Sensing system

Abstract

A sensing system includes a charge sensor that includes two electrodes and a conductive channel connecting the two electrodes. The sensing system further includes a charged molecule attached to the conductive channel. The charged molecule includes an identification site for a marker that reversibly binds a labeled nucleotide; has an unbound favorable conformation associated with an unbound charge configuration; and has a favorable conformation associated with a charge configuration when the identification site binds to the marker. The charge configuration is different from the unbound charge configuration. The sensing system further includes a polymerase attached to the conductive channel or the charged molecule.

Description

[0001] Cross - Reference to Related Applications

[0002] This application claims the benefit of U.S. Provisional Application Serial No. 62 / 783,951, filed Dec. 21, 2018; the content of the U.S. Provisional Application is incorporated herein by reference in its entirety.

[0003] Background

[0004] A variety of protocols in biological or chemical research involve performing a large number of controlled reactions on a local support surface or within a predefined reaction chamber. A specified reaction can then be observed or detected, and subsequent analysis can help identify or reveal the nature of the chemicals involved in the reaction. In some instances, the controlled reaction produces fluorescence, and thus an optical system can be used for detection. In other instances, the controlled reaction alters charge, conductivity, or some other electrical property, and thus an electronic system can be used for detection.

[0005] Introduction

[0006] A first aspect disclosed herein is a sensing system that includes a charge sensor. The charge sensor includes: two electrodes and a conductive channel connecting the two electrodes; a charged molecule attached to the conductive channel, where the charged molecule includes: an identification site for a label that reversibly binds a labeled nucleotide, has an unbound favored conformation associated with an unbound charge configuration, and has a favored conformation associated with a charge configuration when the identification site binds to the label, where the charge configuration is different from the unbound charge configuration; and a polymerase attached to the conductive channel or the charged molecule.

[0007] In an example of this first aspect, the charged molecule is a charged aptamer. In this example, the charged aptamer is selected from the group consisting of DNA aptamers, RNA aptamers, and their analogs.

[0008] In an example of this first aspect, the charged molecule is selected from the group consisting of charged proteins and charged peptides.

[0009] In an example of this first aspect, the charged molecule: further includes a second recognition site for a second label that reversibly binds to a nucleotide of the second label, and has a second favorable conformation associated with a second charge configuration when the second recognition site binds to the second label; further includes a third recognition site for a third label that reversibly binds to a nucleotide of the third label, and has a third favorable conformation associated with a third charge configuration when the third recognition site binds to the third label; and further includes a fourth recognition site for a fourth label that reversibly binds to a nucleotide of the fourth label, and has a fourth favorable conformation associated with a fourth charge configuration when the fourth recognition site binds to the fourth label; and when each of the recognition site, the second recognition site, the third recognition site, and the fourth recognition site is unbound, an unbound favorable conformation associated with an unbound charge configuration occurs.

[0010] In an example of this first aspect, the sensing system further includes a second charged molecule attached to the conductive channel, wherein the second charged molecule: includes a second recognition site for a second label that reversibly binds to a nucleotide of the second label; has an unbound favorable conformation of the second charged molecule associated with the unbound charge configuration of the second charged molecule; and has a favorable conformation of the second charged molecule associated with the charge configuration of the second charged molecule when the second recognition site binds to the second label. In this example, the sensing system may further include a third charged molecule attached to the conductive channel, wherein the third charged molecule: includes a third recognition site for a third label that reversibly binds to a nucleotide of the third label; has an unbound favorable conformation of the third charged molecule associated with the unbound charge configuration of the third charged molecule; and has a favorable conformation of the third charged molecule associated with the charge configuration of the third charged molecule when the third recognition site binds to the third label; and a fourth charged molecule attached to the conductive channel, wherein the fourth charged molecule: includes a fourth recognition site for a fourth label that reversibly binds to a nucleotide of the fourth label; has an unbound favorable conformation of the fourth charged molecule associated with the unbound charge configuration of the fourth charged molecule; and has a favorable conformation of the fourth charged molecule associated with the charge configuration of the fourth charged molecule when the fourth recognition site binds to the fourth label.

[0011] In an example of this first aspect, the charged molecule further comprises a second recognition site for reversibly binding a second label of a labeled nucleotide, and when the second recognition site binds to the second label, has a second favorable conformation associated with a second charge configuration; and the sensing system further comprises a second charged molecule attached to the conductive channel, wherein the second charged molecule: comprises a third recognition site for reversibly binding a third label of a third labeled nucleotide, and a fourth recognition site for reversibly binding a fourth label of a fourth labeled nucleotide; has an unbound favorable conformation of the second charged molecule associated with the unbound charge configuration of the second charged molecule; when the third recognition site binds to the third label, has a third favorable conformation associated with a third charge configuration; and when the fourth recognition site binds to the fourth label, has a fourth favorable conformation associated with a fourth charge configuration.

[0012] In an example of this first aspect, the charged molecule further comprises a second recognition site for reversibly binding a second label of a labeled nucleotide.

[0013] It should be understood that any features of the sensing systems disclosed herein can be combined together in any desired manner and / or configuration.

[0014] A second aspect disclosed herein is a sensing device comprising a flow cell and a sensing system integrated into the flow cell, the sensing system comprising a charge sensor, the charge sensor comprising a conductive channel; a charged molecule attached to the conductive channel, wherein the charged molecule: has an unbound favorable conformation associated with an unbound charge configuration; and when the recognition site of the charged molecule binds to a label of a labeled nucleotide, has a favorable conformation associated with a charge configuration, wherein the charge configuration is different from the unbound charge configuration; and a polymerase attached to the conductive channel or the charged molecule.

[0015] In an example of this second aspect, the sensing device further comprises a reagent delivery system for selectively introducing a reagent into the input end of the flow cell. In some examples, the reagent is in a sample container, and the reagent comprises a labeled nucleotide, the labeled nucleotide comprising: a nucleotide; a linking molecule attached to a phosphate group of the nucleotide; and a recognition site-specific label attached to the linking molecule.

[0016] In an example of this second aspect, the sensing device further comprises a detector for detecting a response from the charge sensor.

[0017] It should be understood that any features of the sensing device can be combined together in any desired manner. Additionally, it should be understood that any combination of features of the sensing system and / or the sensing device can be used together, and / or in combination with any example disclosed herein.

[0018] A third aspect disclosed herein is a method that includes introducing a template polynucleotide strand into a sensing system that includes: a charge sensor that includes two electrodes and a conductive channel connecting the two electrodes; a charged molecule attached to the conductive channel, where the charged molecule includes an identification site; and a polymerase attached to the conductive channel or the charged molecule; introducing a reagent that includes labeled nucleotides into the sensing system, whereby a nucleotide of one of the labeled nucleotides associates with the polymerase and an identification site specific label of the one of the labeled nucleotides associates with the identification site to induce a conformational change of the charged molecule; and detecting a response of the charge sensor in response to the conformational change of the charged molecule.

[0019] In an example of this third aspect, the method further includes correlating the response of the charge sensor with the associated identification site specific label; and identifying the nucleotide of one of the labeled nucleotides based on the associated identification site specific label.

[0020] In an example of this third aspect, the charged molecule includes a plurality of different identification sites, each of which reversibly binds different labels of different labeled nucleotides at different rates. In some examples, the method further includes detecting a plurality of responses of the charge sensor in response to different conformational changes of the charged molecule when different labeled nucleotides respectively associate with the polymerase and different identification site specific labels of the different labeled nucleotides respectively bind to one of the plurality of different identification sites; and identifying the different labeled nucleotides that are respectively associated by different rates.

[0021] In an example of this third aspect, the identification site reversibly binds a plurality of different labels of a plurality of different labeled nucleotides at a plurality of different rates, and wherein the method further includes: detecting a plurality of responses of the charge sensor in response to different conformational changes of the charged molecule when at least some different labeled nucleotides respectively associate with the polymerase and at least some different labels respectively bind to the identification site; and identifying the different labeled nucleotides that are respectively associated by different rates.

[0022] In an example of this third aspect, the identification site reversibly binds up to four different labeled nucleotides, and wherein the method further includes: detecting up to four different responses of the charge sensor in response to different conformational changes of the charged molecule when up to four different labeled nucleotides respectively associate with the polymerase and the identification site, where each of the up to four different responses has a different magnitude; and identifying the different labeled nucleotides that are respectively associated by different magnitudes.

[0023] It should be understood that any features of the method can be combined together in any desired manner. In addition, it should be understood that any combination of the features of the method and / or the sensing system and / or the sensing device can be used together, and / or in combination with any example disclosed herein.

[0024] A fourth aspect disclosed herein is a sensing system, the sensing system including a charge sensor, the charge sensor including: two electrodes and a conductive channel connecting the two electrodes; a charged molecule attached to the conductive channel, wherein the charged molecule: includes an identification site for a marker that reversibly binds a labeled nucleotide; has an unbound favorable conformation associated with an unbound charge configuration; and when the identification site binds to the marker, has a favorable conformation associated with a charge configuration, wherein the charge configuration is different from the unbound charge configuration; and a polymerase attached to at least one of the two electrodes or the substrate, the charge sensor being positioned on the substrate.

[0025] In an example of the fourth aspect, the substrate is a patterned substrate, wherein the charge sensor is positioned in a depression of the patterned substrate, and wherein the polymerase is attached to the surface of the depression.

[0026] It should be understood that any features of the sensing system can be combined together in any desired manner. In addition, it should be understood that any combination of the features of the sensing system and / or the method and / or other sensing systems and / or sensing devices can be used together, and / or in combination with any example disclosed herein.

[0027] A fifth aspect disclosed herein is a sensing device, including a flow cell; and a sensing system integrated into the flow cell, the sensing system including: a charge sensor including a conductive channel; a charged molecule attached to the conductive channel, wherein the charged molecule: has an unbound favorable conformation associated with an unbound charge configuration; and when an identification site of the charged molecule binds to a marker of a labeled nucleotide, has a favorable conformation associated with a charge configuration, wherein the charge configuration is different from the unbound charge configuration; and a polymerase attached to at least one of the two electrodes or the substrate of the flow cell.

[0028] In an example of the fifth aspect, the substrate is a patterned substrate, wherein the charge sensor is positioned in a depression of the patterned substrate, and wherein the polymerase is attached to the surface of the depression.

[0029] It should be understood that any features of the sensing device can be combined together in any desired manner. In addition, it should be understood that any combination of the features of the sensing device and / or the method and / or the sensing system and / or other sensing devices can be used together, and / or in combination with any example disclosed herein.

[0030] In addition, it should be understood that any features of any method and / or any sensing system and / or any sensing device can be combined together in any desired manner and / or can be combined with any of the examples disclosed herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] The features of examples of the present disclosure will become apparent by reference to the following detailed description and the drawings, in which like reference numerals correspond to like but possibly different components. For the sake of brevity, reference numerals or features having a previously described function may be combined with other drawings in which they appear or may not be combined with other drawings in which they appear.

[0033] Figure 1A is a schematic diagram of an example of a sensor disclosed herein when a charged molecule is in its unbound favorable conformation (shown at "(i)") and its favorable conformation (shown at "(ii)");

[0034] Figure 1B is a schematic diagram of another example of a sensor disclosed herein when a charged molecule is in its unbound favorable conformation (shown at "(i)") and its favorable conformation (shown at "(ii)");

[0035] Figure 2 is a schematic diagram of another example of a sensor disclosed herein;

[0036] Figures 3A to 3E is a schematic diagram illustrating another example of a sensor including a charged molecule having four different recognition sites;

[0037] Figure 4 is a schematic perspective view of an example of a sensing system including a flow cell and an example of a sensor disclosed herein;

[0038] Figure 5 schematically illustrates an example of a method disclosed herein;

[0039] Figure 6A and Figure 6B is a graph illustrating a potential response of a sensor disclosed herein;

[0040] Figure 7A is a top view of another example of a flow cell; and

[0041] Figure 7B is located at Figure 7A an enlarged and partially cut-away view of an example of a sensor in the architecture of a flow cell.

[0042] DETAILED DESCRIPTION

[0043] The present disclosure relates to a sensing system that can be used for single molecule detection in a nucleic acid sequencing procedure. The sensing system includes a charged portion attached to a conductive channel of a charge sensor. The charged portion is attached to the conductive channel in such a way that the charged portion is not detected until an event occurs that reconfigures the charged portion to be detectable by the charge sensor (e.g., binding of a target label). More specifically, the charged portion is capable of undergoing reversible binding with a target label that is attached to a nucleotide that can be incorporated by a polymerase. Due to the binding of the target label to the charged portion, the bound charged portion undergoes a conformational change that alters the spatial distribution of the charge. Due to the proximity of the charged portion relative to the conductive channel in the charge sensor, the charge sensor responds to the newly presented charge and generates a detectable signal. The detectable signal is generated even at biologically relevant or physiological concentrations of salt ions, where the Debye screening length is typically less than 1 nm. As an example, a conformation that moves a negatively charged portion closer to the conductive channel can decrease the transconductance, while a conformation that moves a negatively charged portion away from the conductive channel can increase the transconductance. Thus, different conformations of the charged portion result in distinguishable detectable signals. Since the detectable charge resides on the charged portion, the charge does not need to reside on the target label, which can be advantageous.

[0044] In an example disclosed herein, the target label can be customized to be a specific nucleotide that can be incorporated by a polymerase. Since the target label induces the desired conformational change in the charged portion, the resulting signal can be used to identify the specific nucleotide. In addition, in addition to the charge quantity value, the on-and off-rate between the target label and one or more charged portions can be used to generate a unique fingerprint signal that exhibits at a unique frequency, which can be used to identify the corresponding nucleotide attached to the target label.

[0045] Now referring to Figure 1A and Figure 1B , two examples of sensing systems 10, 10' are depicted. Each of the sensing systems 10, 10' includes a charge sensor 11, 11' that includes two electrodes 12, 14 and a conductive channel 16 that connects the two electrodes 12, 14. The sensing systems 10, 10' also include a charged molecule 18 or 18' attached to the conductive channel 16 of the charge sensor 11, 11', and a polymerase 20 attached to the conductive channel 16 ( Figure 1A ) or the charged molecule 18' ( Figure 1B ).

[0046] In other instances of the sensing system, polymerase 20 may be attached to other components of the sensing systems 10, 10' (e.g., attached to electrodes 12 or 14) and / or attached to other components of the flow cell in which the sensing systems 10, 10' are integrated. Polymerase 20 may be attached to any region adjacent to the charge sensors 11, 11' as long as the label of the nucleotide incorporated by polymerase 20 can be reversibly bound to the charged molecule 18 or 18'. In some instances, polymerase 20 is attached within a distance of about 5 nm to about 50 nm of the charged molecule 18 or 18'.

[0047] The charge sensors 11, 11' may be field effect transistors (FETs), such as carbon nanotube (CNT)-based FETs, single-walled carbon nanotube (SWNT)-based FETs, silicon nanowire (SiNW) FETs, silicon nanotube FETs, polymer nanowire FETs, graphene nanoribbon FETs (and related nanoribbon FETs made of 2D materials such as MoS 2 , silicene, etc.), metal oxide semiconductor FETs (MOSFETs), tunnel FETs (TFETs), or any other device having a conductivity that can be modulated by an external field, such as metallic CNTs or multi-walled CNTs. In an FET, the electrodes 12, 14 are the source and drain terminals, and the conductive channel 16 is the gate terminal. The field effect transistor may be a PMOS or p-channel having p-type source and drain terminals in an n-type substrate, or an NMOS or n-channel having n-type source and drain terminals in a p-type substrate.

[0048] The electrodes 12, 14 may include any suitable conductive material. Examples of suitable source and drain materials include cobalt, cobalt silicide, nickel, nickel silicide, aluminum, tungsten, copper, titanium, molybdenum, indium tin oxide (ITO), indium zinc oxide, gold, platinum, carbon, etc.

[0049] The conductive channel 16 may be a nanostructure having at least one nanoscale dimension (ranging from 1 nm to less than 1 μm). In one instance, the at least one dimension refers to the largest dimension.

[0050] The conductive channel 16 may also have any suitable geometry, such as a tubular structure, a wire structure, a planar structure, etc., and may be any suitable semiconductor material or conductive material. As an example, the conductive channel 16 may be selected from the group consisting of semiconductor nanostructures, graphene nanostructures, metal nanostructures, conductive polymer nanostructures, or molecular wires. In some instances, the nanostructure may be a multi-walled nanotube or a single-walled nanotube, a nanowire, a nanoribbon, etc. As a specific example, the nanostructure may be a carbon nanotube, a single-walled carbon nanotube, a silicon nanowire, a silicon nanotube, a polymer nanowire, a graphene nanoribbon, MoS 2Nanoribbons, silicon nanoribbons, etc.

[0051] In systems 10, 10', the charged molecules 18, 18' are covalently or non-covalently attached to the conductive channels 16 of the charge sensors 11, 11'. The charged molecules 18, 18' can be directly bound to the conductive channel 16 or can be indirectly bound to the conductive channel 16 through a tether. The attachment of the charged molecules 18, 18' holds the charged molecules 18, 18' near the conductive channel 16, e.g., within a few Debye lengths. Any suitable charged molecules 18, 18' can be used that can undergo reversible binding to the target marker 24 of the labeled nucleotide 26. More specifically, the charged molecules 18, 18' include an identification site 28 capable of reversibly binding to the marker 24, having an unbound favorable conformation A associated with an unbound charge configuration (see the top portion (labeled (i)) in each of Figure 1A and Figure 1B ), and when the identification site 28 binds to the marker 24, having a favorable conformation B associated with a charge configuration (see the bottom portion (labeled (ii)) in each of Figure 1A and Figure 1B ).

[0052] The term "unbound favorable conformation" refers to a spatial arrangement preferentially presented by the charged molecules 18, 18' when the marker 24 is not bound to the charged molecules 18, 18'. When the marker 24 is not bound, the charged molecules 18, 18' can move dynamically between several different conformations. However, the charged molecules 18, 18' have a preferred spatial arrangement that is more frequently presented than other spatial arrangements when the target marker 24 is not bound. In this example, this preferred spatial arrangement (or preferentially presented spatial arrangement) is the most likely arrangement, e.g., due to molecular stability and / or being in its lowest energy state, and is thus the unbound favorable conformation A. In some cases, the unbound favorable conformation A can be the most stable conformation and / or the lowest energy conformation.

[0053] The unbound favorable conformation A is associated with an unbound charge configuration. The unbound charge configuration is the charge distribution of the charged molecules 18, 18' when they are in their unbound favorable conformation A.

[0054] The term "favorable conformation" refers to a spatial arrangement preferentially presented by the charged molecules 18, 18' when the label 24 binds reversibly to the charged molecules 18, 18'. The favorable conformation B of the charged molecules 18, 18' is different from the unbound favorable conformation A. When the target molecule binds to the charged molecules 18, 18', the charged molecules 18, 18' move to a preferred spatial arrangement which is presented more frequently than other spatial arrangements when the target label is bound. In this example, when the target label is bound, this preferred spatial arrangement is the most likely arrangement, for example due to molecular stability, and is thus the favorable conformation. In one example, the charged molecules 18, 18' have multiple conformations in equilibrium and the label 24 is able to stabilize one of the conformations.

[0055] The favorable conformation B is associated with a charge configuration. The charge configuration is the charge distribution of the charged molecules 18, 18' when they are in their favorable conformation B. The charge configuration associated with the favorable conformation B is detectably different from the unbound charge configuration. The charge configuration can be detected (by a charge sensor) as an increased or decreased magnitude, or a change in frequency, etc.

[0056] As mentioned, the label 24 of the labeled nucleotide 26 is able to bind reversibly to the recognition site 28. Thus, the recognition site 28 is a temporary receptor for the label 24.

[0057] Figure 1A and Figure 1B The top portion (labeled (i)) of Figure 1A and Figure 1B shows the charged molecules 18, 18' in the unbound favorable conformation A. As mentioned herein, the unbound favorable conformation A refers to the preferred orientation or spatial arrangement of the charged molecules 18, 18' when the recognition site 28 does not have the label 24 bound thereto. The unbound conformation A (shown at (i) in Figure 1A and Figure 1B ) is associated with the unbound charge configuration. The unbound charge configuration is the charge distribution of the charged molecules 18, 18' when they are in their unbound favorable conformation A. In Figure 1A and Figure 1B , the centroid of the charge distribution is shown as "·" and the distance between the charge centroid and the surface of the conductive channel 16 is shown as "δ 18A " ( Figure 1A ) and "δ 18’A " ( Figure 1B ). As illustrated in these figures, the charge distribution, centroid, and distances δ 18A , δ 18’A are different for each charged molecule 18, 18' and can change when the charged molecules 18, 18' bind to the target label 24.

[0058] Figure 1A and Figure 1B The bottom portion (labeled (ii)) of Figure 1B shows the charged molecules 18, 18' in the favorable conformation B, i.e., when the target marker 24 binds to the recognition site 28. As mentioned herein, the favorable conformation B (shown at (ii) in Figure 1B and Figure 1A ) refers to the preferred orientation or spatial arrangement of the charged molecules 18, 18' when the marker 24 binds to the recognition site 28. The favorable conformation B is associated with a charge configuration. The charge configuration is the charge distribution of the charged molecules 18, 18' when they are in their favorable conformation B. In Figure 1A (ii), the favorable conformation B of the charged molecule 18 causes the charged molecule 18 to move closer to the surface of the conductive channel 16 when compared to the unbound favorable conformation A. In this example of the favorable conformation B, δ Figure 1A and Figure 1B shown at (ii) in Figure 1B and Figure 1A is the preferred orientation or spatial arrangement of the charged molecules 18, 18' when the marker 24 binds to the recognition site 28. The favorable conformation B is associated with a charge configuration. The charge configuration is the charge distribution of the charged molecules 18, 18' when they are in their favorable conformation B. In Figure 1A (ii), the favorable conformation B of the charged molecule 18 causes the charged molecule 18 to move closer to the surface of the conductive channel 16 when compared to the unbound favorable conformation A. In this example of the favorable conformation B, δ Figure 1A (ii), the favorable conformation B of the charged molecule 18 causes the charged molecule 18 to move closer to the surface of the conductive channel 16 when compared to the unbound favorable conformation A. In this example of the favorable conformation B, δ 18B is less than δ 18A , and the negative or positive charge of the charged molecule 18 is closer to the conductive channel 16. In Figure 1B (ii), the favorable conformation B of the charged molecule 18' causes the charged molecule 18' to move away from the surface of the conductive channel 16 when compared to the unbound favorable conformation A. In this example of the favorable conformation B, δ Figure 1B (ii), the favorable conformation B of the charged molecule 18' causes the charged molecule 18' to move away from the surface of the conductive channel 16 when compared to the unbound favorable conformation A. In this example of the favorable conformation B, δ 18’B is greater than δ 18’A , and the negative or positive charge of the charged molecule 18' is further away from the conductive channel 16. The conductivity of the conductive channel 16 changes in response to the charge movement of the charged molecules 18, 18'. In the examples disclosed herein, the signs of the changes in the two movement directions are opposite to each other; and the actual sign depends on the nature of the sensing systems 10, 10'. As an example, the sign of the response and repulsion or retraction depends on whether the charge sensors 11, 11' are FETs, and if the charge sensors 11, 11' are FETs, whether the FETs are in depletion or inversion mode, and whether the channel carriers are electrons and holes.

[0059] The charged molecules 18, 18' can be charged aptamers, charged proteins, or charged peptides. As used herein, the term "charged aptamer" refers to a structured and charged nucleic acid that is capable of: 1) reversibly binding to a label, and 2) upon reversible binding to the label, changing its favorable conformation and thereby changing the distribution of charge; the term "charged protein" refers to a structured and charged macromolecule that is capable of: 1) reversibly binding to a label, and 2) upon reversible binding to the label, changing its favorable conformation and thereby changing the distribution of charge; and the term "charged peptide" refers to a structured and charged short chain of amino acid monomers linked by peptide (amide) bonds that: 1) reversibly binds to a label, and 2) upon reversible binding to the label, changes its favorable conformation and thereby changes the distribution of charge.

[0060] In some instances, the charged molecules 18, 18' are negatively charged. Examples of suitable negatively charged molecules 18, 18' include negatively charged aptamers, negatively charged proteins, negatively charged peptides, and other negatively charged molecules. Some specific examples of negatively charged aptamers include DNA aptamers, RNA aptamers, or analogs thereof. Some specific examples of negatively charged proteins include HSF1(-17), SHFM1(-21), NFKBIA(-25), RBBP4(-26), APP(-55), PJA2(-87), and many others. Examples of negatively charged peptides include polyglutamates and polyaspartates, as well as more structured peptides such as coiled coils with negatively charged surfaces.

[0061] In some instances, the charged molecules are positively charged. Examples of suitable positively charged molecules 18, 18' include positively charged aptamers, positively charged proteins, positively charged peptides, and other positively charged molecules. Some specific examples of positively charged proteins include H2AFX(+17), PARP1(+21), ELN(+40), TERT(+98), and many others. Examples of positively charged peptides include polylysines and polyarginines, as well as more structured peptides such as coiled coils with positively charged surfaces.

[0062] In an instance, the charged molecules 18, 18' are not polymerases.

[0063] The charged molecules 18, 18' can be attached directly or indirectly and / or via covalent or non-covalent bonds to the conductive channels 16 of the charge sensors 11, 11'. The type of bond formed between the charged molecules 18, 18' and the conductive channels 16 will depend on the molecules 18, 18' and channels 16 used. When aptamers are used as the charged molecules 18, 18', the conductive channels 16 can be silanized to produce amine-terminated silanes, which can bind to thiolated aptamers. Other examples of suitable surface chemistries that can be used to bind the charged molecules 18, 18' can include (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride) (EDC), dibenzocyclooctyne (DBCO), azides that can undergo copper-catalyzed click reactions, etc. In other examples, linkers can be used to attach the charged molecules 18, 18' to the conductive channels 16. The linker can be any of the examples described herein for linker 22.

[0064] In Figure 1A the example shown, the polymerase 20 is immobilized on the conductive channels 16 of the charge sensors 11, 11'. In Figure 1B the example shown, the polymerase 20 is immobilized on the charged molecule 18'. In other examples, the polymerase 20 is immobilized on one of the electrodes 12 or 14 (see Figure 7B ). In still other examples, the polymerase 20 is immobilized on a substrate, such as a substrate that supports the charge sensors 11, 11' (see Figure 7B ). In any case, the polymerase 20 can be immobilized via a linker 22. The linker 22 serves as an anchor for the polymerase 20. Examples of suitable linkers 22 include polyethylene glycol (PEG). In some examples, the linker 22 keeps the polymerase 20 at least 10 nm away from the conductive channels 16 or away from the charged molecule 18. For example, this may be desirable so that conformational changes of the polymerase 20, the charge of the polymerase 20, and / or the charge of the target / template polynucleotide chain held by the polymerase 20 do not interfere with the sensing operation of the charge sensors 11, 11'.

[0065] Any suitable polymerase 20 can be used. Examples include polymerases from family A, such as Bsu polymerase, Bst polymerase, Taq polymerase, T7 polymerase, and many other polymerases; polymerases from family B, such as Phi29 polymerase, Pfu polymerase, KOD polymerase, and many other polymerases; polymerases from family C, such as Escherichia coli DNA PolIII, and many other polymerases; polymerases from family D, such as Pyrococcus furiosus DNA Pol II, and many other polymerases; polymerases from family X, such as DNA Polμ, DNA Polβ, DNA Polσ, and many other polymerases.

[0066] It should be understood that since the target marker 24 binds to the charged molecules 18, 18', the polymerase 20 is not released. Instead, the polymerase 20 remains tethered when and after the binding event occurs, e.g., tethered to the channel 16 or the charged molecules 18, 18' or some other flow cell component.

[0067] In some examples disclosed herein, the charged molecules 18, 18' and the polymerase 20 are different (separate and distinct) entities with different roles / functions that together can achieve single molecule sensing. In an example of single molecule sensing, a signal is detected at the charge sensors 11, 11' when a nucleotide is incorporated into the nascent strand formed along the template strand. In an exemplary nucleotide incorporation event, the polymerase 20 holds the template polynucleotide strand and incorporates a nucleotide into the nascent strand complementary to the nucleotide along the template, while the charged molecules 18, 18' reversibly bind the label (which is attached to the incorporated nucleotide) and undergo a conformational change that produces an identifiable signal at the charge sensors 11, 11'. As mentioned herein, in some examples, it may be desirable to configure the polymerase 20 (e.g., by adjusting the length of the tether 22) such that any conformational change of the polymerase 20 does not interfere with the signal produced by the conformational change of the charged molecules 18, 18'.

[0068] As Figure 1A (ii) and Figure 1B (ii) shows, the labeled nucleotide 26 is introduced into the sensing system 10, 10'. The labeled nucleotide 26 includes a nucleotide 30, a linker molecule 32 attached to the phosphate group of the nucleotide 30, and a recognition site specific marker 24 (also referred to as marker 24 or target marker 24) attached to the linker molecule 32. The labeled nucleotide 26 can be considered a non-natural or synthetic nucleotide since it is structurally or chemically different from a natural nucleotide.

[0069] The nucleotide 30 of the labeled nucleotide 26 can be a natural nucleotide. Natural nucleotides include a nitrogenous heterocyclic base, a sugar, and one or more phosphate groups. Examples of natural nucleotides include, for example, ribonucleotides or deoxyribonucleotides. In ribonucleotides, the sugar is ribose, and in deoxyribonucleotides, the sugar is deoxyribose (i.e., a sugar lacking the hydroxyl group present at the 2'-position in ribose). In an example, the nucleotide 30 is in the form of a polyphosphate as it includes several phosphate groups (e.g., triphosphate (i.e., γ-phosphate), tetraphosphate, pentaphosphate, hexaphosphate (as Figure 5 shown), etc.). The heterocyclic base (i.e., nucleobase) can be a purine base or a pyrimidine base or any other nucleobase analogue. Purine bases include adenine (A) and guanine (G) and their modified derivatives or analogues. Pyrimidine bases include cytosine (C), thymine (T), and uracil (U) and their modified derivatives or analogues. The C-1 atom of deoxyribose binds to N-1 of pyrimidine or N-9 of purine.

[0070] The labeled nucleotide 26 further includes a linker molecule 32. The linker molecule 32 can be any long-chain molecule that can be chemically bonded to the phosphate group of the nucleotide 30 at one end and to the label 24 at the other end. The linker molecule 32 can also be selected such that it will not interact with the polymerase 20. The linker molecule 32 is selected such that it is long enough to allow the label 24 to associate with the recognition site 28 of the charged molecules 18, 18' while, for example, the nucleotide 30 is held by the polymerase 20.

[0071] As an example, the linker molecule 32 can include an alkyl chain, a poly(ethylene glycol) chain, an amido group, a phosphate group, a heterocycle such as triazole, a nucleotide, or a combination thereof. Examples of alkyl chains can include at least 6 carbon atoms, and examples of poly(ethylene glycol) chains can include at least 3 ethylene glycol units.

[0072] The following examples illustrate examples of the labeled nucleotide 26, where the linker molecule 32 includes an alkyl chain, an amide group, a poly(ethylene glycol) chain, and triazole:

[0073]

[0074] The following example illustrates another example of the labeled nucleotide 26, where the linker molecule 32 includes an alkyl chain, an amide group, a poly(ethylene glycol) chain, triazole, and a phosphate group:

[0075]

[0076] The following example illustrates yet another example of the labeled nucleotide 26, where the linker molecule 32 includes an alkyl chain, an amide group, a poly(ethylene glycol) chain, triazole, and a phosphate group:

[0077]

[0078] The following example illustrates another example of a labeled nucleotide 26, where the linker molecule 32 includes an alkyl chain, an amide group, a poly(ethylene glycol) chain, a triazole, a phosphate group, and a polynucleotide chain:

[0079]

[0080] Although several exemplary linker molecules 32 have been described, it should be understood that other linker molecules 32 may be used.

[0081] The recognition site-specific marker 24 is a molecule that can be recognized by charged molecules 18, 18' and can reversibly bind to the charged molecules 18, 18' at the recognition site 28. Examples of suitable recognition site-specific markers 24 include antibiotics such as kanamycin, lividomycin, tobramycin, neomycin, viomycin, streptomycin, etc.; enzyme cofactors such as FMN, NAD, vitamin B12, xanthene, etc.; amino acids such as arginine, citrulline, arginine amide, valine, isoleucine, tryptophan, etc.; and many miscellaneous small molecules such as theophylline, dopamine, sulforhodamine, cellobiose, etc.

[0082] Figure 1A and Figure 1B The figure shows examples of charged molecules 18, 18' that are capable of binding to a target marker 24 of a labeled nucleotide 26. In other examples, a labeled nucleotide 26 may include multiple markers 24 that can bind to a single charged molecule 18, 18' ( Figure 2 ), or a charged molecule 18, 18' may include multiple recognition sites 28, each of which can bind to a corresponding marker 24 of a corresponding nucleotide ( Figures 3A to 3E ).

[0083] In Figure 2 , the charged molecule 18' includes a first recognition site 28A that reversibly attaches to a first marker 24 of a labeled nucleotide 26', and also includes a second recognition site 28B that reversibly attaches to a second marker 24' of a labeled nucleotide 26'. This example includes three different conformational changes - one when the first marker 24 alone is bound, another when the second marker 24' alone is bound, and yet another when both markers 24, 24' are bound simultaneously. In Figure 2In [the context], a specific conformational change of the charged molecule 18’ is achieved by binding two different markers 24, 24’ to two different recognition sites 28A, 28B. As illustrated, a labeled nucleotide 26’ includes both markers 24 and 24’, and these markers 24, 24’ are attached to a nucleotide 30 via respective linker molecules 32 and 32’. Any instance of marker 24 and linker molecule 32 can be used in this instance of labeled nucleotide 26’, as long as markers 24 and 24’ are different from each other and can be recognized by recognition sites 28A and 28B of the charged molecule 18’ either separately or simultaneously. In the illustrated instance, when both markers 24, 24’ are bound, the charged molecule 18’ is in one of its modified conformations B and a detectable signal is produced.

[0084] Although a charged molecule 18’ with a polymerase 20 attached thereto is shown in Figure 2 , it should be understood that the charged molecule 18 and a separately attached polymerase 20 can be used in Figure 2 the illustrated instance.

[0085] In yet another instance not shown in the drawings, the charged molecules 18, 18’ include two recognition sites (e.g., 28A, 28B), either of which can bind to a single marker 24 attached to a nucleotide 30. This instance includes two different favorable conformational changes—one when the marker 24 binds to the first recognition site 28A and another when the marker 24 binds to the second recognition site 28B.

[0086] In Figures 3A to 3E , the charged molecule 18’ includes four different recognition sites 28A, 28B, 28C, 28D, each of which is capable of reversibly binding to a different target marker 24A, 24B, 24C, 24D of a different labeled nucleotide 26A, 26B, 26C, 26D. Although a charged molecule 18’ with a polymerase 20 attached thereto is shown in Figures 3A to 3E , it should be understood that the charged molecule 18 and a separately attached polymerase 20 can be used in Figures 3A to 3E the illustrated instance.

[0087] In this instance, as Figure 3A shown, the charged molecule 18’ includes a first recognition site 28A that reversibly attaches a first marker 24A of a first labeled nucleotide 26A, and has a first favorable conformation B associated with a first charge configuration when the first recognition site 28A binds to the first marker 24A 1 . As Figure 3BAs shown, the charged molecule 18’ also includes a second recognition site 28B for a second label 24B that reversibly attaches a nucleotide 26B with a second label, and when the second recognition site 28B binds to the second label 24B, it has a second favorable conformation B associated with a second charge configuration 2 . As Figure 3C shown, the charged molecule 18’ also includes a third recognition site 28C for a third label 24C that reversibly attaches a nucleotide 26C with a third label, and when the third recognition site 28C binds to the third label 24C, it has a third favorable conformation B associated with a third charge configuration 3 . As Figure 3D shown, the charged molecule 18’ also includes a fourth recognition site 28D for a fourth label 24D that reversibly attaches a nucleotide 26D with a fourth label, and when the fourth recognition site 28D binds to the fourth label 24D, it has a fourth favorable conformation B associated with a fourth charge configuration 4 .

[0088] The unbound favorable conformation A of the charged molecule 18’ is shown in Figure 3E . As depicted, when each of the first recognition site 28A, the second recognition site 28B, the third recognition site 28C, and the fourth recognition site 28C is unbound (i.e., no labeled nucleotides 26A - 26D are bound to sites 28A - 28D), the unbound favorable conformation A of this instance occurs (which is associated with an unbound charge configuration). The centroid of the charge distribution of the unbound favorable conformation A is at a set distance δ from the surface of the conductive channel 16 0 . This distance δ 0 changes according to the labeled nucleotides 26A - 26D that reversibly bind to the charged molecule 18’

[0089] In Figure 3A the instance shown, the labeled nucleotide 26A includes guanine polyphosphate as nucleotide 30A, linker 32A, and a unique label 24A. When polymerase 20 incorporates nucleotide 30A, the effective concentration of the label 24A effectively increases near the charged molecule 18’ (which has a recognition site 24A for the label 24A), resulting in the binding of the charged molecule 18’ to the label 24A. In Figure 3A the instance shown, the binding causes the distance δ between the charge centroid and the channel surface 0 to increase, as indicated by “δ ++ ”.

[0090] In Figure 3BIn the example shown, the labeled nucleotide 26B includes an adenine polyphosphate as nucleotide 30B, a linker 32B, and a unique label 24B. When polymerase 20 incorporates nucleotide 30B, the effective concentration of label 24B effectively increases near the charged molecule 18' (which has an identification site 24B for label 24A), resulting in the binding of the charged molecule 18' to label 24B. In Figure 3B the example shown, the binding results in an increase in the distance δ 0 between the centroid of the charge and the channel surface, as indicated by "δ + ". Although Figure 3A and Figure 3B the favorable conformational changes shown both result in an increased distance δ ++ and δ + , the distances δ ++ and δ + are different and will therefore result in distinguishable measurable signals.

[0091] In Figure 3C the example shown, the labeled nucleotide 26C includes a cytosine polyphosphate as nucleotide 30C, a linker 32C, and a unique label 24C. When polymerase 20 incorporates nucleotide 30C, the effective concentration of label 24C effectively increases near the charged molecule 18' (which has an identification site 24C for label 24C), resulting in the binding of the charged molecule 18' to label 24C. In Figure 3C the example shown, the binding results in a decrease in the distance δ 0 between the centroid of the charge and the channel surface, as indicated by "δ - ".

[0092] In Figure 3D the example shown, the labeled nucleotide 26D includes a thymine polyphosphate as nucleotide 30D, a linker 32D, and a unique label 24D. When polymerase 20 incorporates nucleotide 30D, the effective concentration of label 24D effectively increases near the charged molecule 18' (which has an identification site 24D for label 24D), resulting in the binding of the charged molecule 18' to label 24D. In Figure 3D the example shown, the binding results in a decrease in the distance δ 0 between the centroid of the charge and the channel surface, as indicated by "δ -- ". Although Figure 3C and Figure 3D the favorable conformational changes shown in both result in a decreased distance δ - and δ -- , the distances δ - and δ -- are different and will therefore result in distinguishable measurable signals.

[0093] In Figures 3A to 3E the example shown, a charged molecule 18’ has four different recognition sites 28A - 28D and thus has four different modified configurations which result in four different and distinguishable measurable signals. These different and distinguishable signals enable four different nucleotides 30A - 30D to be identified as they are incorporated into the template strand, respectively.

[0094] Other variants of the multi - recognition - site charged molecule are also contemplated. For example, two charged molecules 18, 18’, each having two different recognition sites 28, can be attached to the conductive channels 16 of the charge sensors 11, 11’. For each of these charged molecules 18, 18’, when the two different recognition sites remain unbound, the unbound favorable conformation A will be presented. In this example, the first of the two charged molecules 18, 18’ includes a first recognition site (e.g., Figure 3A 28A in Figure 3A ) that reversibly attaches a first labeled nucleotide (e.g., Figure 3A 26A in Figure 3A ) and has a first favorable conformation (e.g., 1 B in Figures 3A - 3E ) associated with a first charge configuration when the first recognition site binds to the first labeled nucleotide, and also includes a second recognition site (e.g., Figures 3A - 3E 28B in Figure 3B ) that reversibly attaches a second labeled nucleotide (e.g., Figure 3B 26B in 2 ) and has a second favorable conformation (e.g., Figure 3C B in Figure 3C ) associated with a second charge configuration when the second recognition site binds to the second labeled nucleotide. In this example, the second of the two charged molecules 18, 18’ includes a third recognition site (e.g., Figure 3C 28C in Figure 3D ) that reversibly attaches a third labeled nucleotide (e.g., Figure 3D 26C in Figure 3D ) and a fourth recognition site (e.g., Figure 3C 28D in 3), and when the fourth recognition site binds to the fourth marker, has a fourth favorable conformation associated with the fourth charge configuration (e.g., Figure 3D B in 4 ). In this example, two different charged molecules 18, 18' can be used to identify four different labeled nucleotides 26A - 26D.

[0095] In still other examples, the sensing systems 10, 10' can include a number of charged molecules 18, 18' attached to the conductive channels 16 of the charge sensors 11, 11'. In one example, each charged molecule 18, 18' is capable of reversibly binding to a different labeled nucleotide 26. An example of this sensing system 10" is shown in Figure 4 .

[0096] In this example, four different charged molecules 18A, 18B, 18C, 18D are attached to the conductive channel 16 of the charge sensor 11". In this example, each charged molecule 18A, 18B, 18C, 18D has its own recognition site, unbound favorable conformation, and a favorable conformation (during marker binding) independent of each other charged molecule 18A, 18B, 18C, 18D. More specifically, the first charged molecule 18A attached to the conductive channel 16 includes a first recognition site for a first marker that reversibly binds a first labeled nucleotide, an unbound favorable conformation when the first recognition site is not bound, and a favorable conformation having a charge configuration when the first recognition site is bound to the first marker. In this example, the second charged molecule 18B attached to the conductive channel 16 includes a second recognition site for a second marker that reversibly binds a second labeled nucleotide, an unbound favorable conformation of the second charged molecule, and a favorable conformation of the second charged molecule having the charge configuration of the second charged molecule when the second recognition site binds to the second marker. Also in this example, the third charged molecule 18C attached to the conductive channel 16 includes a third recognition site for a third marker that reversibly binds a third labeled nucleotide, an unbound favorable conformation of the third charged molecule, and a favorable conformation of the third charged molecule having the charge configuration of the third charged molecule when the third recognition site binds to the third marker. Also in this example, the fourth charged molecule 18D attached to the conductive channel 16 includes a fourth recognition site for a fourth marker that reversibly binds a fourth labeled nucleotide, an unbound favorable conformation of the fourth charged molecule, and a favorable conformation of the fourth charged molecule having the charge configuration of the fourth charged molecule when the fourth recognition site binds to the fourth marker.

[0097] As Figure 4As shown, a single polymerase 20 can bind to the conductive channel 16. In this example, the length of each linker molecule 32 of the corresponding labeled nucleotide 26 can be selected such that when the corresponding nucleotide 30 is held by the polymerase 20, the corresponding label 24 can bind to its corresponding charged molecule 18A, 18B, 18C, 18D without binding to adjacent charged molecules 18A, 18B, 18C, 18D.

[0098] Figure 4 An example of a sensing device 40 is also shown. Figure 4 The illustrated example of the sensing device 40 includes a flow cell 41 and a sensing system 10” integrated into the flow cell 41. It should be understood that any example of the sensing systems 10, 10’, 10” can be used in the sensing device 40.

[0099] The flow cell 41 is a container that contains the sensing system 10”. It should be understood that other containers, such as wells, tubes, channels, cuvettes, petri dishes, bottles, etc. can optionally contain the sensing system 10”. Cyclic processes, such as nucleic acid sequencing reactions, are particularly well-suited to the flow cell 41.

[0100] An exemplary flow cell 41 includes a substrate / support 13 and a lid 43 directly or indirectly bonded thereto or integrally formed therewith. The flow cell 41 can include a fluid inlet 45 and a fluid outlet 47 that enable the delivery of bulk reagents to one sensing system 10” or an array of sensing systems 10” contained within the flow cell 41. Any individual flow cell 41 can include dozens, hundreds, thousands, millions, or even billions of individually addressable and readable sensing systems 10, 10’, 10”.

[0101] Figure 7A and Figure 7B The example shown in and is an example of a flow cell 41’ that includes an array of sensing systems 10, 10’, 10”. The array can include a number of sensing systems 10, 10’, 10”, each of which is positioned on the substrate and configured with electronic circuitry such that it is individually addressable and readable. In the example, each sensing system 10, 10’, 10” of the array can be positioned on the substrate in a separate recess. The recesses physically separate each sensing system 10, 10’, 10”.

[0102] In Figure 7AIn an example, the flow cell 41’ includes a flow channel 52. Although several flow channels 52 are shown, it should be understood that any number of channels 52 (e.g., a single channel 52, four channels 52, etc.) may be included in the flow cell 41’. Each flow channel 52 is a region defined between two joined components (e.g., a substrate and a lid or two substrates) into which a fluid (e.g., those described herein) may be introduced and removed. Each flow channel 52 may be isolated from each other flow channel 52 such that the fluid introduced into any particular flow channel 52 does not flow into any adjacent flow channel 52. Some examples of fluids introduced into the flow channel 52 may include reaction components (e.g., labeled nucleotides 26, etc.), wash solutions, etc.

[0103] An example of the architecture within the flow channel 52 of the flow cell 41’ is shown in Figure 7B In the example shown in Figure 7B , the flow cell 41’ includes a substrate 13 that includes a support 54 and a patterned material 56 positioned on the support 54. The patterned material 56 defines recesses 58 separated by gap regions 60. In this example, the surface of the support 54 is exposed at each recess 58, and the sensing systems 10, 10’, 10” are positioned within each recess 58.

[0104] Figure 7B The support 54 in provides support for the other components of the flow cell 41’. The support 54 is generally rigid and insoluble in aqueous liquids. Some examples of suitable supports 54 include epoxy siloxanes, glass, modified glass, plastics, nylon, ceramics / ceramic oxides, silica (silicon oxide (SiO 2 )), fused silica, silica-based materials, aluminosilicates, silicon, modified silicon (e.g., boron-doped p+ silicon), silicon nitride (Si 3 N 4 ), tantalum pentoxide (TaO 5 ), or other tantalum oxides (TaO x ), hafnium oxide (HaO 2 ), inorganic glass, etc. Some examples of suitable plastics for the support 54 include acrylics, polystyrene, copolymers of styrene and other materials, polypropylene, polyethylene, polybutene, polyurethane, polytetrafluoroethylene (e.g., from Chemours ), cycloolefin / cycloolefin polymers (COP) (e.g., from Zeon ), polyimides, etc. The support 54 may also be glass or silicon with a coating of tantalum oxide or another ceramic oxide on the surface.

[0105] The support 54 can be in the form of a wafer, panel, rectangular sheet, die, or any other suitable configuration. In an example, the support 54 can be a circular wafer or panel having a diameter in the range from about 2 mm to about 300 mm. As a more specific example, the support 54 is a wafer having a diameter in the range from about 200 mm to about 300 mm. In another example, the support 54 can be a rectangular sheet or panel having a maximum dimension up to about 10 feet (~3 meters). As a specific example, the support 54 is a die having a width in the range from about 0.1 mm to about 10 mm. Although exemplary dimensions have been provided, it should be understood that the support 54 having any suitable dimensions can be used.

[0106] In Figure 7B the illustrated example, the patterned material 56 is positioned on the support 54. It should be understood that any material that can be selectively deposited or deposited and patterned to form the recess 58 and the gap region 60 can be used for the patterned material 56.

[0107] As an example, an inorganic oxide can be selectively applied to the support 66 by chemical vapor deposition, aerosol printing, or inkjet printing. Examples of suitable inorganic oxides include tantalum oxide (e.g., Ta 2 O 5 ), aluminum oxide (e.g., Al 2 O 3 ), silicon oxide (e.g., SiO 2 ), hafnium oxide (e.g., HfO 2 ), etc.

[0108] As another example, a resin can be applied to the support 54 and then patterned. Suitable deposition techniques include chemical vapor deposition, dip coating, immersion coating, spin coating, spraying, puddle dispensing, ultrasonic spraying, doctor blade coating, aerosol printing, screen printing, microcontact printing, etc. Suitable patterning techniques include photolithography, nanoimprint lithography (NIL), stamping techniques, embossing techniques, molding techniques, microetching techniques, printing techniques, etc. Some examples of suitable resins include polyhedral oligomeric silsesquioxane (POSS)-based resins, non-POSS epoxy resins, poly(ethylene glycol) resins, polyether resins (e.g., ring-opening epoxy resins), acrylic resins, acrylate resins, methacrylate resins, amorphous fluoropolymer resins (e.g., from Bellex ), and combinations thereof.

[0109] As used herein, the term "polyhedral oligomeric silsesquioxane" (POSS) refers to a compound that is intermediate between silica (SiO 2 ) and organosilicon (R 2Hybrid intermediates between SiO) (e.g., RSiO 1.5 ), the chemical composition of which. Examples of POSS can be those described in Kehagias et al., Microelectronic Engineering 86 (2009), pages 776 - 778, which is incorporated herein by reference in its entirety. In an example, the composition is a silicone compound having the chemical formula [RSiO 3 / 2 n , where the R groups can be the same or different. Exemplary R groups for POSS include epoxy, azide / azido, thiol, poly(ethylene glycol), norbornene, tetrazine, acrylate, and / or methacrylate, or further, for example, alkyl, aryl, alkoxy, and / or haloalkyl groups. The resin compositions disclosed herein can include one or more different cage or core structures as monomer units. The polyhedral structure can be a T 8 structure, for example: And is represented by . The monomer unit typically has eight functional group arms R 1 to R 8 .

[0110] The monomer unit can have a cage structure with 10 silicon atoms and 10 R groups, which is called T 10 , for example: Or can have a cage structure with 12 silicon atoms and 12 R groups, which is called T 12 , for example: POSS - based materials can alternatively include T 6 , T 14 or T 16 cage structures. The average cage content can be adjusted during synthesis and / or controlled by purification methods, and the distribution of the cage sizes of the monomer units can be used in the examples disclosed herein.

[0111] As Figure 7B ​As shown, the patterned material 56 includes recesses 58 defined therein and interstitial regions 60 separating adjacent recesses 58. Many different layouts of the recesses 58 can be envisioned, including regular patterns, repeating patterns, and irregular patterns. In an example, the recesses 58 are arranged in a hexagonal grid for close packing and increased density. Other layouts can include, for example, a linear (rectangular) layout, a triangular layout, etc. In some examples, the layout or pattern can be in an x-y format of recesses 58 in rows and columns. In some other examples, the layout or pattern can be a repeating arrangement of recesses 58 and / or interstitial regions 60. In still other examples, the layout or pattern can be a random arrangement of recesses 58 and / or interstitial regions 60. The pattern can include spots, pads, holes, pillars, stripes, swirls, lines, triangles, rectangles, circles, arcs, checkerboards, lattices, diagonals, arrows, squares, and / or cross-hatching.

[0112] The layout or pattern of the recesses 58 can be characterized relative to the density (number of recesses 58) of the recesses 58 in the defined region. For example, the recesses 58 can be present at a density of about 2 million / mm 2 . The density can be adjusted to different densities, including, for example, about 100 / mm 2 , about 1,000 / mm 2 , about 100,000 / mm 2 , about 1 million / mm 2 , about 2 million / mm 2 , about 5 million / mm 2 , about 10 million / mm 2 , about 50 million / mm 2 or more or less density. It should also be understood that the density of the recesses 58 in the patterned material 56 can be between one of the lower values and one of the higher values selected from the ranges above. As an example, a high-density array can be characterized as having recesses 58 with a spacing of less than about 100 nm, a medium-density array can be characterized as having recesses 58 with a spacing of about 400 nm to about 1 μm, and a low-density array can be characterized as having recesses 58 with a spacing greater than about 1 μm. Although exemplary densities have been provided, it should be understood that any suitable density can be used.

[0113] The layout or pattern of the recesses 58 can also or alternatively be characterized according to the average spacing or the separation from the center of a recess 58 to the center of an adjacent recess 58 (center-to-center separation) or the separation from the edge of one recess 58 to the edge of an adjacent recess 58 (edge-to-edge separation). The pattern can be regular such that the coefficient of variation about the average spacing is small, or the pattern can be irregular, in which case the coefficient of variation can be relatively large. In either case, the average spacing can be, for example, about 50 nm, about 0.1 μm, about 0.5 μm, about 1 μm, about 5 μm, about 10 μm, about 100 μm or greater or less. The average spacing of a particular pattern of recesses 58 can be between one of the lower values and one of the higher values selected from the above ranges. In an example, the recesses 58 have a spacing (center-to-center separation) of about 1.5 μm. Although exemplary average spacing values have been provided, it should be understood that other average spacing values can be used.

[0114] The size of each recess 58 can be characterized by its volume, depth, and / or diameter.

[0115] Each recess 58 can have any volume capable of confining a fluid. A minimum or maximum volume can be selected, for example, to accommodate the throughput (e.g., multiplexity), resolution, labeled nucleotide 26, or analyte reactivity expected for a downstream use of the flow cell 41'. For example, the volume can be at least about 1×10 -3 μm 3 、at least about 1×10 -2 μm 3 、at least about 0.1 μm 3 、at least about 1 μm 3 、at least about 10 μm 3 、at least about 100 μm 3 or greater. Optionally or additionally, the volume can be at most about 1×10 4 μm 3 、at most about 1×10 3 μm 3 、at most about 100 μm 3 、at most about 10 μm 3 、at most about 1 μm 3 、at most about 0.1 μm 3 or less.

[0116] The depth of each recess 58 can be large enough to accommodate a sensing system 10, 10', 10". In an example, the depth can be at least about 1 μm, at least about 10 μm, at least about 100 μm or greater. Optionally or additionally, the depth can be at most about 1×10 3μm, up to about 100 μm, up to about 10 μm or less. The depth of each recess 58 can be greater than, less than, or between the values specified above.

[0117] In some cases, the diameter or length and width of each recess 58 can be at least about 50 nm, at least about 0.1 μm, at least about 0.5 μm, at least about 1 μm, at least about 10 μm, at least about 100 μm or greater. Optionally or additionally, the diameter or length and width can be up to about 1×10 3 μm, up to about 100 μm, up to about 10 μm, up to about 1 μm, up to about 0.5 μm, up to about 0.1 μm or less (e.g., about 50 nm). The diameter or length and width of each recess 58 can be greater than, less than, or between the values specified above.

[0118] As Figure 7B shown, each recess 58 in the array includes a corresponding charge sensor 11, 11', 11". It is desirable for each charge sensor 11, 11', 11" in each recess 58 to have a charged molecule 18, 18' attached thereto, and to have a polymerase 20 attached nearby. In some instances, each recess 58 has a charge sensor 11, 11', 11", a charged molecule 18, 18 and a polymerase 20 therein. In other instances, some recesses 58 have a charge sensor 11, 11', 11", a charged molecule 18, 18 and a polymerase 20 therein; while other recesses 58 have a charge sensor 11, 11', 11", a charged molecule 18, 18 and more than one polymerase 20 therein; and still other recesses 58 have a charge sensor 11, 11', 11", a charged molecule 18, 18, and no polymerase 20 therein. In these instances, the number of polymerases 20 that become attached within any given recess 58 can be random and determined by a Poisson distribution.

[0119] In some instances, the charge sensors 11, 11', 11" with the charged molecules 18, 18' attached thereto can be pre-assembled in the recesses 58. To attach the polymerase 20 within the corresponding recesses 58, a fluid containing the polymerase 20 can be introduced into each lane 52 of the flow cell 41'. The polymerase 20 can include a tether 22 attached within the recess 58, or the linker 22 can be pre-attached within the recess 58, and the polymerase 20 can be attached to the linker 22. The fluid can be allowed to incubate for a desired time and at a desired temperature that permits the attachment of the polymerase 20.

[0120] As Figure 7BAs depicted, polymerase 20 can be attached to any component and / or any surface within flow cell 41'. In some instances, polymerase 20 is attached to electrode 12 or 14, the surface of substrate 13 (e.g., the bottom of recess 58, the sidewall of recess 58, etc.), on conductive channel 16, on charged molecules 18, 18', etc.

[0121] Each of charge sensors 11, 11', 11'' is individually electrically addressable and readable. Thus, the signals generated by conformational changes of charged molecules occurring within each recess 58 can be individually detected and analyzed.

[0122] Any instance of sensing device 40 can also include a reagent delivery system 49 to selectively introduce a reagent into the input end of flow cell 41 (e.g., fluid inlet 45) or into lane 52 of flow cell 41', across sensing systems 10, 10', 10'', and then out through fluid outlet 47. Reagent delivery system 49 can include tubing or other fluidics that can be permanently or removably attached to fluid inlet 45. Reagent delivery system 49 can include sample container 51. The reagent (including any instance of labeled nucleotide 26 to be introduced into sensing system 10'') can be stored in the sample container or prepared and introduced into the sample container just prior to use. Reagent delivery system 49 can also include a pump or other suitable device to retrieve the reagent from sample container 51 and deliver it to fluid inlet 45. In other instances, sample container 51 is positioned such that the reagent can flow by gravity to fluid inlet 45, across sensing system 10'', and out through fluid outlet 47.

[0123] When using sensing systems 10, 10', 10'' and sensing device 40, charge sensors 11, 11', 11'' in flow cells 41, 41' can also be operatively connected to detector 15 to detect conductance changes of charge sensors 11, 11', 11''.

[0124] The sensing systems 10, 10', 10'' disclosed herein can be used in sensing methods. An example of the method is schematically shown in Figure 5 and includes:

[0125] introducing a template polynucleotide strand 48 into sensing systems 10, 10', 10'', the sensing systems 10, 10', 10'' including: charge sensors 11, 11', 11'' that include two electrodes 12, 14 and a conductive channel 16 connecting the two electrodes 12, 14; charged molecules 18, 18' attached to conductive channel 16, wherein the charged molecules 18, 18' include recognition sites 28; and a polymerase 20 attached to conductive channel 16 or charged molecules 18, 18';

[0126] A reagent comprising the labeled nucleotide 26 is introduced into the sensing systems 10, 10', 10", whereby the nucleotide 30 of one of the labeled nucleotides 26 associates with the polymerase 20, and the recognition site specific label 24 of one of the labeled nucleotides 26 associates with the recognition site 28 to induce a conformational change in the charged molecules 18, 18'; and

[0127] In response to the conformational change of the charged molecules 18, 18', the response of the charge sensors 11, 11', 11" is detected.

[0128] The template polynucleotide strand 48 can be any sample to be sequenced and can comprise DNA, RNA, or an analogue thereof (e.g., peptide nucleic acid). The source of the template (or target) polynucleotide strand 48 can be genomic DNA, messenger RNA, or other nucleic acids from natural sources. In some cases, the template polynucleotide strand 48 derived from such sources can be amplified prior to use in the methods or systems 40 described herein. Any of a variety of known amplification techniques can be used, including but not limited to polymerase chain reaction (PCR), rolling circle amplification (RCA), multiple displacement amplification (MDA), or recombinase polymerase amplification (RPA). It should be understood that amplification of the template polynucleotide strand 48 prior to use in the methods or systems 40 described herein is optional. Thus, in some embodiments, the template polynucleotide strand 48 will not be amplified prior to use. The template / target polynucleotide strand 48 can optionally be derived from a synthetic library. The synthetic nucleic acid can have a natural DNA or RNA composition or can be an analogue thereof.

[0129] Biological samples from which the template polynucleotide strand 48 can be derived include, for example, biological samples from: mammals such as rodents, mice, rats, rabbits, guinea pigs, ungulates, horses, sheep, pigs, goats, cows, cats, dogs, primates, humans or non-human primates; plants such as Arabidopsis thaliana, corn, sorghum, oats, wheat, rice, rapeseed or soybeans; algae such as Chlamydomonas reinhardtii; nematodes such as Caenorhabditis elegans; insects such as Drosophila melanogaster, mosquitoes, fruit flies, bees or spiders; fish such as zebrafish; reptiles; amphibians such as frogs or Xenopus laevis; Dictyostelium discoideum; fungi such as Pneumocystis carinii, Takifugu rubripes, yeast, Saccharomyces cerevisiae or Schizosaccharomyces pombe; or Plasmodium falciparum. The template polynucleotide strand 48 can also be derived from prokaryotes such as bacteria, Escherichia coli, staphylococci or Mycoplasma pneumoniae; archaea; viruses such as hepatitis C virus, Ebola virus or human immunodeficiency virus; or viroids. The template polynucleotide strand 48 can be derived from a homogeneous culture or population of the above organisms, or alternatively from a collection of several different organisms in, for example, a community or ecosystem.

[0130] In addition, the template polynucleotide strand 48 may not be from a natural source, but can be synthesized using known techniques. For example, gene expression probes or genotyping probes can be synthesized and used in the examples described herein.

[0131] In some instances, the template polynucleotide strand 48 can be obtained as a fragment of one or more larger nucleic acids. Fragmentation can be performed using any of a variety of techniques known in the art, including, for example, nebulization, sonication, chemical cleavage, enzymatic cleavage, or physical shearing. Fragmentation can also result from the use of specific amplification techniques that produce amplicons by replicating only a portion of a larger nucleic acid strand. For example, PCR amplification produces fragments having a size determined by the length of the nucleotide sequence on the original template between the positions where the flanking primers hybridize during amplification. The length of the template polynucleotide strand 48 can be based on the number of nucleotides or on a metric length (e.g., nanometers).

[0132] A population of template / target polynucleotide strands 48 or their amplicons can have an average strand length that is desired or appropriate for a particular application of the methods or systems 40 described herein. For example, the average strand length can be less than about 100,000 nucleotides, about 50,000 nucleotides, about 10,000 nucleotides, about 5,000 nucleotides, about 1,000 nucleotides, about 500 nucleotides, about 100 nucleotides, or about 50 nucleotides. Optionally or additionally, the average strand length can be greater than about 10 nucleotides, about 50 nucleotides, about 100 nucleotides, about 500 nucleotides, about 1,000 nucleotides, about 5,000 nucleotides, about 10,000 nucleotides, about 50,000 nucleotides, or about 100,000 nucleotides. The average strand length of a population of target polynucleotide strands 48 or their amplicons can be within a range between the maximum and minimum values set forth above.

[0133] In some cases, a population of template / target polynucleotide strands 48 can be generated or otherwise configured under certain conditions to have a maximum length for its members. For example, the maximum length of the members can be less than about 100,000 nucleotides, about 50,000 nucleotides, about 10,000 nucleotides, about 5,000 nucleotides, about 1,000 nucleotides, about 500 nucleotides, about 100 nucleotides, or about 50 nucleotides. Optionally or additionally, a population of template polynucleotide strands 48 or their amplicons can be generated or otherwise configured under certain conditions to have a minimum length for its members. For example, the minimum length of the members can be greater than about 10 nucleotides, about 50 nucleotides, about 100 nucleotides, about 500 nucleotides, about 1,000 nucleotides, about 5,000 nucleotides, about 10,000 nucleotides, about 50,000 nucleotides, or about 100,000 nucleotides. The maximum and minimum strand lengths of the template polynucleotide strands 48 in the population can be within a range between the maximum and minimum values set forth above.

[0134] As Figure 5 shown, the template polynucleotide strand 48 introduced into the sensing system 10 (or 10', 10") can be held in place by the polymerase 20, which in this example is tethered to the conductive channel 16. Figure 5 The template polynucleotide strand 48 shown in is a template strand of DNA. The template polynucleotide strand 48 can be introduced into a biostable solution together with reagents such as labeled nucleotides 26. The biostable solution can be any buffer suitable for polymerase base incorporation reactions such as polymerase chain reaction (PCR) or linear amplification. As an example, the biostable solution can include a buffer having a pH close to 7, a salt concentration above a few millimolar, and millimolar concentrations of Mg 2+ ions.

[0135] Also as Figure 5 shown, the labeled nucleotide 26 can include a base complementary to the target nucleic acid of the template polynucleotide strand 48. The labeled nucleotide 26 will be held in place in part by the polymerase 20, which also binds to the template polynucleotide strand 48. As an example, the polymerase 20 can incorporate a specific nucleotide 30, which can remain for a period of time ranging from a few milliseconds (e.g., 2 milliseconds) to several hundred milliseconds.

[0136] The interaction between the labeled nucleotide 26 and the polymerase 20 and the length of the linker molecule 32 enable the target label 24 to associate near the charged molecule 18. When the sensing systems 10, 10', 10" are present in an array and include individually addressable and individually readable charge sensors 11, 11', 11", it should be understood that once the labeled nucleotide 26 interacts with the polymerase 20 of a particular sensing system 10, 10', 10", the length of the linker molecule 32 can also prevent any individual target label 24 from associating with adjacent sensing systems 10, 10', 10".

[0137] In some instances, the association of the target marker 24 results in an increase in the effective concentration of the marker 24, leading to the binding of the charged molecule 18 to the target marker 24. The charged molecule 18 can dynamically change its conformation at equilibrium and, in the absence of the target marker 24, can spend most of its time in a particular conformation (i.e., the unbound favorable conformation). The binding of the target marker 24 will cause the charged molecule 18 to move to a different favorable conformation (from the unbound favorable conformation). The favorable conformation during binding is different from the unbound favorable conformation (e.g., the conformation most exhibited by the charged molecule 18 in the absence of the bound marker 24). The charge distribution in the unbound favorable conformation is different from the charge distribution in the favorable conformation (e.g., when the charged molecule 18 is bound to the marker 24). The change in the charge distribution of the charged molecule 18 in turn changes the conductance in the channel 16.

[0138] The responses of the charge sensors 11, 11', 11'' can indicate the base of the labeled nucleotide 26 incorporated because the target marker 24 is nucleotide-specific (i.e., a particular marker 24 is selected for a particular base) and because the recognition site 28 of the charged molecule 18 is marker-specific. Thus, the method can also include correlating the responses of the charge sensors 11, 11', 11'' with the associated recognition-site-specific marker 24 (i.e., the marker 24 that has changed the conformation of the charged molecule 18) and identifying, based on the associated recognition-site-specific marker 24, the nucleotide (e.g., base) of the associated labeled nucleotide 26 (i.e., the labeled nucleotide 26 that has been associated with the polymerase 20 and the recognition site 28).

[0139] It should be understood that the binding rate and dissociation rate between the charged molecules 18, 18' and the marker 24 can be adjusted such that a unique fingerprint signal is generated.

[0140] For a marker 24 with a slow dissociation rate, the marker 24 will remain bound for a significant duration, e.g., throughout the nucleotide incorporation cycle. This extended binding will produce a change in the DC level of the current flowing through the channel 16 of the charge sensors 11, 11'. This is in Figure 6Ais schematically shown, where different markers 24 with slow dissociation rates are used for four different nucleotides, generating four different and distinguishable detectable signals. These signals can be detected by a single charged molecule 18, 18' having four different recognition sites 28; or by up to four different charged molecules 18, 18', each charged molecule 18, 18' having a marker-specific recognition site 28; or by a single charged molecule 18, 18' having a single recognition site 28, which single recognition site 28 can bind up to four different nucleotides at different slow dissociation rates.

[0141] For markers 24 having fast association and dissociation rates, the marker 24 can associate / dissociate with the charged molecules 18, 18' multiple times during the entire nucleotide incorporation cycle. This rapid association and dissociation binding will generate a signal similar to a tremor (e.g., DC level, amplitude, frequency, percentile level, characteristic distribution, etc.) from the charge sensors 11, 11'. This is shown schematically in Figure 6B is schematically shown, where different markers 24 with fast association and dissociation rates are used for four different nucleotides, generating four different and distinguishable detectable signals. These signals can be detected by a single charged molecule 18, 18' having four different recognition sites 28; or by up to four different charged molecules 18, 18', each charged molecule 18, 18' having a marker-specific recognition site 28; or by a single charged molecule 18, 18' having a single recognition site 28, which single recognition site 28 can bind one to four different nucleotides at different association and dissociation rates.

[0142] The frequency of conformational state changes of the charged molecules 18, 18' can also be monitored.

[0143] The magnitude of the charge sensor response can also be different. In some instances, the recognition site 28 reversibly binds up to four different labeled nucleotides 26. When one of the four different labeled nucleotides 26 associates with the polymerase 20 and the recognition site 28, the response of the charge sensors 11, 11', 11" has a different magnitude that can be used to identify one of the four different labeled nucleotides 26. Each of the four different labeled nucleotides 26 can also have a different magnitude (e.g., a magnitude different from the magnitude associated with each of the other four different labeled nucleotides 26).

[0144] In other instances, from Figure 6A and Figure 6BThe modalities can be combined in some form. For example, among the labeled nucleotides 26 exposed to the sensing systems 10, 10', 10'', some labels 24 with slow binding and dissociation rates can be used, and other labels 24 with fast binding and dissociation rates can be used.

[0145] As a result of the incorporation cycles described herein, the bases of the relevant labeled nucleotides 26 will be incorporated into the nascent strand 50 hybridized to the template polynucleotide chain 48. When the base is fully incorporated and the sugar backbone of the nascent strand 50 is extended, the linker 32 between the nucleotide 30 and the label 24 is cleaved naturally. This results in the effective concentration of the label 24 being reduced back to the background level. The target label 24 dissociates, and the charged molecules 18, 18' return to their unbound (sometimes referred to as "wild-type") conformation, in which it preferentially exhibits a favorable unbound conformation.

[0146] The methods disclosed herein can be repeated for a desired number of sequencing cycles.

[0147] The labeled nucleotides 26 and sensing systems 10, 10', 10'' disclosed herein can be used for any of a variety of applications. As referenced Figure 5 As described, a particularly useful application is nucleic acid sequencing, such as sequencing by synthesis (SBS). In SBS, the extension of a nucleic acid sequencing primer along a template nucleic acid 48 is monitored to determine the sequence of nucleotides in the template. The underlying chemical process can be polymerization (e.g., as catalyzed by polymerase 20 as described herein). In a particular polymerase-based SBS instance, nucleotides (e.g., bases) are added to the sequencing primer in a template-dependent manner (thereby extending the sequencing primer) such that detection of the order and type of nucleotides added to the primer to form the nascent strand can be used to determine the sequence of the template. Multiple different templates 48 at different sensing systems 10, 10', 10'' in the array can undergo SBS techniques. The events occurring at different templates 48 can be distinguished in part due to the location of the specific sensing systems 10, 10', 10'' in the array. The charge sensors 11, 11' of each sensing system 10, 10', 10'' in the array can be individually addressable and readable, and thus can detect the signal at each sensor 11, 11'.

[0148] Other suitable applications of the labeled nucleotides 26 and sensing systems 10, 10', 10'' disclosed herein include ligation sequencing and hybridization sequencing.

[0149] It should be understood that all combinations of the foregoing concepts and additional concepts discussed in more detail below (provided that such concepts are not mutually inconsistent) are considered to be part of the inventive subject matter disclosed herein. In particular, all combinations of the claimed subject matter appearing at the end of this disclosure are considered to be part of the inventive subject matter disclosed herein. It should also be understood that terms explicitly used herein that may also appear in any disclosure incorporated by reference should be given the meaning most consistent with the particular concepts disclosed herein.

[0150] References throughout the specification to "one example", "another example", "examples", etc., mean that a particular element (e.g., feature, structure, and / or property) described in connection with the example is included in at least one example described herein, and may or may not be present in other examples. In addition, it should be understood that, in various examples, the elements described for any example may be combined in any suitable manner, unless the context clearly dictates otherwise.

[0151] The terms "substantially" and "about" as used throughout this disclosure, including the claims, are used to describe and account for small fluctuations, such as due to variations in processing. For example, they may refer to less than or equal to ±5%, such as less than or equal to ±2%, such as less than or equal to ±1%, such as less than or equal to ±0.5%, such as less than or equal to ±0.2%, such as less than or equal to ±0.1%, such as less than or equal to ±0.05%.

[0152] In addition, it should be understood that the ranges provided herein include the stated range and any value or sub-range within the stated range, as if they were explicitly recited. For example, a range represented as from 1 nm to less than 1 μm should be interpreted to include not only the explicitly recited limits of from 1 nm to less than 1 μm, but also individual values such as about 15 nm, 22.5 nm, 45 nm, etc., and sub-ranges such as from about 20 nm to about 48 nm, etc.

[0153] Although several examples have been described in detail, it should be understood that the disclosed examples may be modified. Accordingly, the foregoing description is considered to be non-limiting.

Claims

1. A sensing system, comprising: a charge sensor, the charge sensor comprising: two electrodes; a conductive channel connecting the two electrodes; a polymerase attached to the conductive channel by a first tether, the polymerase holding a template polynucleotide chain to be sequenced; and a charged molecule, which is separate and distinguishable from the polymerase, the charged molecule i) being directly attached to the conductive channel or indirectly attached to the conductive channel via a second tether and ii) attached within a distance of 5 nm to 50 nm from the polymerase, wherein the charged molecule: comprises an identification site for a marker that reversibly binds a labeled nucleotide incorporated into the template polynucleotide chain, wherein the marker is selected from the group consisting of antibiotics, amino acids, and small molecules, and the small molecules are selected from the group consisting of theophylline, dopamine, sulforhodamine, and cellobiose; has an unbound favorable conformation associated with an unbound charge configuration; when the identification site binds to the marker, has a favorable conformation associated with a charge configuration different from the unbound charge configuration; and is a charged aptamer selected from the group consisting of DNA aptamers, RNA aptamers, and their analogs.

2. The sensing system according to claim 1, wherein: the charged molecule: further comprises a second identification site for a second marker that reversibly binds a second labeled nucleotide, and when the second identification site binds to the second marker, has a second favorable conformation associated with a second charge configuration; further comprises a third identification site for a third marker that reversibly binds a third labeled nucleotide, and when the third identification site binds to the third marker, has a third favorable conformation associated with a third charge configuration; and further comprises a fourth identification site for a fourth marker that reversibly binds a fourth labeled nucleotide, and when the fourth identification site binds to the fourth marker, has a fourth favorable conformation associated with a fourth charge configuration; and when each of the identification site, the second identification site, the third identification site, and the fourth identification site is unbound, the unbound favorable conformation associated with the unbound charge configuration occurs.

3. The sensing system according to claim 1, further comprising: a second charged molecule attached to the conductive channel, wherein the second charged molecule: comprises a second identification site for a second marker that reversibly binds a second labeled nucleotide; has an unbound favorable conformation of the second charged molecule associated with an unbound charge configuration of the second charged molecule; and when the second identification site binds to the second marker, has a favorable conformation of the second charged molecule associated with a charge configuration of the second charged molecule.

4. The sensing system according to claim 3, further comprising: a third charged molecule attached to the conductive channel, wherein the third charged molecule: comprises a third identification site for a third marker that reversibly binds a third labeled nucleotide; An unbound favorable conformation of the third charged molecule related to the unbound charge configuration of the third charged molecule; and When the third recognition site binds to the third marker, a favorable conformation of the third charged molecule related to the charge configuration of the third charged molecule; and A fourth charged molecule attached to the conductive channel, wherein the fourth charged molecule: Includes a fourth recognition site for a fourth marker that reversibly binds a fourth labeled nucleotide; Has an unbound favorable conformation of the fourth charged molecule related to the unbound charge configuration of the fourth charged molecule; and When the fourth recognition site binds to the fourth marker, a favorable conformation of the fourth charged molecule related to the charge configuration of the fourth charged molecule.

5. The sensing system according to claim 1, wherein: The charged molecule further includes a second recognition site for a second marker that reversibly binds a second labeled nucleotide, and when the second recognition site binds to the second marker, has a second favorable conformation related to the second charge configuration; and The sensing system further includes a second charged molecule attached to the conductive channel, wherein the second charged molecule: Includes: A third recognition site for a third marker that reversibly binds a third labeled nucleotide; and A fourth recognition site for a fourth marker that reversibly binds a fourth labeled nucleotide; Has an unbound favorable conformation of the second charged molecule related to the unbound charge configuration of the second charged molecule; When the third recognition site binds to the third marker, has a third favorable conformation related to the third charge configuration; and When the fourth recognition site binds to the fourth marker, has a fourth favorable conformation related to the fourth charge configuration.

6. The sensing system according to claim 1, wherein the charged molecule further includes a second recognition site for a second marker that reversibly binds the labeled nucleotide.

7. A sensing device, comprising: A flow cell; and The sensing system according to claim 1, which is integrated into the flow cell.

8. The sensing device according to claim 7, further comprising a reagent delivery system for selectively introducing a reagent into the input end of the flow cell.

9. The sensing device according to claim 8, wherein the reagent is in a sample container, and the reagent includes the labeled nucleotide, and the labeled nucleotide includes: A nucleotide; A linking molecule attached to the phosphate group of the nucleotide; and A recognition site specific marker attached to the linking molecule.

10. The sensing device according to claim 7, further comprising a detector for detecting the response from the charge sensor.

11. A sensing method, comprising: Introduce a template polynucleotide strand into the sensing system according to claim 1, and introduce a reagent comprising a labeled nucleotide into the sensing system, whereby a nucleotide of one of the labeled nucleotides associates with the polymerase, and an identification site specific marker of the one of the labeled nucleotides associates with the identification site to induce a conformational change of the charged molecule; and In response to the conformational change of the charged molecule, detect the response of the charge sensor.

12. The sensing method according to claim 11, further comprising: Associate the response of the charge sensor with the relevant identification site specific marker; and Based on the relevant identification site specific marker, identify the nucleotide of the one of the labeled nucleotides.

13. The sensing method according to claim 11, wherein the charged molecule comprises a plurality of different identification sites, and each identification site reversibly binds different markers of different labeled nucleotides at different rates.

14. The sensing method according to claim 13, further comprising: When different labeled nucleotides associate with the polymerase respectively and different identification site specific markers of the different labeled nucleotides bind to one of the plurality of different identification sites respectively, detect a plurality of responses of the charge sensor in response to different conformational changes of the charged molecule; and Identify the different labeled nucleotides that are respectively associated by the different rates.

15. The sensing method according to claim 11, wherein the identification site reversibly binds up to four different labeled nucleotides, and the method further comprising: When at least some of the different labeled nucleotides associate with the polymerase and the identification site respectively and at least some of the different markers bind to the identification site respectively, detect a plurality of responses of the charge sensor in response to different conformational changes of the charged molecule; and Identify the different labeled nucleotides that are respectively associated by the different rates.

16. The sensing method according to claim 11, wherein the identification site reversibly binds up to four different labeled nucleotides, and the method further comprising: When the up to four different labeled nucleotides associate with the polymerase and the identification site respectively, detect up to four different responses of the charge sensor in response to different conformational changes of the charged molecule, wherein each of the up to four different responses has a different magnitude; and Identify the different labeled nucleotides that are respectively associated by the different magnitudes.

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