analyte / molecule is absent from the sample, this can be detected by the detection unit as the absence of a signal change corresponding to the barcode signal, resulting in a negative test result.

[0044] A nanopore is a nanometer-sized hole that can be formed by pore-forming proteins or exist as a pore in a synthetic material such as silicon or graphene. Nanopores formed by pore-forming proteins are organic nanopores, or they can be inorganic solid-state nanopores, such as those formed in membranes of silicon compounds. When nanopores are present in electrically insulating membranes, they have been shown to function as single-molecule detectors, where a voltage is applied across the membrane and the ionic current passing through the nanopore is monitored. The membrane containing the nanopore (either biological or solid-state) is surrounded by an electrolyte solution, which is divided into two compartments by the membrane. Applying a bias voltage across the membrane creates an electric field, which in turn drives the movement of charged particles (ions). Preferably, any voltage drop is concentrated near and within the nanopore, so that charged particles in the solution only experience the force of the electric field when they are close to the pore region (also called the capture region). Nanoscale polymers (such as DNA or proteins with a net charge) placed in one of the compartments experience the force of the electric field when they are close to the pore region. As a result, molecules are attracted to the pore capture region, enter the nanopore, and are transported within the nanopore by a combination of electrophoretic, electroosmotic, and sometimes thermophoretic forces.

[0045] For molecular-sized nanopores, the passage of a molecule such as DNA results in a disruption in the pore current level, generating a signal as the molecule is transported through the pore. Within the pore, the molecule occupies a certain volume, partially restricting the flow of ions, which manifests as a decrease in the ionic current. The characteristics of this current interruption or disturbance can then be used to determine the sequence or identity of the molecule passing through the pore. The amplitude of the ionic current changes and the duration of the transport will vary based on a variety of factors such as geometry, size, and chemical composition. Different molecules can then be sensed and potentially identified based on this ionic current modulation, which is called nanopore sensing.

[0046] Nanopore sequencing is a sequencing technology based on nanopore sensing that can be used for sequencing of DNA and RNA, wherein single molecules of DNA or RNA can be sequenced without the need for PCR amplification or chemical labeling. The DNA or RNA sequence can be determined by detecting the bases when RNA or single-stranded DNA (ssDNA) is transported through the hole. There are biological nanopores, such as α-hemolysin (αHL) and Mycobacterium smegmatis porin A (MspA), and it has been demonstrated that all four bases can be identified using ionic currents measured through the αHL hole. Alternatively, solid-state nanopores are used, which do not contain proteins in their systems, but instead use various metal or metal alloy substrates with nanoscale pores to allow DNA or RNA to pass through. For example, measuring electron tunneling through bases when ssDNA is transported through a nanopore is used as a solid-state nanopore sequencing method. Alternatively, solid-state nanopores and fluorescence can be used for DNA sequencing, wherein each base is converted into a characteristic representation of multiple nucleotides that are bound to a fluorescent probe chain to form dsDNA, and each base can be identified by two or four separate fluorescences. The advantages of biological nanopores lie in their low transport velocity—i.e., transit speed slow enough to be measured—and the reproducibility of pore size. Solid-state nanopores have the advantages of tolerance to environmental conditions, long lifespan, and ease of fabrication for large-scale production. To enable the use of short recognition molecules disclosed herein, nanopore sensors have field-effect transistors (FETs) embedded in the nanopore, with specifically adapted pore size measurements.

[0047] Although it was previously impossible to determine the complete DNA sequence of an unmodified DNA molecule transported through a solid-state nanopore from the ionic current passing through it, solid-state nanopores and nanochannels can now detect differential electrical signals from different DNA structures (e.g., DNA hairpins of different lengths) and regions with different DNA structures (e.g., ssDNA versus ssDNA hybridized to an oligonucleotide). The spacing and type of these DNA structures can be modified / designed so that the electrical signal read out using a solid-state nanopore resembles a barcode.

[0048] like Figure 2A As shown in , the DNA carrier can be modified with side chains, i.e., backbone modifications, which block the current passing through the solid-state nanopore as shown in 2B, for example, by causing the DNA to protrude from the double strand (e.g., through a dumbbell or hairpin sequence), resulting in peaks in the electrical signal. By varying the position and type of the dumbbell or hairpin, a combination of peaks constituting a barcode pattern can be generated, also as shown in Figure 2B shown. Figure 2C Also shown is the relationship between the side chain modifications and the peak pattern obtained and the resulting identification signal 111010110. One such example is shown in FIG3 . Figure 3Aand B show a backbone A, such as double-stranded DNA, with modifications B and C thereon, generating a value of "1" or "0," respectively, through a nanopore sensor with chambers D and E.

[0049] Therefore, if Figure 4A and 4B As shown, barcodes can be used for nucleic acid detection and protein detection. A DNA hairpin structure (recognition / barcode molecule) can be attached to an RNA capture molecule (assay molecule), thereby allowing hybridization with a target RNA in a sample, and a photocleavable DNA hairpin structure (recognition molecule) can be attached to a protein capture molecule (antibody assay molecule), thereby allowing binding to a target protein. After photocleavage, the recognition molecule is released, allowing the barcode to be read out in a solid-state nanopore platform. Using a strategy similar to the nCounter technology developed by Nanostring, gene expression analysis can be performed by reading out the hairpin barcode unique to each capture probe. Figure 1A An example of a nanopore readout using two different types of modifications (hairpins) with a certain spacing is shown in , to generate peaks that can be assigned specific values ​​(0 or 1) to produce a unique signal.

[0050] To provide an improved sensing method that avoids the shortcomings of the prior art, the inventors discovered that by using a nanopore FET with a specific measurement value, it is possible to read the densely spaced backbone modifications of the recognition molecules, thereby allowing the backbone molecules used to be shorter. This solves the problem of long recognition molecules forming bundles, resulting in some recognition molecules being unreadable. Because shorter recognition molecules are more rigid, the problem of unreadable portions of the recognition molecules is overcome.

[0051] As described above, using solid-state nanopores and nanochannels, it is possible to detect differential electrical signals from different modifications (different DNA structures, such as DNA hairpins with different hairpin lengths) of recognition molecules (with modified backbones) and regions with different DNA structures (such as ssDNA and ssDNA hybridized to the same oligonucleotide). The spacing of these modifications / DNA structures can be modified / designed so that the electrical signals read out using solid-state nanopores are similar to barcodes. However, to date, these DNA structures have primarily been made using DNA origami methods and long DNA molecules. The inventors have now recognized that using shorter recognition molecules has multiple advantages, such as short oligonucleotides as continuous double-helical DNA backbones, onto which different DNA structures can be implanted, thereby generating different electrical signals in solid-state nanopores (more particularly nanopore-FETs). The high bandwidth of nanopore-FETs enables them to distinguish closely spaced DNA structures, so that more and more densely spaced modifications can be accommodated on a shorter backbone than before, thereby generating different electrical signals (signal modulation) when these DNA structures pass through the nanopore-FET. The nanopore-FET for use in the present invention is also able to distinguish a large number of different types of DNA structures, thus having a higher barcoding potential for DNA of the same length compared to conventional solid-state nanopores.

[0052] Methods for producing solid-state nanopore-readable DNA hairpins have been described in Chen K et al., Nano Lett. 2019 Feb 13; 19(2): 1210-1215, wherein the DNA hairpin is used to generate a signal in the nanopore readout that is labeled as a bit (0 or 1) and thus enables data encoding in the DNA structure. Thus, it has been recognized that different bits can be used as "barcodes" (recognition signals / sequences) for recognition, and using recognition molecules containing side chain modifications, for example, a DNA hairpin structure that produces a solid-state nanopore readout of 1001101110 can actually be used as a different barcode sequence than a DNA hairpin structure that produces a nanopore readout of 1111101110, and thus different barcode sequences (arrays / sequences of recognition values) are unique recognition signals.

[0053] In the field of molecular biology, DNA sequence-based barcodes (e.g., nucleotide barcodes) are often used to identify biological molecules. Compared to the identification molecules of the present invention, these DNA sequence-based barcodes cannot currently be distinguished by measuring the ionic current when the DNA sequence-based barcode is transported through a solid-state nanopore, and their application in protein-based biological nanopores is limited because the error rate of DNA sequencing in protein-based nanopores is currently high, and therefore long DNA sequences are required to account for nanopore sequencing errors. By converting DNA sequence-based barcodes into DNA hairpin structure-based barcodes, solid-state-based nanopores can be used to perform the same types of assays that can be performed using DNA sequence-based barcodes on current DNA sequencing platforms. This includes a wide range of application areas, such as the use of DNA-encoded chemical libraries, where chemical molecules can be labeled with DNA hairpin barcodes.

[0054] The design of nanopore-readable recognition molecules / barcodes is not limited to the use of hairpin DNA sequences as backbone / side chain modifications of the DNA backbone: any bulky DNA structure (cruciform DNA, DNA origami, quadruplex DNA) or DNA modification (biotin, oligonucleotides, peptides, PNA) or different DNA structures (such as ssDNA and ssDNA hybridized with oligonucleotides) and combinations of the above can be used to design solid-state nanopore-readable recognition signals, as shown in Figure 5. Figure 5A Schematic diagram showing different side chain modifications on the backbone that form the recognition molecule, including the use of dumbbells and three different sizes of DNA linker structures (4-way linker, 6-way linker, and 12-way linker) to generate a quaternary encoding system; other modifications (such as DNA hairpins, 3-way linkers, 8-way linkers, etc.) are also feasible. Figure 5B Shows Figure 5A The readout of the recognition molecule in the nanopore, i.e., the nanopore readout, shows distinguishable signals for each of these four structures. Depending on the structure and resolution of the nanopore-FET, there may be more than four distinguishable nanopore encoding systems. A single dumbbell unit is 14 nucleotides long, and 11 dumbbell units (220 nucleotides) provide sufficient barcode readout signal.

[0055] Compared to DNA sequence-based barcode reading, reading DNA structure-based recognition molecules / barcodes on solid-state nanopores has the advantages of extremely low measurement costs and extremely high and rapid throughput per measurement. Since very long DNA molecules can be transported through solid-state nanopores, and since the distance between DNA hairpins or DNA structures determines the barcode / value array (recognition signal / barcode signal) of the recognition molecule, in addition to extremely long recognition signals (1010110101…), very short recognition signals (such as 01) can also be used. Different recognition systems can be used: for example, the length of the recognition signal can be different (for example, a barcode with 2 components, represented here by 0 and 1, can produce recognition signals such as 0011010 or 1110000000), and complex barcodes can also be designed in which multiple components can be used (producing a barcode with a sequence of 0a2&, where 0, a 2 , & represent different DNA structures).

[0056] However, when long recognition molecules (e.g., long DNA molecules) are transported through solid-state nanopores, reading out the barcode structure can be problematic. Long recognition molecules can form tertiary structures, such as bundles, during transport, and thus large portions of the recognition molecule may be obscured and thus not correctly read. According to some sources, up to 85% of nanopore transport events are excluded due to incomplete or folded DNA being transported through the nanopore, thereby preventing correct barcode reading (e.g., see Bell and Keyser, Nat. Nanotech 2014, Digitally encoded DNA nanostructures for multiplexed, single-molecule protein sensing with nanopores). This therefore presents significant problems when using such recognition molecules for detection / sequencing of target molecules.

[0057] Therefore, a solution to this problem could be to use shorter recognition molecules that are more rigid and less prone to forming bundles. However, due to the distance between modifications required to read the correct barcode signal, this is not possible using conventional solid-state nanopores. However, by using nanopore FETs (NPFETs) with optimized bandwidth and reading aperture parameters, it has now been demonstrated that it is possible to read modifications with spacings as close as 10 bp or nt, while the conventional spacing is typically, for example, 114-1032 bp. This allows for more modifications per backbone length; therefore, the same number of modifications can be suitable for much shorter recognition molecules.

[0058] Conventional solid-state nanopores also have lower sensitivity for detecting side chains of different sizes / charges than NPFET nanopores, which means that more different types of barcodes can be detected on oligonucleotides or DNA sequences of the same length using NPFETs compared to conventional solid-state nanopores. In addition, the high bandwidth of the nanopore-FET enables it to distinguish DNA structures that are more closely spaced, which means that shorter DNA sequences can be used than those used in conventional solid-state nanopores. The combination of these features suggests that there may be multiple advantages to using short oligonucleotides (e.g., 200-400 nucleotides) in nanopore-FETs and setting up hairpin DNA barcode structures on them. The higher bandwidth advantage offered by nanopore-FETs means that millions of barcodes can be generated on very short oligonucleotides (e.g., 200nt). For example, molecular modeling suggests that modifications of every 10 nucleotides can be detected by nanopore-FETs. This means that 20 binary (or higher order) modifications can be detected on every 200nt oligonucleotide, resulting in at least about 1 million barcodes (2 20 ).

[0059] The generation of barcoded oligonucleotides can be performed using several methods, such as Figure 6 As shown. For example, customized azido-nucleotides (for side chain implantation based on click chemistry to generate barcodes) can be introduced during oligonucleotide synthesis. Smaller oligonucleotides can be modified with different side chain modifications in a separate reaction and then linked together to generate barcode oligonucleotides. Alternatively, Taq1 methyltransferase (4nt recognition sequence) can be used to enzymatically modify oligonucleotide DNA. Synthetic oligonucleotides can be designed so that the spacing of its recognition sequence is every 10 or X nucleotides, where X can vary between, for example, 10 to 40nt. Different methyltransferases with different target sequences can be used to generate barcodes by introducing different side chains.

[0060] In some embodiments, the oligonucleotide of the present invention is synthesized by phosphoramidite chemistry, and the oligonucleotide of the present invention is synthesized by phosphoamidite chemistry.Although there is the problem that product forms incomplete when preparing the long oligonucleotide of size above 100nt, multiple strategies can be adopted to solve this problem, for example, the long oligonucleotide (300nt) prepared by phosphoramidite method can be carried out for the purification of size by PAGE or HPLC.Further, the DNA assembly technology based on commercial synthetic biology can be used to produce the oligonucleotide of correct size.Example includes the BioXP technology from CODEX DNA company, and this technology uses synthetic biology (Gibson assembling (Gibson assembly)) that short 20-30nt oligonucleotide is assembled into the longer fragment of correct size.Similar techniques from other companies (such as Elegen company, Evonetix company etc.) can also be used.

[0061] In one example, a DNA ligation-based approach, also known as a "mini-origami" approach, can be used, such as Figure 7 The method involves generating a single "base" oligonucleotide of 200 nucleotides, purifying it to the correct length, and generating a set of 10 purified complementary oligonucleotide molecules (approximately 20-30 nt in size). These complementary oligonucleotides hybridize with the base oligonucleotide to generate barcodes, linking the fragments together. This method requires the generation of 21 purified oligonucleotides, which allows the generation of millions of barcodes using a 0-1 bit encoding system.

[0062] Short oligonucleotides, with a barcode structure of 10 nucleotides per oligonucleotide, transported through a nanopore-FET offer several advantages in nanopore sensing. One advantage is faster speed and shorter transport time, allowing more barcodes to be read in the same amount of time, resulting in higher throughput and fully leveraging the advantages of the NPFET. For example, an 11-unit dumbbell structure requires 880 nucleotides to generate a barcode structure of 01 or 10 (these 880 nucleotides include space for two side chain structures to determine orientation during transport through the nanopore). Another advantage is the uninterrupted double-helix backbone and more rigid DNA, which prevents the backbone and barcode structure from folding, reducing errors (DNA with secondary structure entering the pore can make the barcode unreadable) and noise (less Brownian motion). Another advantage is lower cost (fewer oligonucleotides are required to assemble the barcode). Compared to conventional solid-state nanopores, the higher sensitivity and bandwidth of the nanopore-FET also allow for the detection of more different barcode types (more bits per barcode) on oligonucleotides or DNA sequences of the same length. In addition, it reduces the molecular weight of the barcode (which is beneficial for assay reagent kinetics and assay time), for example, at approximately 0.6 kDa / bp, the molecular weight of 2 kbp DNA is approximately 1200 kDa, while that of nanoantibodies is 12-15 kDa; aptamers are 5-15 kDa; antibodies: 150 kDa; it avoids origami bending points, has no origami folding errors, no backbone folding, reduces dehybridization and reduces errors. Figure 8 The recognition molecules of the present disclosure are shown to have side chain modifications at a distance of 10 nt from each other on the backbone molecule.

[0063] In the present disclosure, recognition molecules can be used to identify targets in samples. In some aspects, these recognition molecules are designed as nanopore-readable barcodes, wherein the recognition molecules are coupled to assay molecules (such as antibodies) for detecting target biomolecules in samples, thereby indirectly identifying and quantifying biomolecules by transporting the recognition molecule-assay-target complex, or by separating the recognition molecule / barcode molecule from the assay-target complex and transporting only the recognition molecule through the nanopore, so as to be able to identify the specific barcode connected to the biomolecule and thereby determine the presence of the biomolecule or a specific part of the biomolecule. By measuring the quantity (number) of transported recognition molecules associated with a specific biomolecule, the quantity or concentration can be determined, such as the absolute concentration or the relative concentration relative to another measured biomolecule in the sample. Quantification can be performed by counting the number of unique signals obtained, which can be associated with the concentration of the target sample. In some aspects, the counting of barcodes can indicate the concentration of the target biomolecule, such as in a scenario where "one barcode corresponds to one target molecule".

[0064] The structure used as the recognition molecule (barcode molecule) in the present disclosure does not only need to be a DNA structure, but can also contain any type of backbone (with backbone / side chain modifications added thereto) so that these modifications generate a barcode signal when read out in a nanopore (preferably a nanopore FET). The backbone can be any linear structure, typically a polymer, which can be used to imprint a backbone structure (backbone / side chain modifications) thereon so that when transported through the nanopore, a barcode-like signal is observed, that is, a unique recognition signal containing an array / sequence of identification values. Spherical structures (such as nanoparticles) or nonlinear structures have limitations in their potential for generating a large number of barcodes (unless connected together to form a linear chain), but any type of linear structure can be used for this purpose as long as it can contain the backbone structure. In some embodiments, the backbone is generated by charged molecules, such as biopolymers and synthetic polymers. Alternatively, the backbone is generated by uncharged molecules, for example polyethers, such as polyethylene glycol (PEG) or its variants. Alternatively, structures such as nanotubes or nanowires can be used. In a preferred embodiment, the backbone molecule is DNA-based, such as ss DNA or ds DNA.

[0065] In some embodiments, the backbone is a biopolymer backbone, such as an RNA, peptide, or carbohydrate biopolymer backbone, to which one or more backbone or side chain modifications are added, such that these modifications generate a barcode signal in the nanopore FET. In other embodiments, the backbone is a synthetic polymer backbone, such as polyesters, polyamides, rigid rod polymers, and modified polymers with carbon linkers. In a preferred embodiment, the backbone comprising modifications is a DNA backbone, including modifications in the form of DNA hairpins and DNA dumbbells.

[0066] These recognition molecules / barcode molecules or their side chain modifications (barcode structures) can then be chemically coupled to another biopolymer to barcode the biopolymer, and after measurement and optional removal of the recognition molecules, the current signal amplitude modulation (e.g., peak pattern) generated by the recognition molecules / barcode structures upon transport can be read using a nanopore FET.

[0067] Therefore, the present invention relates to providing (e.g., generating) short recognition molecules comprising a backbone with densely spaced modifications, which, when transported through a nanopore sensor, generate a recognition signal that can be read directly from the recognition molecule or by assigning a value to the signal modification / peak obtained from the modification of the recognition molecule. The backbone or side chain modifications can have a variety of modification types, which, due to their different effects on the electric field, will produce different signals / peaks when read out on the nanopore sensor. Each peak can be assigned an identification value, for example, when two modification types are used, the identification value can be a binary barcode value of "0" or "1", and the complete readout of the identification values ​​of the multiple modifications of the recognition molecule and the corresponding peaks is called a recognition signal, for example, 001, 101, or 111 in the case of a three-peak readout. Therefore, all the recognition values ​​of the recognition molecule form an array or sequence of identification values ​​in the order of each peak obtained when the recognition molecule is transported through the nanopore, and the array / sequence is the recognition signal. Therefore, when using two modification types with identification values ​​of 0 or 1, the identification signal will be a binary signal of 0 and 1 corresponding to the backbone modification of the recognition molecule, where the identification value assigned to the peak is predetermined. Therefore, each recognition molecule contains a unique identification signal.

[0068] Recognition molecules can be connected with assay molecules to form assay-recognition molecules. The assay molecules disclosed herein are molecules that bind target molecules in assays (such as immunoassays), wherein each type of assay molecule is designed to bind a certain target molecule. The assay molecule can be, for example, DNA, protein, peptide or polysaccharide, and the assay molecule can be connected to the recognition molecule by a variety of methods, such as direct conjugation, affinity-based conjugation, hybridization, ionic interaction or cross-linking, such as cross-linking including click chemistry. Therefore, since it is predetermined to assign recognition values ​​to different peaks, the recognition signal of each recognition molecule is known accordingly, and since the connection of specific (one or more) recognition molecules to specific assay molecules that bind specific target molecules is also known, one or more recognition signals can be associated with specific target molecules. In addition, when using other methods other than intrinsic barcodes to identify recognition molecules, the association between the unique identifier of the recognition molecule and the assay molecule that binds to a specific target is known, which can be used to detect the target after identifying the recognition molecule in the nanopore sensor.

[0069] In some embodiments, the present invention relates to a method for the determination of the target molecule of the present invention. The method comprises the steps of: determining the target molecule of the present invention and determining the target molecule of the present invention. The method comprises the steps of: determining the target molecule of the present invention and determining the target molecule of the present invention. The method comprises the steps of: determining the target molecule of the present invention and determining the target molecule of the present invention. The method comprises the steps of: determining the target molecule of the present invention and determining the target molecule of the present invention. The method comprises the steps of: determining the target molecule of the present invention and determining the target molecule of the present invention. The method comprises the steps of: determining the target molecule of the present invention and determining the target molecule of the present invention. The method comprises the steps of: determining the target molecule of the present invention and determining the target molecule of the present invention. The method comprises the steps of: determining the target molecule of the present invention and determining the target molecule of the present invention. The method comprises the steps of: determining the target molecule of the present invention and determining the target molecule of the present invention. The method comprises the steps of: determining the target molecule of the present invention and determining the target molecule of the present invention. The method comprises the steps of: determining the target molecule of the present invention and determining the target molecule of the present invention. By transporting the complex through the nanopore, or by separating the recognition molecule from the target-assay-recognition molecule in the sample (e.g., using a photocleavable recognition molecule and cleaving the recognition molecule by photolysis, wherein the separation may include separating the recognition molecule from the sample, i.e., removing the recognition molecule from the sample), the recognition molecule can be passed through the nanopore sensor, wherein different types of backbone / side chain modifications of the recognition molecule will produce a signal or peak pattern, which can be converted into a recognition / barcode pattern, for example, by assigning an identification value to each peak, wherein the recognition pattern (sequence of identification values) is called a recognition signal. In some embodiments, the recognition signal is based on the size or transit time / duration of the recognition molecule.

[0070] The recognition of a specific recognition molecule in a nanopore can be related to the presence of a target in the sample used in the assay. In some aspects, the recognition molecule itself can be an identifier, such as its length can be related to a unique identifier. In some aspects, the recognition molecule can carry information (such as modifications) that can be detected as a peak pattern in the nanopore and assigned an identification value (such as a barcode value). It should be noted that the "barcode molecules" and "barcode values" of the present invention are different from typical DNA barcodes, the barcodes of which are present in the nucleobase sequence. The unique identifier can also be based on the transition time between side chain modifications in the nanopore sensor. If the target molecule is not present, then when incubated with the target sample, no assay molecule will bind, and therefore no recognition molecule will be detected in the nanopore.

[0071] The recognition molecule can be transported through the nanopore in both directions. For example, if it enters in one direction, the signal will be 10001111, while entering in the opposite direction will produce 11110001. To enable the use of a large number of recognition molecules, a specific signal can be tagged on one of the two ends of the recognition molecule to determine directionality.

[0072] In some aspects, each end of the recognition molecule contains a small portion that generates a predetermined array of values ​​for detecting the orientation of the recognition molecule during transport through the nanopore, thereby determining the directionality of the recognition molecule during passage through the nanopore sensor. For example, a "barcode region" specific for orientation purposes can be present in the backbone of the recognition molecule. Therefore, in order to be able to infer the orientation of the molecule transported through the nanopore, smaller recognition molecules or backbone / side chain modifications can be attached to the corresponding sides / ends of the molecule (the "left side" and "right side", or the front end / end) of the molecule, wherein the small recognition molecules contain backbone / side chain modifications that are converted into recognition patterns (recognition signals) each containing, for example, 10 modifications, and wherein the patterns / signals on the "right side" and the "left side" are different and do not appear in the main recognition signal of the target molecule to be detected.

[0073] In a preferred embodiment, a field-effect transistor (FET) is embedded in the nanopore, forming a so-called FET nanopore sensor, also known as a nanopore FET (NPFET). A field-effect transistor is a transistor that uses an electric field to control the flow of current in a semiconductor. A FET is a device with three terminals: source, gate, and drain. A FET controls current flow by applying a voltage to the gate, which in turn changes the conductivity between the drain and source. The most common type of FET is the MOSFET (metal oxide semiconductor field effect transistor), which utilizes an insulator (typically SiO2) between the gate and the body. Using nanopore FET arrays for barcode readout is highly efficient, offering exceptional throughput, dynamic range, and multiplexing capabilities. Multiplexing allows for longer barcodes to generate billions of labels, while fluorescent labels, for example, are limited to 16. In terms of throughput, nanopore arrays can achieve a throughput of up to 1 million molecules per second per pore, giving NPFETs a bandwidth advantage (>1 MHz). In terms of dynamic range, they can detect even very low-abundance species, offering high throughput and single-molecule sensitivity.

[0074] The nanopore FET disclosed herein should have a high bandwidth, such as 1 MHz or higher. In order to provide a high SNR for short recognition molecules at high bandwidth, the pore size of the nanopore FET should be of a specific size. The pore height should be approximately 3,4-14 nm, roughly corresponding to the resolution of modifications on the recognition molecule that are 10-40 nt apart. Preferably, the pore height is less than 10 nm, i.e., 3,4-10 nm. The pore diameter should also be controlled, preferably in the range of 1-20 nm, for example, 3-10 nm, preferably less than 10 nm.

[0075] Figure 9A schematic representation of a cross-section of a nanopore FET device is shown in Figure 1. Here, the pore size (1) is defined as the narrowest constriction of the nanopore device. The pore size has nanometer-scale dimensions. Its diameter (2) and height (3) largely determine the signal intensity generated when a molecule passes through the nanopore device. The pore size also determines the resolution of the nanopore device, which is defined here as the shortest distance between labels that the device can still distinguish between different labels on the double-stranded DNA backbone.

[0076] Thus, the present disclosure provides a recognition molecule for use in nanopore sensing, wherein the recognition molecule is capable of generating a unique recognition signal when transported through the nanopore sensor, the recognition molecule comprising a backbone molecule having a plurality of backbone modifications, wherein each backbone modification causes a modulation of the signal amplitude when transported through the nanopore sensor, and wherein the distance between the backbone modifications of the recognition molecule is 3.4-13.6 nm, corresponding to a distance of 10-40 nucleotides (nt) or base pairs (bp) (wherein the average nt length is 0,34 nm).

[0077] Therefore, the spacing between the backbone modifications on the backbone molecules of the recognition molecules should be relatively close, 10-40 nucleotides apart in the DNA backbone. In a preferred embodiment, the spacing between the modifications is less than 10 nm, such as 6.8 nm, 5.1 nm or 3.4 nm, corresponding to a distance of 30 nt, 20 nt, 15 nt or 10 nt, such as preferably 10 or 15 nt apart.

[0078] The length of the recognition molecule disclosed herein is much shorter than that in the prior art, and this is realized by using the modification of close intervals. The length of the recognition molecule disclosed herein or its skeleton is within the range of 6.8-136nm, corresponding to 20-400nt (wherein average nt length is 0,34nm). Preferably, length is less than 102nm (corresponding to 300nt), and even more preferably is less than 68nm (corresponding to 200nt). Length can be, for example, 34nm, 27.2nm, 20.4nm, 13.6nm or 6.8nm, corresponding to 100nt, 80nt, 60nt, 40nt or 20nt. If the length of molecule is in a lower range, then the modification distance is also in a lower range, such as at intervals of 10nt, so that the length of skeleton is always greater than the distance between skeleton modifications.

[0079] In the recognition molecules of the present invention, the backbone modifications are selected from a plurality of modification types, and when read in the nanopore sensor, each signal amplitude modulation obtained from the plurality of modifications is assigned an identification value, and the total number of identification values ​​corresponding to the modifications yields an array of identification values ​​corresponding to the unique recognition signal of the recognition molecule. The assignment of these identification values ​​is based on i) the distance between the modifications, or ii) the modification type.

[0080] In some embodiments, two modification types are used, and each signal amplitude modulation of these two modification types is assigned an identification value of "0" or "1", and all identification values ​​of these corresponding modifications produce a binary identification value array corresponding to the unique identification signal of the identification molecule.

[0081] In preferred embodiments, the recognition molecule has a biopolymer backbone, such as a DNA backbone, preferably a double-stranded DNA molecule. Backbone modifications are selected from bulky DNA structures, such as DNA hairpins, cruciform DNA, DNA origami, and quadruplex DNA; DNA modifications, such as biotin, oligonucleotides, peptides, PNA, carbohydrates, or organic molecules; and differentiated DNA structures, such as ssDNA differentiated into ssDNA hybridized to oligonucleotides. In some embodiments, the backbone is encoded as a block chain structure formed by one or more layers of nucleic acids, peptides, or proteins.

[0082] In a preferred embodiment, the nanopore sensor has a field effect transistor (FET) embedded in the nanopore.

[0083] According to the present disclosure, methods for using the recognition molecules of the present invention in assays are also provided. For example, methods for detecting or quantifying a target molecule using the recognition molecules of the present invention are provided. In one example, a method for detecting or quantifying one or more target molecules in a sample using a nanopore sensor comprises: providing (S1) a sample comprising one or more target molecules to be detected / quantified; providing (S2) at least one (short) recognition molecule as described above and below for the corresponding target molecule, wherein the recognition molecule is capable of generating a unique recognition signal when transported through the nanopore sensor, and wherein the association between the unique recognition signal of the recognition molecule and the bound target molecule is known; incubating (S4) the at least one recognition molecule with the sample that may contain one or more target molecules, thereby allowing the at least one recognition molecule to bind to the one or more target molecules to form at least one target-recognition molecule, or alternatively, linking the recognition molecule to an assay molecule and allowing the assay molecule to bind to the target molecule to form one or more target-assay-recognition molecules; transporting the target-recognition molecule (or alternatively the target-assay-recognition molecule or merely the isolated recognition molecule) through the nanopore sensor (S5) to obtain at least one unique recognition signal; and detecting (S6) any presence of the one or more target molecules in the sample by associating the obtained at least one unique recognition signal with the one or more target molecules. Preferably, the nanopore sensor has a field effect transistor (FET) embedded in the nanopore.

[0084] Further provided is a system for target molecule sensing and sequencing using a nanopore sensor, the system comprising: a first electrolyte reservoir and a second electrolyte reservoir, the first and second reservoirs being separated by a barrier, the barrier comprising one or more FET-embedded nanopores, the nanopore comprising an pore having a predetermined height and a predetermined diameter; and optionally, an electrode for transporting molecules from the first electrolyte reservoir to the second electrolyte reservoir through the nanopore, wherein at least one of the first and second electrolyte reservoirs comprises a recognition molecule of the present disclosure, wherein the recognition molecules are capable of generating a unique recognition signal when transported through the nanopore.

[0085] The pore is the opening at the bottom of the nanopore channel that reads the recognition molecule. The narrow diameter and low height of the pore produce a strong signal when reading the recognition molecule.

[0086] In embodiments, the pore height h of the FET-embedded nanopore is predetermined based on the distance between the backbone modifications of the recognition molecule, such that a shorter distance between the backbone modifications determines a shorter pore height. The pore height can be in the range of 3,4-14 nm, preferably 7 nm or less. This corresponds to a spacing between modifications of 10-40 nt, preferably 20 nt or less.

[0087] The pore diameter is in the range of 3-20 nm, preferably 10 nm or less.The nanopore FET preferably has a bandwidth of 1 MHz or higher.

[0088] The system can be used to perform a method for detecting one or more target molecules in a sample using a nanopore sensor, the method comprising: providing (S1) a sample containing one or more target molecules to be detected; providing (S2) at least one recognition molecule according to the present disclosure for the corresponding target molecule, wherein the recognition molecule is capable of generating a unique recognition signal when transported through the nanopore sensor, and the association between the unique recognition signal of the recognition molecule and the bound target molecule is known; optionally linking the at least one recognition molecule to an assay molecule (S3); incubating the at least one recognition molecule with a sample that may contain one or more target molecules (S4), thereby allowing the at least one recognition molecule to bind to the one or more target molecules to form at least one target-recognition molecule or target-assay-recognition molecule, and allowing the recognition molecule to be transported through the nanopore sensor (S5) to obtain at least one unique recognition signal, wherein the recognition molecule is in a complex or optionally separated from the target-recognition molecule or target-assay-recognition molecule; and detecting (S6) any presence of the one or more target molecules in the sample by associating the obtained at least one unique recognition signal with the one or more target molecules.

[0089] In the drawings and description, exemplary aspects of the present disclosure have been disclosed. However, many variations and modifications may be made to these aspects without departing substantially from the principles of the present disclosure. Therefore, the present disclosure should be considered illustrative rather than restrictive, and is not limited to the specific aspects discussed above. Therefore, although specific terms are employed, these terms are used in a generic and descriptive sense only and not for purposes of limitation.

[0090] The description of the example embodiments provided herein has been presented for illustrative purposes. This description is not intended to be exhaustive or to limit the example embodiments to the precise form disclosed, and modifications and variations are possible in accordance with the above teachings or can be obtained from the practice of various alternatives to the embodiments provided. The examples discussed herein are selected and described to explain the principles and properties of the various example embodiments and their practical applications so that those skilled in the art can utilize the example embodiments in various ways and with various modifications suitable for the intended specific purposes. The features of the embodiments described herein can be combined in all possible combinations of methods, products, and systems. It should be understood that the example embodiments presented herein can be practiced in any combination with each other. The present invention has been primarily described above with reference to several embodiments, however, as will be readily appreciated by those skilled in the art, within the scope of the present invention as defined by the appended patent claims, other embodiments other than the embodiments disclosed above are equally possible.

[0091] It should be noted that the word "comprising" does not necessarily exclude the presence of other elements or steps than those listed, and the word "a" or "an" preceding an element does not exclude the presence of a plurality of such elements. It should also be noted that any reference signs do not limit the scope of the claims, and example embodiments can be implemented in the broadest sense of the claims. All cited references are incorporated by reference to the extent permitted.

Claims

1. A recognition molecule for use in nanopore sensing, wherein the recognition molecule is capable of generating a unique recognition signal when transported through the nanopore sensor, the recognition molecule comprising a backbone molecule having multiple backbone modifications, wherein each backbone modification causes signal amplitude modulation when transported through the nanopore sensor, and wherein the distance between the backbone modifications of the recognition molecule is 3.4-13.6 nm, corresponding to a distance of 10-40 nucleotides (nt).

2. The recognition molecule according to claim 1, wherein the length of the backbone molecule is 6.8-136 nm, corresponding to 20-400 nt, wherein the length of the backbone molecule is greater than the distance between the backbone modifications.

3. The recognition molecule according to claim 1-2, wherein the distance between the backbone modifications of the recognition molecule is less than 10 nm, such as 6.8 nm, 5.1 nm or 3.4 nm.

4. The recognition molecule according to claim 2-3, wherein the length of the backbone molecule is 102 nm or less, such as 68 nm, 34 nm, 27.2 nm, 20.4 nm, 13.6 nm or 6.8 nm.

5. A recognition molecule according to any one of claims 1-4, wherein the backbone modifications are selected from a plurality of modification types, and wherein each signal amplitude modulation obtained from the plurality of modifications is assigned an identification value when read out in the nanopore sensor, and wherein all identification values ​​of the corresponding modifications produce an array of identification values ​​corresponding to a unique recognition signal of the recognition molecule.

6. The recognition molecule according to claim 5, wherein the assignment of the recognition values ​​is based on i) the distance between the modifications, or ii) the type of modification.

7. A recognition molecule according to any one of claims 1 to 6, wherein two modification types are used and each signal amplitude modulation of these two modification types is assigned a recognition value of "0" or "1", and wherein all recognition values ​​of these corresponding modifications produce an array of binary recognition values ​​corresponding to the unique recognition signal of the recognition molecule.

8. The recognition molecule according to any one of claims 1 to 7, wherein the backbone molecule is a biopolymer backbone, such as a double-stranded DNA molecule, or wherein the backbone is encoded as a block chain structure formed by one or more layers of nucleic acids, peptides, peptide nucleic acids or proteins.

9. A recognition molecule according to any of the preceding claims, wherein the one or more backbone modifications are selected from bulky DNA structures, such as DNA hairpin structures, cruciform DNA, DNA origami and quadruplex DNA; DNA modifications, such as biotin, oligonucleotides, peptides, PNA, carbohydrates or organic molecules; and differentiated DNA structures, such as ssDNA differentiated into ssDNA hybridized to oligonucleotides, or a combination thereof.

10. The recognition molecule of any preceding claim, wherein the nanopore sensor has a field effect transistor (FET) embedded in the nanopore, or comprises a remotely extended FET wherein electrodes surrounding the nanopore are connected to a remote gate sensor.

11. A system for target molecule sensing and sequencing using a nanopore sensor, the system comprising: a first electrolyte reservoir and a second electrolyte reservoir, the first and second reservoirs separated by a barrier, the barrier comprising one or more FET-embedded nanopores comprising an pore having a predetermined height and diameter; and Optionally, an electrode for transporting molecules from the first electrolyte reservoir to the second electrolyte reservoir through the nanopore, in, At least one of the first and second electrolyte reservoirs comprises recognition molecules according to any one of claims 1-10, wherein the recognition molecules are capable of generating a unique recognition signal when transported through the nanopore.

12. The system according to claim 11, wherein the pore height is in the range of 3,4-14 nm, preferably 7 nm or less.

13. The system according to claims 11-12, wherein the pore diameter is in the range of 3-20 nm, preferably 10 nm or less.

14. The system of claims 11-13, wherein the bandwidth of the FET-embedded nanopore is at least 1 MHz.

15. A method for detecting one or more target molecules in a sample using a nanopore sensor, the method comprising: Providing (S1) a sample containing one or more target molecules to be detected; Providing (S2) at least one recognition molecule according to claims 1 to 10 for the corresponding target molecule, wherein the recognition molecule is capable of generating a unique recognition signal when transported through the nanopore sensor, and the correlation between the unique recognition signal of the recognition molecule and the bound target molecule is known; optionally linking the at least one recognition molecule to a determination molecule (S3); incubating the at least one recognition molecule with a sample that may contain one or more target molecules (S4), thereby allowing the at least one recognition molecule to bind to the one or more target molecules to form at least one target-recognition molecule or target-assay-recognition molecule, transporting the recognition molecule through the nanopore sensor (S5) to obtain at least one unique recognition signal, wherein the recognition molecule is in complex or optionally separated from the target-recognition molecule or target-assay-recognition molecule; and Any presence of the one or more target molecules in the sample is detected (S6) by associating the obtained at least one unique recognition signal with the one or more target molecules.

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