RU2791444C2 - Reading systems - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to biotechnology, namely to reading systems. The reading system includes a pH sensor. The pH sensor includes two electrodes and a conducting channel operatively connected to two electrodes. The complex is attached to the conductive channel of the pH sensor. The complex includes a polymerase associated with at least one pH-altering fragment, which should participate in generating a pH change near the conducting channel as a result of the consumption of a secondary substrate in the liquid affected by the pH sensor. At least one pH-altering fragment is selected from a group consisting of an enzyme, a metal coordination complex, a cofactor and an activator.

EFFECT: expanding the arsenal of means for a specific purpose.

30 cl, 7 dwg

Description

Cross-reference to related application

[0001] This application claims priority to U.S. Provisional Patent Application Serial Number 62/806,545, filed February 15, 2019, the contents of which are incorporated herein by reference in their entirety.

State of the art

[0002] Various protocols in biological or chemical research involve performing a large number of controlled reactions on local support surfaces or in predetermined reaction chambers. These reactions can then be observed or detected, and subsequent analysis can help identify or reveal the properties of the chemicals involved in the reaction. In some examples, controlled reactions generate luminescence and therefore an optical system can be used for detection. In other examples, controlled reactions change charge, conductivity, or some other electrical property, and therefore an electronic system can be used for detection.

Introduction

[0003] The first aspect disclosed herein is a reading system comprising a pH sensor including two electrodes and a conductive channel operatively connected to the two electrodes; and a complex attached to the conduction channel of the pH sensor, wherein the complex includes a polymerase associated with at least one pH-changing component (pH-changing component) that is to be involved in generating a pH change in the vicinity of the conductive channel by virtue of secondary consumption. a substrate in a fluid that is disclosed for a pH sensor, wherein at least one pH change component is selected from the group consisting of an enzyme, a metal coordination complex, a cofactor, and an activator.

[0004] In an example of this first aspect, at least one pH change component is an enzyme, wherein the enzyme forms an acid or base in reaction with a secondary substrate. In one specific example, the enzyme is selected from the group consisting of hydrolases and oxidases.

[0005] In an example of this first aspect, the kinetics of at least one pH change component is at least 10 times faster than the polymerase kinetics.

[0006] In an example of this first aspect, at least one pH change component is an enzyme, and the complex further comprises a nucleic acid hairpin/enzyme inhibitor conjugate attached to the enzyme.

[0007] In an example of this first aspect, at least one pH change component is an enzyme; and the complex further includes a second enzyme attached to the polymerase.

[0008] In an example of this first aspect, the complex is a fusion protein or protein chimera.

[0009] In an example of this first aspect, the conductive channel of the pH sensor is selected from the group consisting of a semiconductive nanostructure, a graphene nanostructure, a metal nanostructure, and a conductive polymer nanostructure.

[0010] An example of this first aspect further comprises a support including a plurality of recesses separated by intervening regions, wherein at least a pH sensor conduit is at the bottom of one of the plurality of recesses; and a plurality of additional pH sensors, wherein at least the conductive channel of each of the plurality of additional pH sensors is at the bottom of a respective one of the plurality of recesses. In one specific example, each of the plurality of recesses includes side walls, wherein the side walls include a pH buffer material.

[0011] It should be understood that any features of the reading system disclosed herein may be combined with each other according to any desired method and/or configuration.

[0012] A second aspect disclosed herein is a kit comprising a pH sensor including two electrodes and a conductive channel operatively connected to the two electrodes; and a fluid comprising a carrier fluid and a complex in the carrier fluid, wherein the complex includes a polymerase associated with at least one enzyme that is to create a pH change in the vicinity of the conductive channel due to the consumption of a secondary substrate in the second fluid, which is disclosed for the pH sensor.

[0013] An example of this second aspect further comprises a second fluid including a second carrier fluid and a labeled nucleotide that includes a nucleotide that has a binding molecule (linker molecule) attached to the terminal phosphate group of the nucleotide and a label attached to binding molecule, and the label is selected from the group consisting of the first group, which improves the kinetics of the enzyme, and the second group, which slows down the kinetics of the enzyme. In one particular example, the secondary substrate is in a second fluid and is a single molecule relative to the labeled nucleotide, and the first or second group is to change the kinetics of the acid or base formation reaction comprising the enzyme and the secondary substrate. In another specific example, the secondary substrate is in the second fluid and is a single molecule relative to the labeled nucleotide, the label is the second group that retards enzyme kinetics, and the second group is selected from the group consisting of an allosteric inhibitor, a steric exclusion group, and a buffering group. . In yet another specific example, the secondary substrate is in the second fluid and is a single molecule relative to the labeled nucleotide, the label is the first moiety that improves enzyme kinetics, and the first moiety is the enzyme's cofactor.

[0014] Another example of this second aspect further comprises a second fluid including a second carrier fluid and a labeled nucleotide that includes a nucleotide and a secondary substrate attached to a base or sugar of the nucleotide, wherein the kinetics of the secondary substrate is at least , 10 times faster than polymerase kinetics.

[0015] It should be understood that any features of the kit can be combined with each other in any desired way. In addition, it should be understood that any combination of the features of this kit and/or reader system can be used in conjunction with and/or combined with any of the examples disclosed herein.

[0016] A third aspect disclosed herein is a kit comprising a pH sensor including two electrodes and a conductive channel operatively connected to the two electrodes; and a fluid comprising a carrier fluid and a complex in the carrier fluid, the complex including a polymerase associated with a metal coordination complex that is to create a pH change in the vicinity of the conductive channel due to the consumption of a secondary substrate in the second fluid, which disclosed for the pH sensor.

[0017] An example of this third aspect further comprises a second fluid including a second carrier fluid; a secondary substrate (in which case the secondary substrate must form an acid or base through reaction with a metal coordination complex) and a labeled nucleotide that includes a nucleotide binding molecule attached to the terminal phosphate group of the nucleotide and a label attached to the binding molecule, where the label represents is a ligand for the metal of the metal coordination complex, wherein the ligand changes the catalytic property of the metal coordination complex.

[0018] It should be understood that any features of this kit can be combined with each other in any desired way. In addition, it should be understood that any combination of features of this kit and/or reading system, and/or other kit can be used in conjunction and/or combined with any of the examples disclosed herein.

[0019] The fourth aspect disclosed herein is a labeled nucleotide comprising a nucleotide, a binding molecule attached to the terminal phosphate group of the nucleotide, and a catalytic label attached to the binding molecule, wherein the catalytic label should create a pH change due to consumption of secondary substrate in a labeled nucleotide fluid.

[0020] In the example of the fourth aspect, the catalytic label is selected from the group consisting of hydrolases and oxidases.

[0021] It should be understood that any features of this labeled nucleotide can be combined with each other in any desired manner. In addition, it should be understood that any combination of features of this labeled nucleotide and/or reading system and/or kits can be used in conjunction and/or combined with any of the examples disclosed herein.

[0022] The fifth aspect disclosed herein is a kit containing a labeled nucleotide of the fourth aspect and a reading system including two electrodes, a conduction channel operably connected to the two electrodes, and a complex attached to the conduction channel, the complex comprising a polymerase conjugated to a cofactor or catalytic tag activator of the labeled nucleotide.

[0023] It should be understood that any features of this kit can be combined with each other in any desired way. In addition, it should be understood that any combination of features of this kit and/or reading system, and/or other kits, and/or labeled nucleotide can be used in conjunction and/or combined with any of the examples disclosed herein.

[0024] The sixth aspect disclosed herein is a labeled nucleotide comprising a nucleotide having a 3'-OH blocking group, a cleavable binding molecule attached to the base or sugar of the nucleotide, and a label attached to the cleavable binding molecule, wherein the label must participate in the reaction of changing the pH, which contains a secondary substrate.

[0025] It should be understood that any features of this labeled nucleotide can be combined with each other in any desired manner. In addition, it should be understood that any combination of features of this labeled nucleotide and/or reading system and/or kits and/or other labeled nucleotide can be used in conjunction with and/or combined with any of the examples disclosed herein.

[0026] The seventh aspect disclosed herein is a kit containing a labeled nucleotide of the sixth aspect and a reading system including two electrodes, a conduction channel operably connected to the two electrodes, and a complex attached to the conduction channel, the complex comprising into itself a polymerase conjugated with a cofactor or an activator of the labeled nucleotide catalytic label.

[0027] It should be understood that any of the features of this kit can be combined with each other in any desired way. In addition, it should be understood that any combination of the features of this kit and/or reading system and/or kits and/or labeled nucleotides can be used in conjunction with and/or combined with any of the examples disclosed herein.

[0028] An eighth aspect disclosed herein is a method comprising injecting a fluid into a sensor array including a plurality of separately addressable conductive channels, thereby attaching the complex to at least some of the plurality of separately addressable conductive channels. , and the complex includes a polymerase and a pH change component associated with the polymerase, and the pH change component is selected from the group consisting of an enzyme that should catalyze the consumption of a secondary substrate in a solution that should be available to the array of sensors, a metal coordination complex that must catalyze the consumption of a secondary substrate in solution, which must be available to the array of sensors, and a cofactor or activator of the catalytic label attached to the labeled nucleotide, which must be introduced into the array of sensors.

[0029] It should be understood that any of the features of this method can be combined with each other in any desired way. Furthermore, it should be understood that any combination of features of this method and/or reading system and/or kits and/or labeled nucleotides may be used in conjunction with and/or combined with any of the examples disclosed herein.

[0030] The ninth aspect disclosed herein is a method comprising introducing a template polynucleotide strand into a sensor having a polymerase bound to a conductive channel; introducing a fluid comprising a secondary substrate and labeled nucleotides into the sensor, whereby a nucleotide of one of the labeled nucleotides binds to a polymerase, and a label of one of the labeled nucleotides participates in a pH change reaction involving a secondary substrate that is located in the vicinity of conducting channel; and detecting a conductive channel response.

[0031] In an example of this ninth aspect, the sensor polymerase is part of a complex with an enzyme catalyst, the label is a group that improves or retards the kinetics of the enzyme catalyst, and the method further comprises detecting a charge change from base charge.

[0032] In an example of this ninth aspect, the sensor polymerase is part of an enzyme catalyst complex, the label is a secondary substrate, and the method further comprises detecting a change in charge from base charge.

[0033] In an example of this ninth aspect, the sensor polymerase is part of a complex with a metal coordination complex, the label is a ligand for the metal of the metal coordination complex, and the method further comprises detecting a change in charge from base charge.

[0034] In an example of this ninth aspect, the sensor polymerase is part of a complex with an enzyme catalyst, the nucleic acid hairpin/enzyme inhibitor conjugate is attached to the enzyme catalyst, the label is an oligonucleotide sequence that is complementary to the nucleic acid hairpin/enzyme inhibitor conjugate, and the method further contains the detection of a charge change compared to the base charge.

[0035] Any one or more examples of the ninth aspect may further comprise identifying the nucleotide associated with the polymerase from charge change and/or charge change rate.

[0036] In the example of this ninth aspect, the labeled nucleotides have different incorporation rates, and the method further comprises identifying the bound labeled nucleotide by its different incorporation rate.

[0037] It should be understood that any of the features of this method can be combined with each other in any desired way. In addition, it should be understood that any combination of features of this method and/or other methods and/or reading system and/or kits and/or labeled nucleotides may be used in conjunction with and/or combined with any of the examples disclosed herein. .

[0038] The tenth aspect disclosed herein is a method comprising selecting a pH change component from the group consisting of an enzyme that is to catalyze consumption of a secondary substrate in solution, a metal coordination complex that is to catalyze consumption of a secondary substrate in solution, and a cofactor or activator for a catalytic label attached to a labeled nucleotide; conjugating the polymerase with the pH change component to form a complex; and attaching the complex to a conductive channel operably connected to the two electrodes.

[0039] It should be understood that any of the features of this method can be combined with each other in any desired way. In addition, it should be understood that any combination of features of this method and/or other methods and/or reading system and/or kits and/or labeled nucleotides may be used in conjunction with and/or combined with any of the examples disclosed herein. .

[0040] The eleventh aspect disclosed herein is an inclusion mixture comprising a carrier liquid; a complex comprising a polymerase and a pH change component associated with the polymerase, wherein the pH change component is selected from the group consisting of an enzyme that is to catalyze the consumption of the secondary substrate, a metal coordination complex that is to catalyze the consumption of the secondary substrate, and a cofactor or activator, which should catalyze the consumption of the secondary substrate; and a labeled nucleotide comprising a nucleotide having a binding molecule attached to a terminal phosphate group of the nucleotide and a label attached to the binding molecule, which label must participate in a pH change reaction comprising a secondary substrate.

[0041] In the example of this eleventh aspect, the pH change component is an enzyme, and the label is selected from the group consisting of a first group that improves the kinetics of the enzyme and a second group that slows down the kinetics of the enzyme; the pH change component is a metal coordination complex, and the label is a ligand for the metal of the metal coordination complex, wherein the ligand changes the catalytic property of the metal coordination complex; or the pH change component is a cofactor or activator, and the label is a catalytic label that is activated by the cofactor or activator.

[0042] In the example of this eleventh aspect, the pH change component is an enzyme; the complex further includes a nucleic acid hairpin/enzyme inhibitor conjugate attached to the enzyme; and the label is an oligonucleotide sequence that is complementary to the nucleic acid hairpin/enzyme inhibitor conjugate.

[0043] It should be understood that any features of this inclusion mixture may be combined with each other in any desired manner. In addition, it should be understood that any combination of features of the mixture for inclusion and/or methods and/or reading system and/or kits and/or labeled nucleotides can be used together and/or combined with any of the examples disclosed herein. .

[0044] The twelfth aspect disclosed herein is a kit containing a mixture to include the eleventh aspect and a secondary substrate mixture comprising a second carrier fluid and a secondary substrate.

[0045] An example of the twelfth aspect further comprises a flow cell including a substrate including a plurality of recesses separated by intermediate regions; a conductive channel at the bottom of each of the plurality of recesses; and at least one primer attached in each of the recesses.

[0046] It should be understood that any of the features of this kit can be combined with each other in any desired way. In addition, it should be understood that any combination of the features of this kit and/or inclusion mix and/or methods and/or reading system and/or other kits and/or labeled nucleotides can be used in conjunction with and/or combined with any from the examples disclosed in this document.

[0047] The thirteenth aspect disclosed herein is an inclusion mixture containing a carrier liquid comprising a buffer; a complex comprising a polymerase and a pH change component associated with the polymerase, wherein the pH change component is selected from the group consisting of an enzyme that is to catalyze the consumption of the secondary substrate, a metal coordination complex that is to catalyze the consumption of the secondary substrate, and a cofactor or activator, which should catalyze the consumption of the secondary substrate; and a labeled nucleotide comprising a nucleotide having a 3'-OH blocking group, a cleavable binding molecule attached to the base or sugar of the nucleotide, and a label attached to the binding molecule, wherein the label must participate in a pH change reaction involving a secondary substrate.

[0048] In the example of the thirteenth aspect, the pH change component is an enzyme, and the label is selected from the group consisting of a first group that improves the kinetics of the enzyme, a second group that slows down the kinetics of the enzyme, and a secondary substrate; or the pH change component is a metal coordination complex, and the label is a ligand for the metal of the metal coordination complex, wherein the ligand changes the catalytic property of the metal coordination complex; or the pH change component is a cofactor or activator, and the label is a catalytic label that is activated by the cofactor or activator.

[0049] In the example of the thirteenth aspect, the pH change component is an enzyme; the complex further includes a nucleic acid hairpin/enzyme inhibitor conjugate attached to the enzyme; and the label is an oligonucleotide sequence that is complementary to the nucleic acid hairpin/enzyme inhibitor conjugate.

[0050] It should be understood that any of the features of this inclusion kit can be combined with each other in any desired manner. In addition, it should be understood that any combination of features of this mixture for inclusion and/or methods and/or reading system and/or kits and/or labeled nucleotides can be used together and/or combined with any of the examples disclosed in this document.

[0051] The fourteenth aspect disclosed herein is a kit containing the inclusion mixture defined in the thirteenth aspect and a secondary substrate mixture comprising a second carrier fluid and a secondary substrate.

[0052] An example of the fourteenth aspect further comprises a flow cell including a substrate including a plurality of recesses separated by intermediate regions; a conductive channel at the bottom of each of the plurality of recesses; and a primer attached in each of the recesses.

[0053] An example of the fourteenth aspect further comprises a solution of a deblocking agent.

[0054] It should be understood that any features of this kit can be combined with each other in any desired way. In addition, it should be understood that any combination of the features of this kit and/or methods and/or reading system and/or other kits and/or labeled nucleotides and/or incorporation mixture can be used together and/or combined with any from the examples disclosed in this document.

[0055] Still further, it should be understood that any features of any of the aspects may be used alone or in combination in any desired manner and/or may be combined with any of the examples disclosed herein at least to achieve the benefits as described in this document.

Brief description of the drawings

[0056] Features of examples of the present disclosure should become apparent with reference to the following detailed description and drawings, in which like reference numerals correspond to similar, although perhaps not identical, components. For brevity, reference numbers or features having the above function may or may not be described in connection with the other drawings in which they appear.

[0057] FIG. 1A-1C are schematic illustrations of various examples of labeled nucleotides disclosed herein;

[0058] FIG. 2 is a schematic illustration illustrating one example of a reading system disclosed herein and an example of a labeled nucleotide and secondary substrate that can be used in this example of a reading system;

[0059] FIG. 3A is a schematic illustration illustrating another example of a reading system disclosed herein and an example of labeled nucleotides and a secondary substrate that can be used in this example of a reading system;

[0060] FIG. 3B illustrates an example of the reader system of FIG. 3A used in another example of a labeled nucleotide disclosed herein;

[0061] FIG. 4 is a schematic illustration illustrating another example of a reading system disclosed herein and an example of a labeled nucleotide and secondary substrate that can be used in this example of a reading system;

[0062] FIG. 5 is a schematic illustration illustrating yet another example of a reading system disclosed herein and an example of a labeled nucleotide and secondary substrate that can be used in this example of a reading system;

[0063] FIG. 6A is a schematic illustration illustrating yet another example of the reader system disclosed herein;

[0064] FIG. 6B illustrates an example of the reader system of FIG. 6A used in another example of a labeled nucleotide disclosed herein;

[0065] FIG. 7A is a top view of an example flow cell;

[0066] FIG. 7B is an enlarged partial sectional view of an example of a flow cell suitable for use in single molecule readings;

[0067] FIG. 7C is an enlarged partial sectional view of an example flow cell suitable for use in ensemble sequencing; And

[0068] FIG. 7D is an enlarged partial sectional view of yet another example of a flow cell suitable for use in ensemble sequencing.

Detailed description of the invention

[0069] Some exemplary reading systems disclosed herein can be used to detect single molecules in nucleic acid sequencing procedures. Each of these reading systems includes one conducting channel with one polymerase or one polymerase-containing complex attached to it. This conductive channel can detect a change in charge as a result of a localized change in pH (hydrogen ion concentration). When used, labeled nucleotides and a secondary substrate are introduced into the reading system. As the nitrogenous base of one of the labeled nucleotides is incorporated into the resulting strand by polymerase, the secondary substrate is consumed in an acid or base formation reaction (which increases or decreases the concentration of charged ions). The acid or base formation reaction also includes a pH change component, which, in some examples, is immobilized on the conductive channel as part of the complex; and, in other examples, is a label of the included labeled nucleotide. Some acid or base formation reactions involve multiple pH changing components, for example one as part of the complex and another as a tag for the labeled nucleotide. These configurations allow the pH change component to be positioned on/near the surface of the conductive channel; and because of this, the reaction of formation of acid or base is localized on the surface of the conducting channel. In some cases, charged ions react with the surface groups of the conductive channel, causing protonation or deprotonation of the surface groups, which changes the charge of the surface groups. In these cases, the charge of the surface groups is read out. Since the charges are directly on the surface of the conductive channel, they cannot be effectively shielded by the ions in solution and therefore can cause large changes in the threshold voltage and current. In other cases, charged ions resulting from the formation of an acid or base are read.

[0070] Some other exemplary reading systems disclosed herein may be used for array nucleic acid sequencing procedures. Each of these reading systems includes a conductive channel with a lawn of primers (eg, oligopares) on it. This conductive channel can detect a change in charge as a result of a localized change in pH. When used, library templates are introduced and hybridized into primers. Clustering is performed in order to form multiple template polynucleotide chains. Polymerase-containing complexes, labeled nucleotides and secondary substrates are then introduced into the reading system. During or after the nitrogenous base of the respective labeled nucleotides is incorporated (via the respective complex polymerases) into the respective nascent strands formed along the respective template polynucleotide strands, secondary substrates are consumed in an acid or base formation reaction (which increases or decreases the concentration of charged ions). Similar to reading single molecules, the acid or base formation reaction also includes a pH change component that is part of the polymerase-containing complex or is a label of the included labeled nucleotides. These configurations allow the pH change component to be positioned on/near the surface of the conductive channel; and because of this, the reaction of formation of acid or base is localized on the surface of the conducting channel. Charged ions or the reaction of charged ions with the surface groups of the conducting channel is read.

[0071] In any of the examples disclosed herein, acid or base formation reactions can form hundreds or thousands or even more protons or base molecules, thereby generating a large and localized change in pH.

[0072] In any of the examples disclosed herein, the pH change component can be any chemical species that can participate in an acid or base forming reaction with a secondary substrate or modify the activity of another pH change component that acts on the secondary substrate. As examples, the pH change component can catalyze (cause or speed up) an acid or base forming reaction containing a secondary substrate, can inhibit an acid or base forming reaction containing a secondary substrate, can improve or slow down the kinetics of an acid or base forming reaction or may otherwise participate in an acid or base formation reaction containing a secondary substrate. Some examples of the pH change component are immobilized on the conduction channel as part of the complex; and other examples of the pH change component is the label of the included labeled nucleotide. In some cases, the two components of the pH change may interact to form the pH change.

[0073] Acid or base formation reactions (pH changes) disclosed herein involve a secondary substrate. As used herein, the term "secondary substrate" means chemical species that are consumed in a reaction that forms an acid or a base, where the chemical species is a separate molecule from a nucleotide whose base allows interaction with the polymerase of the complex, or is a different molecule, attached to the sugar or base of a nucleotide whose base allows interaction with the polymerase of the complex, and is not a by-product of the nitrogenous base incorporation event. The use of a different secondary substrate allows the kinetics of the detected pH change to be decoupled from the kinetics of nucleotide incorporation. In some examples, the pH change component that interacts with the secondary substrate may be kinetically significantly faster than the polymerase that interacts with the nucleotide. This makes it possible to observe a larger change in pH for a single turn-on event or for a group of turn-on events.

[0074] Desirably, acid or base formation reactions (pH changes) take place in the vicinity of the conductive channel. "Proximity" generally means any distance that a formed acid or base can diffuse. In an array with repetitive/periodic reading systems, "nearby" can be specified in terms of the pitch of the sensor/reading system. Sensor pitch or reader system pitch refers to the distance between two consecutive sensors/reader systems along a line. In an example, the reaction zone (in which the acid or base is formed) of the reading system may be within 1/2 of the sensor pitch from the conductive channel of the reading system.

[0075] Several examples of a single molecule reading system 10A-10E are disclosed herein and shown and described in FIG. 2, fig. 3A, FIG. 3B, FIG. 4, fig. 5, fig. 6A and FIG. 6b. An example of array sequencing read systems 40A and 40B is shown in FIG. 7C and FIG. 7D. Each example of reading system 10A-10E or 40A-40B can be used with a labeled nucleotide. Some labeled nucleotides are naturally cleavable after base incorporation, and this example is shown schematically in FIG. 1A. Some other labeled nucleotides include a reversible terminator that allows only single base insertion to occur in each sequencing run, and examples of the chemical structure of some of these labeled nucleotides are shown in FIG. 1b. Any of these examples can also include a secondary substrate as a different molecule attached to a sugar or base (FIG. 1C). Each of the labeled nucleotides is described below.

[0076] Labeled nucleotides

[0077] As illustrated in FIG. 1A, labeled nucleotide 12 includes nucleotide 14, a binding molecule 16 attached to the terminal phosphate group of nucleotide 14, and a label 18 attached to binding molecule 16. Labeled nucleotide 12 may be considered an unnatural or synthetic nucleotide because it is structurally or chemically different from natural nucleotide.

[0078] Nucleotide 14 of labeled nucleotide 12 may be a natural nucleotide. Natural nucleotides include a nitrogen-containing heterocyclic base (or nitrogenous base), a sugar, and one or more phosphate groups. Examples of natural nucleotides include, for example, ribonucleotides or deoxyribonucleotides. In a ribonucleotide, the sugar is a ribose, and in a deoxyribonucleotide, the sugar is a deoxyribose (i.e., a sugar with a missing hydroxyl group that is present at the 2' position in the ribose). In the example, nucleotide 14 is in the form of a polyphosphate because it includes several phosphate groups (eg, triphosphate (ie, gamma phosphate), tetraphosphate, pentaphosphate, hexaphosphate, etc.). The heterocyclic base may be a purine base or a pyrimidine base, or any other nucleobase analog. 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 modified derivatives or analogs thereof. The C-1 atom of deoxyribose binds to the N-1 of the pyrimidine or the N-9 of the purine.

[0079] Labeled nucleotide 12 also includes binding molecule 16. Binding molecule 16 can be any long chain molecule that can chemically bond, at one end, to the phosphate group(s) of nucleotide 14, and that can chemically bond, at the other end, labeled 18. In some cases, the binding molecule 16 can also be chosen so that it does not interact with polymerase 28 (see, for example, Fig. 2, Fig. 3A, Fig. 4, Fig. 5, Fig. 6A and Fig. 6B). In other cases, the binding molecule 16 may also be chosen such that it interacts weakly with the polymerase 28, since this weak interaction can help direct the label 18 to a position close to the conduction channel. Binding molecule 16 may be chosen such that it is long enough to allow label 18 to bind to the pH change component and/or secondary substrate 34 (see, for example, FIG. 2), while, for example, nucleotide 14 retained by polymerase 28 during the on event.

[0080] As examples, linking molecule 16 may include an alkyl chain, a poly(ethylene glycol) chain, an amido group, a phosphate group, a heterocycle such as a triazole, nucleotides, or combinations of the above. Examples of an alkyl chain may include at least 6 carbon atoms, and examples of a poly(ethylene glycol) chain may include at least 3 ethylene glycol units.

[0081] The following example illustrates an example of labeled nucleotide 12, in which binding molecule 16 contains an alkyl chain, an amide group, a poly(ethylene glycol) chain, and a triazole:

The following example illustrates another example of labeled nucleotide 12 in which binding molecule 16 contains alkyl chains, an amide group, poly(ethylene glycol) chains, a triazole, and a phosphate group:

The following example illustrates yet another example of labeled nucleotide 12, in which binding molecule 16 contains alkyl chains, amide groups, poly(ethylene glycol) chains, a triazole, and a phosphate group:

The following example illustrates a further example of labeled nucleotide 12, in which binding molecule 16 contains alkyl chains, an amide group, poly(ethylene glycol) chains, a triazole, a phosphate group, and a polynucleotide chain:

[0082] [0083] Although several exemplary binding molecules 16 are described, it should be understood that other binding molecules 16 may be used. molecules 16 can be chosen so that when the corresponding nucleotide 14 is retained by the polymerase of a separate reading system, the corresponding label 18 can participate in the reaction of forming an acid or base on the surface of the conductive channel 26 (see, for example, Fig. 2).

[0084] In each exemplary labeled nucleotide 12, tag 18 is attached (via binding molecule 16) to the terminal phosphate end of nucleotide 14. Attachment at the terminal phosphate end may be desirable in some sequencing technologies because, after incorporating the nucleotide base, the bond between the alpha-phosphate and binding molecule 16 or between alpha-phosphate and beta-phosphate is naturally cleaved. This natural cleavage allows label 18 (and binding molecule 16) to dissociate from the incorporated nucleotide base and diffuse away from the reading system 10A-10E such that another labeled nucleotide 12 can bind to polymerase 28 of the reading system 10A-10E, and thus that the previously turned on base does not produce sustained signals that interfere with the detection of the next turned on base.

[0085] The label 18 that is included in the labeled nucleotide 12 may depend, in part, on the reading system 10A-10E or 40A-40B to be used with the labeled nucleotide 12. The various labels 18 in relation to each of the reading systems are described in more detail below. 10A-10E and 40A-40B.

[0086] As illustrated in FIG. 1B, each labeled nucleotide 12A, 12B, 12C includes a nucleotide 14' having a 38A, 38B, 38C 3'-OH blocking group, a cleavable binding molecule 16' attached to the base or sugar of nucleotide 14', and a tag 18 attached to the binding molecule 16'.

[0087] Nucleotide 14' may be any of the examples set forth for nucleotide 14. In the examples disclosed herein, nucleotide 14' has a 3'-OH blocking group attached. The blocking group 38A, 38B, 38C 3'-OH can be linked to the oxygen atom of the sugar molecule at nucleotide 14'. The 3'-OH blocking group 38A, 38B, 38C may be a reversible terminator that allows only single base insertion to occur in each sequencing run. The reversible terminator prevents the incorporation of additional bases into the resulting strand, which is complementary to the template polynucleotide strand. This ensures the detection and identification of one included base. The 38A, 38B, 38C 3'-OH blocking group can then be removed, allowing for additional sequencing rounds in each template polynucleotide strand. Examples of various 38A, 38B, 38C 3'-OH blocking groups are shown in FIG. 1B, including the 3'-ONH ₂ reversible terminator (shown in 38A), the 3'-O-allyl reversible terminator (-CH=CHCH ₂ , shown in 38B), and the 3'-O-azidomethyl reversible terminator (-CH ₂ N ₃ shown at 38C). Other suitable reversible terminators include o-nitrobenzyl ethers, alkyl o-nitrobenzyl carbonate, ester moieties, other allyl moieties, acetals (e.g., t-butoxyethoxy), MOM-(-CH ₂ OCH ₃ )- moieties, 2,4 -dinitrobenzenesulphenyl, tetrahydrofuranyl ether, 3'-phosphate, ethers, -F, -H ₂ , -OCH ₃ , -N ₃ , -HCOCH ₃ and 2-nitrobenzene carbonate.

[0088] In the examples shown in FIG. 1B, binding molecule 16' is attached to the base (eg, purine base or pyrimidine base) of nucleotide 14'. In some examples, binding molecule 16' includes a cleavage site, identified by arrow 42 in FIG. 1b. Some examples of suitable binding molecules 16' are shown in FIG. 1B, although it should be understood that any suitable cleavable binding agent can be used that can attach the label 18 to the base or sugar of nucleotide 14'.

[0089] The label 18 that is included in the labeled nucleotide 12A, 12B, 12C may depend, in part, on the reading system 10A-10E or 40A-40B to be used with the labeled nucleotide 12A, 12B, 12C. In the following, the various marks 18 are described in more detail with respect to each of the reading systems 10A-10E and 40A-40B.

[0090] Yet another example of labeled nucleotide 12' is shown in FIG. 1C. As illustrated in FIG. 1C, labeled nucleotide 12' includes nucleotide 14 and a secondary substrate 34 attached to the base or sugar of nucleotide 14. Either nucleotide 14 or 14' can be used in this case. Secondary substrate 34 can be attached directly to the base or sugar of nucleotide 14 or 14'. In one example, secondary substrate 34 can be attached directly to the carbon or nitrogen atom of the base of nucleotide 14 or 14'. In another example, secondary substrate 34 can be attached directly to the oxygen or carbon atom of the sugar molecule of nucleotide 14 or 14'. If secondary substrate 34 is attached to the 3'-OH of nucleotide 14 as shown in FIG. 1C, it should block the addition of the next base (similar to labeled nucleotides 12A-12C). This positioning on the sugar molecule may be desirable as it results in consumption of the secondary substrate 34 prior to the next inclusion event and does not require a separate deblocking agent in order to remove the blocking group. If nucleotide 14' (which includes the blocking group 38A, 38B, 38C 3'-OH) is used, secondary substrate 34 can be attached directly to the 2' position of the sugar molecule of nucleotide 14'. Alternatively, secondary substrate 34 may be attached indirectly to the base or sugar of nucleotide 14 or 14', for example via a binding molecule. Examples of suitable binding molecules for attaching secondary substrate 34 to the base or sugar of nucleotide 14 or 14' include a polyethylene glycol chain, an alkyl group, biotin/streptavidin, proparglyamino, or any group attached to a commercially available functionalized nucleobase.

[0091] In this exemplary labeled nucleotide 12', secondary substrate 34 is selected such that the kinetics of consumption of secondary substrate 34 is at least ten (10) times faster than the kinetics of polymerase 28, which is used upon incorporation of the nucleotide base. Thus, the consumption of the secondary substrate 34 occurs faster than the turn on event, and therefore the change in pH can be detected before or as the turn on event occurs. In an example, secondary substrate 34 may be a hydrolase substrate such as polyester chain (which can be consumed by esterase), cellulose (which can be consumed by cellulase), peptide (which can be consumed by protease), starch (which can be consumed by amylase), etc. In another example, secondary substrate 34 is an additional polyphosphate chain. In this example, the additional polyphosphate chain is not blocked and its reactivity should depend on the higher local concentration when polymerase 28 reacts on nucleotide 12'. Also in this example, the terminal phosphate of nucleotide 14 or 14' may include a blocking group thus that the polyphosphate chain of nucleotide 14 or 14' is not involved in the acid or base formation reaction.

[0092] Detection of single molecules

[0093] Referring now to FIG. 2, fig. 3A, FIG. 3B, FIG. 4, fig. 5, fig. 6A and FIG. 6B, each of the reading systems 10A-10E can be used in single molecule readings. Each of the systems includes a pH sensor 20 which includes two electrodes 22, 24 and a conductive channel 26 connecting the two electrodes 22, 24.

[0094] Each pH sensor 20 may be a field effect transistor (FET). In FET, electrodes 22, 24 are the source and drain terminals, and the conductive path 26 is the gate terminal. The FET can be a p-type channel or an n-type channel, which affects the polarity of the response, but not the sense principle. Ion-sensitive FETs (ISFETs), in-plane gate FETs (JFETs), metal-semiconductor FETs (MESFETs), metal-oxide-semiconductor FETs (MOSFETs), or junctionless field devices are suitable detectors in examples disclosed in this document.

[0095] The electrodes 22, 24 may comprise 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. d.

[0096] Conductive channel 26 may include any electrically conductive or semi-conductive and pH sensitive material. In one example, the conductive path 26 is an electrically conductive path. In one example, an electrically conductive or semi-conductive and pH sensitive material is capable of reading charged ions on its surface. In another example, an electrically conductive or semi-conductive and pH sensitive material includes surface groups or is coated with another material that includes surface groups that are capable of being protonated and/or deprotonated in response to charged ions formed by reacting the secondary substrate 34 with pH change component. The electrically conductive or semi-conductive and pH sensitive material may comprise an organic material, an inorganic material, or both.

[0097] In some examples, the electrically conductive or semi-conductive and pH-sensitive material may include a semiconductor material such as silicon that is capable of charge detection. The semi-conductive and pH-sensitive material may also have a gate dielectric on at least a portion of its surface. In some examples, the gate dielectric may surround the entire outer surface of the semiconductive and pH sensitive material (excluding the contact points with electrodes 22, 24), and in other examples, the gate dielectric may be positioned on a portion of the outer surface of the semiconductive and pH sensitive material. In one example, the gate dielectric serves to prevent leakage of current into the environment (eg, fluid) and may also provide surface groups that are susceptible to being protonated and/or deprotonated. Examples of the gate dielectric include silicon dioxide (SiO ₂ ), silicon oxynitride (SiON), silicon nitride (Si ₃ N ₄ ), tantalum pentoxide (Ta ₂ O ₅ ), hafnium oxide (HfO ₂ ), zirconium oxide (ZrO ₂ ) , aluminum oxide (Al ₂ O ₃ ), hafnium or zirconium silicates (HfSiO ₄ , ZrSiO ₄ ), etc. The surface groups may be silanol groups (Si—OH), Si—NH ₂ groups, carboxyl groups (—COOH) or hydroxyl (—OH) groups.

[0098] In other examples, the electrically conductive or semi-conductive and pH sensitive material may include a carbon material (eg, carbon nanotubes, vitreous carbon, graphene) without a gate dielectric. These electrically conductive or semi-conductive and pH sensitive materials may include carboxyl (-COOH) groups, hydroxyl (-OH) groups, or other surface functionality that is pH responsive or allows pH sensitive functionality to be attached.

[0099] In yet other examples, the electrically conductive or semi-conductive and pH sensitive material may be a biomolecule. Examples of suitable biomolecules include peptides and deoxyribonucleic acids. These materials may have a low pKa value at pH formed as a result of an acid or base formation reaction, and therefore may also include a conductance modulator that has an almost neutral pKa value. For example, the conductivity of peptides can be modulated by pH-sensitive amino acids (histidine, etc.). As another example, DNA conductivity can be modulated with a pH-responsive intercalator such as doxorubicin (pKa ~ 8) and its analogues.

[00100] In single molecule readout systems 10A-10E, conductive channel 26 may have any suitable geometry such as tubular structure, wire structure, planar structure, and so on. In some examples, the conductive channel 26 may also be a nanostructure that has at least one nanoscale dimension (ranging from 1 nm to less than 1 µm). In one example, this dimension means the largest dimension. In an example, the conductive channel 26 of the pH sensor is selected from the group consisting of a semiconductive nanostructure, a graphene nanostructure, a metal nanostructure, and a conductive polymer nanostructure. The nanostructure can be a multi- or single-walled nanotube, nanowire, nanoribbon, etc.

[00101] Each of the reading systems 10A-10E suitable for use in reading single molecules includes a polymerase 28 attached to the surface of the conductive channel 26. In some examples, only polymerase 28 is attached to the conductive channel 26 (see, for example, Fig. 2), and in other examples, polymerase 28 is part of the complex 30A-30D (see, for example, Fig. 3A, Fig. 3B, Fig. 4, Fig. 5, Fig. 6A and Fig. 6B), which includes another component associated with polymerase 28.

[00102] It should be understood that polymerase 28 allows retention of the template polynucleotide strand and incorporation of one nucleotide (from labeled nucleotide 12, 12A, 12B, 12C, or 12') at a time into the resulting strand that forms along the template. It should be understood that polymerase 28 and its functions are separate and distinct from the acid-base formation reaction(s) comprising the secondary substrate 34. In particular, polymerase 28 is involved in a separate acid formation reaction from the acid-base formation reaction(s). containing secondary substrate 34. For example, polymerase 28 generates a small amount of acid as a result of incorporation of a nucleotide base (e.g., 1 molecule per incorporation event), while acid or base formation reaction(s) generate hundreds or thousands or more the number of protons or base molecules. The acid formed by polymerase 28 must be overwhelmed by the large amount of acid or base formed from the reaction involving the secondary substrate 34. In some cases, the local pH change resulting from acid or base formation reactions can affect the kinetics of polymerase 28. In some cases, changing the pH may turn off polymerase 28 activity until the base pH is restored. This may be advantageous in order to create a delay between turn-on events (eg when nucleotide 12 or 12' is used). Other factors, such as choice of pH change component, buffer, temperature changes during onset, etc., can also be adjusted to further improve or balance a particular effect with respect to polymerase kinetics.

[00103] When using any of the reading systems 10A-10E, a method for reading single molecules includes introducing a template polynucleotide strand 44 (see, for example, Fig. 2) into a sensor 20 having a polymerase 28 (single or as part of a complex ) attached to the conductive channel 26; introducing a fluid comprising secondary substrate 34 and labeled nucleotides 12, 12A, 12B, 12C, or 12' into sensor 20, whereby a nucleotide of one of the labeled nucleotides 12, 12A, 12B, 12C, or 12' binds to polymerase 28 , and a label 18 of one of the labeled nucleotides 12, 12A, 12B, 12C, or 12' participates in a pH change reaction involving a secondary substrate 34 that is adjacent to the conduction channel 26; and detecting the response of the conductive channel 26. In some cases, the detected charge change must be relative to the base charge. In addition, different nucleotides 12, 12A-12C, or 12' may have different incorporation rates, which may affect, for example, how long an acid or base is formed, or how long an acid or base formation reaction is inhibited. This, in turn, should affect the local pH and charge on the surface of the conductive channel 26. The turn-on rate can be used to distinguish between different nucleotides.

[00104] Each of the reader systems 10A-10E and any methods associated with the reader systems 10A-10E are described in more detail below with reference to the individual drawings in which they are shown.

[00105] 10A Reader System

[00106] Referring specifically to FIG. 2, the reading system 10A includes a pH sensor 20 and a polymerase 28 connected to a conductive channel 26 via a tie 32.

[00107] In this exemplary reading system 10A, any polymerase 28 that can accept labeled nucleotides 12 or 12A-12C (FIG. 2) and that can successfully incorporate a nucleotide base into the resulting strand 46 along the template 44 can be used. In the single molecule reading technologies disclosed herein, it is desirable if polymerase 28 is highly processive such that multiple inclusion events can occur. Exemplary polymerases include those polymerases from the A family such as Bsu polymerase, Bst polymerase, Taq polymerase, T7 polymerase, and many others; polymerases from families B and B2, such as Phi29 polymerase and other highly processive polymerases (B2 family), Pfu polymerase (B family), KOD polymerase (B family), 9oN (B family) and many others; family C polymerases such as Esterichia Coli type III DNA polymerase and many others; family D polymerases such as Pyrococcus furiosus type II DNA polymerase and many others; family X polymerases such as DNA polymerase m, DNA polymerase b, DNA polymerase y, and many others. In one example, it may be desirable to select a polymerase 28 that is known to function for at least 2 pH units, which is sufficient to provide a 100 mV range in the signal. For four different nucleotides and an off state, this range may include distinctive 20 mV changes that are detectable.

[00108] In this exemplary reading system 10A, tether 32 is used as an anchor for polymerase 28. An example of a suitable tether 32 includes polyethylene glycol (PEG). In readout system 10A, tether 32 is long enough to keep polymerase 28 close enough to channel 26 such that any acid or base formed is allowed to diffuse into channel 26 before it is neutralized in solution. In an example, tether 32 may have a length ranging from about 2 nm to about 200 nm, or from about 5 nm to about 150 nm, or from about 10 nm to about 100 nm. In some examples, tether 32 keeps polymerase 28 at least 10 nm away from conduction channel 26. This may be desirable, for example, such that conformal changes in polymerase 28, polymerase 28 charges, and/or target/template charges polynucleotide chain held by polymerase 28 does not interfere with the read operation of the pH sensor 20. However, it should be understood that this approximate minimum distance (at least 10 nm) may not be required, for example, when pH changes dominate the signal which is being read.

[00109] Although not shown, the readout system 10A may also include a detector to detect voltage and/or current changes that correspond to a change in pH occurring at/near the surface of the pH readout channel 26.

[00110] Also, although not shown, the reader system 10A may be positioned on a support such as a silicon chip or a complementary metal-oxide-semiconductor (CMOS) structure. The electrodes 22, 24 can be connected to an electronic circuit that makes them work (for example, after being connected to a detector and a power source).

[00111] In some examples, sensor 10A may come pre-assembled.

[00112] In other examples, the sensor components may be part of a kit, and the kit components may be used to assemble the sensor 10A. An example kit includes a pH probe 20 on a support and a separate polymerase solution. The polymerase solution includes a carrier fluid and any example of polymerase 28. In some examples, polymerase 28 is attached to tether 32, and in other examples, tether 32 is attached to pH readout channel 26 as part of readout system 10A. As examples, the polymerase solution carrier fluid can be water or an ionic salt buffer fluid such as citrate saline at millimolar-molar concentrations.

[00113] When using the kit, the user can deposit the polymerase solution on the support and allow the polymerase solution to remain on the support for a suitable time to attach, via tie 32, to the conductive channel 26, or to attach, via polymerase 28, to the tie 32 on conducting channel 26. The support can be flushed with an appropriate buffer to remove any unbound polymerases 28.

[00114] FIG. 2, kit examples may also include the fluid to be used with sensor 10A during single molecule readings. This fluid includes a carrier fluid, labeled nucleotides 12 or 12A-12C, and a secondary substrate 34. In some cases, this fluid also includes a template polynucleotide strand 44. In other cases, one solution includes a carrier fluid , labeled nucleotides 12 or 12A-12C and secondary substrate 34, and another solution includes template polynucleotide strand 44.

[00115] Template polynucleotide strand 44 may be any sample to be sequenced and may be composed of DNA, RNA, or analogues thereof (eg, peptide nucleic acids). Template polynucleotide strand 44 may be a circular array. The source of template (or target) polynucleotide strand 44 may be genomic DNA, messenger RNA, or other nucleic acids from native sources. In some instances, template polynucleotide strand 44 that is derived from such sources may be amplified prior to use in the method or system 40 disclosed herein. Any of a variety of known amplification techniques may be used, including, but not limited to, polymerase chain reaction (PCR), rolling ring amplification (RCA), multiple displacement amplification (MDA), or random primer amplification (RPA). It should be understood that amplification of template polynucleotide strand 44 prior to use in the method or system set forth herein is optional. As such, template polynucleotide strand 44 is not amplified prior to use in some examples. Template/target polynucleotide chains 44 may optionally be retrieved from synthetic libraries. Synthetic nucleic acids may have native DNA or RNA compounds, or may be analogues thereof.

[00116] Labeled nucleotides 12 or 12A-12C include any example of nucleotide 14 or 14' and binding molecule 16 or 16' disclosed herein. Label 18 of labeled nucleotide 12 or 12A-12C shown in FIG. 2 is catalytic tag 18A. As used herein, "catalyst tag" means chemical species that initiate or accelerate an acid or base formation reaction comprising secondary substrate 34. In this regard, catalytic tag 18A is one example of a pH change component that is attached to labeled nucleotide 12 or 12A-12C. For example, catalytic label 18A may be an enzyme that reacts with secondary substrate 34 to form an acid, thereby lowering the pH. A specific example of an acid forming pair includes acetylcholinesterase as a catalytic label 18A and an ester support (e.g. acetylcholine) as a secondary substrate 34. Another specific example of an acid forming pair includes carbonic anhydrase as a catalytic label 18A and a bicarbonate ion in as secondary substrate 34. As another example, catalytic label 18A may be an enzyme that reacts with secondary substrate 34 to form a base, thereby increasing pH. In some examples, the surface groups on the surface of channel 26 are sensitive to acidic or basic ions that are formed and can be protonated or deprotonated, thereby changing the charge density directly on the surface of channel 26. In other examples, charged ions formed during reactions of acid formation or base formation are read through the conduction channel 26.

[00117] The carrier fluid that can be used with the 10A sensor during single molecule readings is water or a low buffer fluid (10 mM or less) having a pH in the range of about 6 to about 9 or about 7 to about up to 8. The pH of the fluid depends on the polymerase 28 that is used and all the conditions that help maximize the signal when a change in pH occurs. Because catalytic tag 18A and secondary substrate 34 are present in the fluid in this example, some reaction(s) of acid or base formation may occur in the fluid at a great distance from conduction channel 26. However, the ions formed in these reactions may be neutralized in the fluid (by the low buffer fluid) and therefore may not result in a detectable signal. This prevents large global pH changes in the fluid. However, it should be understood that when labeled nucleotide 12 or 12A-12C is retained by polymerase 28, catalytic label 18A is retained more closely to the pH readout channel 26 (e.g., compared to when labeled nucleotide 12 or 12A- 12C floats in solution). In turn, any change in pH resulting from the reaction involving secondary substrate 34 and temporarily bound catalytic tag 18A is also in closer proximity to the pH reading channel 26. In addition, the kinetics of catalytic tag 18A can be faster than the kinetics of polymerase 28. For example, the turnover rate of the acid-forming enzyme, acetylcholinesterase, can be as high as at least 25,000 s ^-1 while the turn-on event can be any value from about 0.1 s ^-1 to about 100 s ^-1 . In this example, catalytic label 18A may be formed from about 250 to about 250,000 acid molecules per nucleotide that is included (per one inclusion event). When the catalytic mark 18A and acid or base formation is localized on the surface of the conductive channel 26, a pH gradient can form around the pH readout channel 26, which can generate detectable charge signals in the pH readout channel 26. These signals can be recorded.

[00118] In an example, the buffer concentration in the low buffer fluid may be less than about 10 mM, which, as described herein, prevents global pH changes but allows local pH changes near the conduction channel 16 to be detected. In some cases, the buffer concentration is well below 10 mM, examples of which include about 5 mM, about 2.5 mM, about 2 mM, about 1 mM, about 0.5 mM, or about 0.25 mM. In other examples, solid phase buffers can be used to help maintain global pH without buffering the local environment.

[00119] In one example of a fluid that can be used with the reading system 10A, four different labeled nucleotides 12 or 12A-12C can be included. The four different labeled 12 or 12A-12C nucleotides include different 14 or 14' nucleotides (eg, T, A, G, or C) and different 18A catalytic labels. Different 18A catalytic labels may have different kinetics such that acid or base is formed at different rates, which in turn should result in different charge densities in channel 26. Since different 10A catalytic labels are specific to nucleotides (e.g. 18A is selected for a specific base), the response of the pH sensor 20 may indicate the included base of the labeled nucleotide 12 or 12A-12C.

[00120] The following description relates to an exemplary method that includes labeled nucleotides 12 and a reading system 10A. In this example, template polynucleotide strand 44 may be introduced into reader system 10A in a fluid such as a biologically stable solution, together with or separately from labeled nucleotides 12 and secondary substrate 34. Polymerase 28 binds to template polynucleotide strand 44 such that strand 44 is held in place. The complementary base of one of the labeled nucleotides 12 must be incorporated into the nascent strand 46 (which is formed along the template polynucleotide strand 44) by polymerase 28. During the incorporation event, the 18A catalytic label of labeled nucleotide 12 appears in the vicinity of channel 26 and any secondary substrate 34 in the fluid , which is also in the vicinity of channel 26. Because the kinetics of catalytic tag 18A may be faster than the onset event, an acid or base formation reaction with secondary substrate 34 occurs before base onset is complete. Charged ions from an acid or base forming reaction may be found in the conductive channel 26 or, if reactive surface groups are present, may react with the surface groups to change the state of charge of the surface groups. The changed charge is detected by the pH sensor 20.

[00121] In this exemplary method, after incorporation of a nucleotide base into the resulting strand 46, the bond between alpha phosphate and binding molecule 16 or between alpha phosphate and beta phosphate is naturally cleaved. This natural cleavage allows the 18A label (and binding molecule 16) to dissociate from the incorporated nucleotide base and diffuse away from the 10A reading system. Polymerase 28 can then accept another labeled nucleotide 12 that includes a nucleotide base that is complementary to the next nucleotide in the 44 template polynucleotide strand.

[00122] The following description relates to an exemplary method that includes labeled nucleotides 12A-12C and a 10A readout system. In this example, template polynucleotide strand 44 may be introduced into reader system 10A in a fluid such as a biologically stable solution, together with or separately from labeled nucleotides 12A-12C and secondary substrate 34. Polymerase 28 binds to template polynucleotide strand 44 such that chain 44 is held in place. The complementary base of one of the labeled nucleotides 12A-12C must be incorporated into the nascent strand 46 (which is formed along the template polynucleotide strand 44) by polymerase 28. In this example, the labeled nucleotide 12A-12C includes a reversible terminator. In this regard, the inclusion of the labeled nucleotide 12A-12C serves as a polymerization terminator. This allows acid or base formation and charge detection to be carried out in a manner similar to that described above.

[00123] In this exemplary method, after incorporation of a nucleotide base into the resulting strand 46, fluid, which includes any non-incorporated nucleotides, may be removed from the reading system 10A. This can be achieved using a washing solution (eg water). The deblocking agent may then be introduced into the reader system 10A. The deblocking agent may allow i) cleavage of all 16' cleavable binding molecules from the included 12A-12C labeled nucleotide (which also removes the 18A catalytic tag), and ii) removal of the 38A, 38B, 38C 3'-OH blocking group (see FIG. 1B) from the included labeled nucleotide 12A-12C. Removal of the 38A, 38B, 38C 3'-OH blocking group allows a subsequent sequencing run to be performed. Examples of 3'-OH blocking groups and suitable deblocking agents include: o-nitrobenzyl ethers and alkyl o-nitrobenzyl carbonate, which can be removed photolytically; ester components that can be removed by base hydrolysis; allyl components which can be removed with Nal, chlorotrimethylsilane and Na2S2O ₃ or with Hg(II) in acetone/water; azidomethyl, which can be cleaved with phosphines such as tris-(2-carboxyethyl)-phosphine (TCEP) or tri-(hydroxypropyl)-phosphine (THP); acetals such as tert-butoxyethoxy, which can be cleaved under acidic conditions; MOM-(-CH ₂ OCH ₃ )-components, which can be split with LiBF ₄ and CH ₃ CN/H ₂ O; 2,4-dinitrobenzenesulphenyl, which can be cleaved by nucleophiles such as thiophenol and thiosulfate; tetrahydrofuranyl ether which can be cleaved with Ag(I) or Hg(II); and 3'-phosphate, which can be cleaved by phosphatase enzymes (eg, polynucleotide kinase). Other useful reversible components include ethers, -F, -H ₂ , -OCH ₃ , -N ₃ , -HCOCH _{3 ,} and 2-nitrobenzene carbonate, and useful deblocking treatments include irradiation with light (for example, to cause photodegradation), heating, exposure to chemical reactants, exposure to catalysts, exposure to electric current (for example, to cause electrolysis), etc.

[00124] 10B Reader System

[00125] Referring specifically to FIG. 3A and FIG. 3B, the reading system 10B includes a pH sensor 20 and a complex 30A connected to a conductive channel 26 of the pH sensor 20.

[00126] Complex 30A includes polymerase 28 associated with a pH change component (shown in these examples as 50A, 50B, or 50C) that would be involved in generating a pH change in the vicinity of the conduction channel 26 due to the consumption of secondary substrate 34 in the fluid. environment, which is disclosed for the pH sensor 20.

[00127] Polymerase 28 of complex 30A can be any example of the polymerase described with reference to FIG. 2.

[00128] In this example, the pH change component is selected from the group consisting of 50A enzyme, 50B cofactor, and 50C activator.

[00129] Enzyme 50A forms an acid or base in reaction with secondary substrate 34. As examples, enzyme 50A can be selected from the group consisting of hydrolases and oxidases. Examples of suitable hydrolases include phosphatases, esterases (eg, acetylcholinesterase), sequence-specific proteases (eg, TEV protease or thrombin), and glycosidases (which do not degrade ribose, such as cellulose or amylase). Another suitable hydrolase is carbonic anhydrase. Examples of suitable oxidases include glucose oxidase, monoamine oxidase, xanthine oxidase, and the like. Although several examples are provided, it is believed that any available 50A enzyme or engineered 50A enzyme may be used, as long as the enzymatic kinetics are faster than the polymerase kinetics.

[00130] Cofactor 50B is a substance whose presence is essential for the activity of an enzyme that forms an acid or base in reaction with a secondary substrate 34. Some examples of suitable 50B cofactors for oxidases include flavin adenine dinucleotide (FAD), nicotine adenine dinucleotide (NAD), nicotine adenine dinucleotide phosphate (NADP), etc.

[00131] A 50C activator is a substance that initiates or stimulates an acid or base formation reaction with secondary substrate 34. In some cases, the 50C activator functions similarly to the 50B cofactor, and as such, any examples of the 50B cofactor can be used for the 50C activator. For metal dependent hydrolases, examples of suitable 50C activators include divalent metals.

[00132] In complex 30A, polymerase 28 and any example of component 50A-50C pH changes are conjugated to each other. In one example, this 30A complex is a fusion protein or protein chimera. The 30A complex can be formed using conjugation methods such as SpyTag/SpyCatcher coupling, β-fixer mediated cysteine conjugation, biotin/streptavidin, SUMO (small ubiquitin-like modifier) protein, His6/NTA metal chelation, cysteine /maleimide, dibenzocyclooctyne (DBCO)/azide or any other suitable conjugation method.

[00133] Complex 30A may be connected to conduction channel 26 via anchor 32, examples of which are described with reference to FIGS. 2. Alternatively, complex 30A can be directly attached via a bond between the pH change component 50A-50C and the groups on the surface of the conduction channel 26.

[00134] Although not shown, the readout system 10B may also include a detector to detect voltage and/or current changes that correspond to a change in pH occurring at/near the surface of the pH readout channel 26.

[00135] Also, although not shown, the reader system 10B may be positioned on a support, such as a silicon chip. The electrodes 22, 24 can be connected to an electronic circuit that makes them work (for example, after being connected to a detector and a power source).

[00136] In some examples, sensor 10B may come pre-assembled.

[00137] In other examples, the sensor components may be part of a kit, and the kit components may be used to assemble the sensor 10B. An exemplary kit includes a pH sensor 20 on a support and a fluid that is used to inject complex 30A into pH sensor 20. The fluid includes a carrier fluid and complex 30A. In some examples, the fluid complex 30A is attached to tether 32. In other examples, tether 32 is attached to pH readout channel 26 as part of readout system 10B. In still other examples where there is a direct link created between the pH change component 50A-50C and the surface groups of the channel 26, it should be understood that the 30A fluid complex does not attach to anchor 32 and anchor 32 does not attach to the pH-reader. channel 26. In an example, the carrier fluid is water or an example of a low buffer fluid such as citrate salt at millimolar concentrations.

[00138] In one example of this kit, complex 30A in the carrier fluid includes polymerase 28 associated with at least one enzyme 50A. In another example of this kit, complex 30A in the carrier fluid includes polymerase 28 conjugated to cofactor 50B. In yet another example of this kit, the 30A complex in the carrier fluid includes polymerase 28 conjugated to the 50C activator. In each of these examples, assembly 30A may also include a tie 32. Using these set examples, the user may deposit fluid on the support and allow the fluid to remain on the support for a suitable time to attach, via tie 32, assembly 30A to conductive channel 26, to attach, via complex 30A, to anchor 32 on channel 26, or to bond, via pH change component 50A-50C, to channel 26 surface groups. The support may be washed with a suitable buffer to remove all unbound complexes 30A.

[00139] Some examples of the kit may also include fluid(s) to be used with the 10B reader system during single molecule readings. The fluid content may vary depending on which pH change component 50A-50C is part of the complex 30A of the readout system 10B and which labeled nucleotides 12, 12A-12C or 12' are used. Several examples of these fluids are described below.

[00140] In the examples shown in FIG. 3A, the fluid(s) to be used during single molecule sequencing includes any example of the carrier fluid disclosed herein, labeled nucleotides 12 or 12A-12C, and secondary substrate 34 (which, in these examples, is is a separate molecule relative to labeled nucleotides 12 or 12A-12C).

[00141] The 18B or 18C label that attaches to labeled nucleotides 12 or 12A-12C would depend on which pH change component is included in the 30A complex.

[00142] When reading system 10B includes enzyme 50A as part of complex 30A, label 18B is used. As mentioned herein, enzyme 50A forms an acid or base in reaction with secondary substrate 34 when they are in proximity to each other. To correlate the acid or base formation reaction between enzyme 50A and secondary substrate 34 with labeled nucleotide 12 or 12A-12C incorporated, the 18B label on labeled nucleotides 12 or 12A-12C can be individually tailored to improve the kinetics of the 50A enzyme or slow down the kinetics of the 50A enzyme. In this regard, label 18B is one example of a pH change component that attaches to labeled nucleotide 12 or 12A-12C. This example system 10B therefore includes two different examples of the pH change component. Thus, in one example, the 18B label is selected from the group consisting of a first group that improves the kinetics of the 50A enzyme and a second group that slows down the kinetics of the 50A enzyme.

[00143] When the 18B label improves the kinetics of the 50A enzyme, it also improves (e.g., initiates or accelerates) the kinetics of the acid or base formation reaction comprising the 50A enzyme and the secondary substrate 34. Examples of 18B labels that improve the kinetics of the 50A enzyme include themselves cofactors of the enzyme. Examples of other 18B labels that improve the 50A enzyme kinetics include labels that have a crowding effect. This type of label 18B can be a crowding agent that can reduce the amount of water available to other molecules and effectively increase the local concentration of secondary substrate 34 (which in turn increases the reaction rate).

[00144] In contrast, when the 18B label retards the kinetics of the 50A enzyme, it also retards the kinetics of the acid or base formation reaction comprising the 50A enzyme and the secondary substrate 34. Examples of 18B labels that retard the kinetics of the 50A enzyme may be selected from the group consisting of an allosteric inhibitor, a steric exclusion group, and a buffering group. The allosteric inhibitor binds to the allosteric site of the 50A enzyme and changes the conformation of the active site of the 50A enzyme. As a result, enzyme 50A is no longer able to react with secondary substrate 34 and becomes inactive. It may be desirable if the binding between the allosteric inhibitor and the 50A enzyme is weaker than the binding between polymerase 28 and the base of the labeled nucleotide 12 or 12A-12C such that the labeled nucleotides 12 or 12A-12C are not linked only through the label 18B. The steric exclusion group may be a molecule that is bulkier than secondary substrate 34. The steric exclusion group of the included labeled nucleotide may block secondary substrate 34 from coming close enough to enzyme 50A to react. In other words, the steric exclusion group can reduce access to the 50A enzyme without actually binding to the 50A enzyme. The buffering group can act as a local buffer to absorb protons formed during the acid formation reaction or base molecules formed during the base formation reaction and thereby neutralize the pH. By way of example, dendrimers having multiple acid or base groups can serve to buffer the local vicinity of the conduction channel 26.

[00145] In an example, 18B labels that attach to different nucleotides 12 or 12A-12C (eg, A, T, C, G) can be selected to increase or decrease acid or base formation at different rates. In this example, when one of the labeled nucleotide 12 or 12A-12C is turned on by polymerase 28, label 18B moves to a position in the vicinity of enzyme 50A and secondary substrate 34 and enhances or slows down the response at a unique label-dependent rate. In turn, the pH in the conductive channel 26 changes. The change in pH is correlated with increased or decreased acid or base formation and can therefore be used to identify the included labeled nucleotide 12 or 12A-12C.

[00146] When reading system 10B includes cofactor 50B or activator 50C as part of complex 30A, label 18C is used. The 18C label may be an enzyme that forms an acid or base in reaction with secondary substrate 34. In this regard, the 18C label is one example of a pH change component that attaches to a labeled nucleotide 12 or 12A-12C. This example system 10B therefore includes two different examples of the pH change component. When a labeled 12 or 12A-12C nucleotide is incorporated by polymerase 28, the 18C enzyme tag moves to a position adjacent to the 50B cofactor or 50C activator and secondary substrate 34. The attached 50B cofactor or 50C activator can enhance or initiate the reaction between the 18C enzyme tag and secondary substrate 34 in a fluid environment.

[00147] Because labeled nucleotides 12 or 12A-12C do not include secondary substrate 34, the fluid(s) provided in the example of FIG. 3A also includes secondary substrate 34. Secondary substrate 34 can be any secondary substrate 34 that can react with enzyme 50A to form an acid or base, or whose reaction with enzyme label 18C can be enhanced or initiated by cofactor 50B. or activator 50C.

[00148] The following description relates to an exemplary method that includes labeled 12 nucleotides and a 10B readout system. In this example, template polynucleotide strand 44 may be introduced into reader system 10B in a fluid such as a biologically stable solution, together with or separately from labeled nucleotides 12 and secondary substrate 34. Polymerase 28 binds to template polynucleotide strand 44 such that strand 44 is held in place. The complementary base of one of the labeled nucleotides 12 must be incorporated into the nascent strand 46 (which is formed along the template polynucleotide strand 44) by polymerase 28. During the inclusion event, the label 18B or 18C of the labeled nucleotide 12 appears in the vicinity of channel 26, the pH change component 50A-50C and any secondary substrate 34 in a fluid that is also in the vicinity of the pH change component 50A-50C. The reaction of acid or base formation with secondary substrate 34 may be enhanced, retarded, or inhibited depending on the complex 30A and labels 18B or 18C that are used, which changes the pH of the fluid and the charge on the surface of the channel 26. In this exemplary method, after incorporation of the nucleotide base into the resulting chain 46, the bond between alpha-phosphate and binding molecule 16 or between alpha-phosphate and beta-phosphate is naturally cleaved. This natural cleavage allows the 18B or 18C label (and binding molecule 16) to dissociate from the incorporated nucleotide base and diffuse away from the 10B reader system. Polymerase 28 can then accept another labeled nucleotide 12, which includes a nucleotide base that is complementary to the next nucleotide in the 44 template polynucleotide strand.

[00149] The following description relates to an exemplary method that includes labeled nucleotides 12A-12C and a 10B readout system. In this example, template polynucleotide strand 44 may be introduced into reader system 10B in a fluid such as a biologically stable solution, together with or separately from labeled nucleotides 12A-12C and secondary substrate 34. Polymerase 28 binds to template polynucleotide strand 44 such that chain 44 is held in place. The complementary base of one of the labeled nucleotides 12A-12C must be incorporated into the nascent strand 46 (which is formed along the template polynucleotide strand 44) by polymerase 28. The reaction of acid or base formation with secondary substrate 34 may be enhanced, retarded, or inhibited depending on the 30A complex and labels 18B or 18C are used, which changes the pH of the fluid and the charge on the surface of the channel 26. In this example, the labeled nucleotide 12A-12C includes a reversible terminator. In this regard, the inclusion of the labeled nucleotide 12A-12C serves as a polymerization terminator. This provides improved, delayed or inhibited acid or base formation and charge detection similar to those described above. In this exemplary method, washing and exposure to a deblocking agent may be used to prepare the 10B read system for another sequencing run.

[00150] In any of the examples described with reference to FIG. 3A, it should be understood that the distance between polymerase 28 and the pH change component 50A-50C and the length of the binding molecule 16, 16' can be chosen such that the label 18B or 18C moves to a position proximate the pH change component 50A-50C when labeled nucleotide 12 or 12A-12C is incorporated by polymerase 28. This provides predictable effects on the acid or base formation reaction.

[00151] As mentioned herein, the reading system 10B can also be used with labeled nucleotide 12', which, as described with reference to FIG. 1C includes secondary substrate 34 attached to nucleotide 14. FIG. 3B illustrates system 10B and labeled nucleotide 12'.

[00152] In this example, the fluid in the kit includes labeled nucleotides 12', not labeled nucleotides 12 or 12A-12C. Unlike the fluids described with reference to FIG. 3A, this fluid does not include a separate secondary substrate 34 because secondary substrate 34 attaches to nucleotide 14.

[00153] When reading system 10B includes enzyme 50A as part of complex 30A, the fluid available to system 10B may include carrier fluid and labeled nucleotides 12'. When polymerase 28 includes the base of labeled nucleotides 12', secondary substrate 34 moves to a position in the vicinity of enzyme 50A and an acid or base formation reaction can proceed.

[00154] When reading system 10B includes cofactor 50B or activator 50C as part of complex 30A, the fluid available to system 10B may include carrier fluid and labeled nucleotides 12'. This example kit may also include an enzyme fluid that is separate from the fluid containing the labeled nucleotides 12'. The enzyme fluid is included because neither labeled nucleotide 12' nor complex 30A contains an enzyme that is involved in an acid or base formation reaction with secondary substrate 34. The enzyme fluid may include a carrier fluid (e.g., any of the buffers disclosed herein) and an enzyme 54 which is to participate in the consumption of the secondary substrate 34. The enzyme 54 used depends on the secondary substrate 34, which is attached to the labeled nucleotide 12'.

[00155] When fluid(s) containing the 44 template polynucleotide strand and the 12' labeled nucleotide, and, in some cases, the enzyme fluid, are introduced (successively or simultaneously) into the 10B reader system, the 44 template polynucleotide strand binds to polymerase 28, and one complementary labeled nucleotide 12' is included in the growing nascent strand 46. In this example, enzyme 50A of complex 30A or free-floating enzyme 54 reacts to consume secondary substrate 34 in order to form an acid or base, and this reaction must be faster nucleotide incorporation. In these examples, secondary substrate 34 is consumed, and upon consumption, the pH should return to baseline and the nucleotide base should be included.

[00156] 10C Reader System

[00157] Referring specifically to FIG. 4, the reading system 10C includes a pH sensor 20 and a complex 30B connected to a conductive channel 26 of the pH sensor 20.

[00158] Complex 30B includes polymerase 28 attached to two different enzymes 50A and 50D. Polymerase 28 of complex 30A may be any example of the polymerase described with reference to FIG. 2.

[00159] Enzymes 50A, 50D may be any example of the enzyme described with reference to FIG. 3A and FIG. 3B, provided that the two enzymes 50A, 50D are different. Enzymes 50A and 50D each form an acid or base in reaction with secondary substrate 34; however, enzymes 50A and 50D can be chosen such that they have a different response when in the vicinity of a particular 18D nucleotide tag. As one example, the 50A enzyme may respond to corresponding 18D labels attached to two types of labeled nucleotides 12 or 12A-12C (e.g., A and T) and may not respond to corresponding 18D labels attached to the other two types of labeled nucleotides 12 or 12A-12C (eg C and G). On the other hand, the 50D enzyme may respond to the corresponding 18D labels attached to the other two types of labeled nucleotides 12 or 12A-12C (for example, C and G), but may not respond to the corresponding 18D labels attached to two types of labeled nucleotides 12 or 12A-12C (eg, A and T). In this example, each 18D label, respectively, attached to nucleotides 12 or 12A-12C may be different from each other 18D label, respectively, attached to other nucleotides 12 or 12A-12C.

[00160] In complex 30B, polymerase 28 and two different enzymes 50A, 50D are conjugated together. In one example, this 30B complex is a protein fusion or chimera. The 30B complex can be formed using conjugation methods such as SpyTag/SpyCatcher coupling reaction, β-fixer mediated cysteine conjugation, biotin/streptavidin, SUMO (small ubiquitin-like modifier) protein, His6/NTA metal chelation, cysteine /maleimide, dibenzocyclooctyne (DBCO)/azide or any other suitable conjugation method.

[00161] In one example (as shown in FIG. 4), complex 30B can be attached to pathway 26 via anchors 32, 32' attached to each of enzymes 50A, 50D. In another example, one binding 32 may attach polymerase 28 to pathway 26 and enzymes 50A, 50D, respectively, may attach to polymerase 28. enzymes 50A, 50D can bind directly to the surface groups of the conduction channel 26 and to polymerase 28.

[00162] Although not shown, the readout system 10C may also include a detector to detect voltage and/or current changes that correspond to a change in pH occurring at/near the surface of the pH readout channel 26.

[00163] Also, although not shown, the reader system 10C may be positioned on a support, such as a silicon chip. The electrodes 22, 24 can be connected to an electronic circuit that makes them work (for example, after being connected to a detector and a power source).

[00164] In some examples, the sensor 10C may come pre-assembled.

[00165] In other examples, the sensor components may be part of a kit, and the kit components may be used to assemble the 10C sensor. An exemplary kit includes a pH sensor 20 on a support and a fluid that is used to inject complex 30B into pH sensor 20. The fluid includes a carrier fluid and complex 30B. In some examples, the fluid complex 30B is attached to the anchors 32, 32'. In other examples, the 30B fluid complex includes one binding 32 attached to polymerase 28 and enzymes 50A and 50D are attached to polymerase 28. In some other examples, attachment(s) 32, 32' are attached to the pH read -channel 26 as part of the reading system 10C. In still other examples where there is a direct bond created between the enzymes 50A, 50D and channel surface groups 26, it should be understood that the 30B complex in fluid does not attach to anchors 32, 32' and anchors 32, 32' do not attach. to the pH reading channel 26. In an example, the carrier fluid is water or an example of a low buffer fluid such as citrate saline at millimolar concentrations.

[00166] When using these kit examples, the user can deposit fluid on the support and allow the fluid to remain on the support for a suitable time to attach, via the bindings 32, 32', the assembly 30B to the conduit 26, to attach, by binding 32 polymerase 28 of complex 30B to conduction channel 26, or to bind, via enzymes 50A and 50D, to the surface groups of channel 26. The support can be washed with an appropriate buffer to remove any unbound 30B complexes.

[00167] Some examples of the kit may also include fluid(s) to be used with the 10C reader system during single molecule readings. In this example, the fluid(s) includes any example of the carrier fluid disclosed herein, labeled nucleotides 12 or 12A-12C and secondary substrate 34 (which, in these examples, is a separate molecule from labeled nucleotides 12 or 12A-12C).

[00168] The 18D tag that attaches to labeled nucleotides 12 or 12A-12C must be dependent on the two enzymes 50A, 50D of the 30B complex. As mentioned herein, enzymes 50A, 50D each form an acid or base in reaction with secondary substrate 34. To correlate a particular acid or base formation reaction between enzyme 50A and secondary substrate 34 or between enzyme 50D and secondary substrate 34, each label 18D at labeled nucleotides 12 or 12A-12C can be individually tailored to improve (eg, initiate or accelerate) the kinetics of the 50A enzyme or the 50D enzyme, or slow down the kinetics of the 50A enzyme or the 50D enzyme. In this regard, the tag 18D is one example of a pH change component that attaches to the labeled nucleotide 12 or 12A-12C. This example system 10B therefore includes two different examples of the pH change component. Any of the 18B labels can be used for the 18D labels, and each of the four nucleotides (eg, A, T, C, G) can be labeled with a different label that affects acid or base formation reactions differently. In one example, a label 18D selected for labeled nucleotide 12 or 12A-12C with "A" as the nitrogenous base can improve first rate acid formation when in proximity to enzyme 50A and secondary substrate 34; the 18D label selected for labeled nucleotide 12 or 12A-12C with "T" as the nitrogenous base can slow acid formation at the second rate when in the vicinity of enzyme 50A and secondary substrate 34; tag 18D selected for labeled nucleotide 12 or 12A-12C with "C" as the nitrogenous base can improve third rate base formation when in proximity to enzyme 50D and secondary substrate 34; and label 18D selected for labeled nucleotide 12 or 12A-12C with "G" as the nitrogenous base can slow base formation at speed four when in the vicinity of enzyme 50A and secondary substrate 34. When various labeled nucleotides are included in the resulting strand 46 , different pH levels should result locally on the surface of the charge sensitive channel 46.

[00169] Because labeled nucleotides 12 or 12A-12C do not include secondary substrate 34, the fluid(s) provided in the example of FIG. 4 also includes secondary substrate 34. Secondary substrate 34 can be any secondary substrate 34 that can react with enzyme 50A or 50D to form an acid or base.

[00170] The following description relates to an exemplary method that includes labeled 12 nucleotides and a 10C reading system. In this example, template polynucleotide strand 44 may be introduced into the 10C reading system in a fluid such as a biologically stable solution, together with or separately from labeled nucleotides 12 and secondary substrate 34. Polymerase 28 binds to template polynucleotide strand 44 such that strand 44 is held in place. The complementary base of one of labeled nucleotides 12 must be incorporated into the nascent strand 46 (which is formed along the template polynucleotide strand 44) by polymerase 28. During the inclusion event, label 18D of labeled nucleotide 12 appears in the vicinity of channel 26, enzymes 50A and 50D, and any secondary substrate 34 in a fluid that is also in the vicinity of enzymes 50A and 50D. The acid or base formation reaction with secondary substrate 34 may be enhanced, retarded, or inhibited depending on the 18D label and its effect on the reaction containing the 50A or 50D enzyme. This changes the pH of the fluid and the charge on the surface of the channel 26. In this exemplary method, after incorporation of a nucleotide base into the resulting strand 46, the bond between alpha phosphate and binding molecule 16 or between alpha phosphate and beta phosphate is naturally cleaved. This natural cleavage allows the 18D label (and binding molecule 16) to dissociate from the incorporated nucleotide base and diffuse away from the 10C reader system. Polymerase 28 can then accept another labeled nucleotide 12 that includes a nucleotide base that is complementary to the next nucleotide in the 44 template polynucleotide strand.

[00171] The following description relates to an exemplary method that includes labeled nucleotides 12A-12C and a 10C reading system. In this example, template polynucleotide strand 44 may be introduced into the 10C readout system in a fluid such as a biologically stable solution, together with or separately from labeled nucleotides 12A-12C and secondary substrate 34. Polymerase 28 binds to template polynucleotide strand 44 such that chain 44 is held in place. The complementary base of one of the labeled nucleotides 12A-12C must be incorporated into the nascent strand 46 (which is formed along the template polynucleotide strand 44) by polymerase 28. The acid or base formation reaction with secondary substrate 34 may be enhanced, slowed down, or inhibited depending on the 18D label and its effect on the reaction containing the enzyme 50A or 50D. This changes the pH of the fluid and the charge on the surface of the channel 26. In this example, the labeled nucleotide 12A-12C includes a reversible terminator. In this regard, the inclusion of the labeled nucleotide 12A-12C serves as a polymerization terminator. This provides improved, delayed or inhibited acid or base formation and charge detection similar to those described above. In this exemplary method, washing and exposure to a deblocking agent can be used to prepare the 10C read system for another sequencing run.

[00172] In any of the examples described with reference to FIG. 4, it should be understood that the distance between polymerase 28 and the corresponding enzymes 50A, 50D and the length of the binding molecule 16, 16' can be chosen such that the tag 18D moves to a position in the vicinity of enzyme 50A and/or and 50D when its labeled nucleotide 12 or 12A-12C is incorporated by polymerase 28. This provides predictable effects on the acid or base formation reaction.

[00173] 10D Reader System

[00174] Referring specifically to FIG. 5, the reading system 10D includes a pH sensor 20 and a complex 30C connected to a conductive channel 26 of the pH sensor 20.

[00175] Complex 30C includes a polymerase 28 associated with a metal coordination complex ("M"), which should create a pH change in the vicinity of the conductive channel 26 due to the consumption of secondary substrate 34 in the fluid that is opened to the pH sensor 20. In complex 30C, the ratio of polymerase 28 to metal coordination complex M is 1:1. The metal coordination complex M may attach directly to polymerase 28, or the binding may link them together.

[00176] Polymerase 28 of complex 30C can be any example of the polymerase described with reference to FIG. 2.

[00177] Metal coordination complex 50E is similar to enzyme 50A in that it forms an acid or base in reaction with secondary substrate 34. Examples of metal coordination complex include copper(II) complexes with ligands such as bis-(2-pyridylmethyl )-amine or pyridine-functionalized cyclodextrin.

[00178] In the 30C complex, polymerase 28 and the 50E metal coordination complex are conjugated together. The 30C complex can be formed using any number of conjugation methods such as SpyTag/SpyCatcher coupling reaction, p-fixer mediated cysteine conjugation, biotin/streptavidin, SUMO (small ubiquitin-like modifier) protein, His6/NTA metal chelation , cysteine/maleimide, dibenzocyclooctyne (DBCO)/azide, or any other suitable conjugation method.

[00179] Complex 30C may be attached to conduction channel 26. In the example shown in FIG. 5, the 50E metal coordination complex is attached to the surface of the channel 26, and polymerase 28 is attached to the 50E metal coordination complex. In this example, the attachment of the 30C complex can be via tether 32 (not shown in FIG. 5) or the terminal group of the 50E metal coordination complex. In another example, polymerase 28 attaches to the channel surface 26 and the 50E metal coordination complex attaches to polymerase 28. In this example, attachment of the 30C complex can be via tether 32 (not shown in FIG. 5) attached to polymerase 28.

[00180] Although not shown, the readout system 10D may also include a detector to detect voltage and/or current changes that correspond to a change in pH occurring on/near the surface of the pH readout channel 26.

[00181] Also, although not shown, the reader system 10D may be positioned on a support, such as a silicon chip. The electrodes 22, 24 can be connected to an electronic circuit that makes them work (for example, after being connected to a detector and a power source).

[00182] In some examples, the sensor 10D may come pre-assembled.

[00183] In other examples, the sensor components may be part of a kit, and the kit components may be used to assemble the sensor 10D. An exemplary kit includes a pH sensor 20 on a support and a fluid that is used to inject the 30C complex into the pH sensor 20. The fluid includes a carrier fluid and the 30C complex. In an example, the carrier fluid is water or an example of a low buffer fluid such as saline citrate at millimolar concentrations.

[00184] When using these kit examples, the user can deposit a fluid on the support and allow the fluid to remain on the support for a suitable time to attach, by tying 32 or the terminal end of the metal coordination complex 50E complex 30C to the conductive channel 26. Support may be washed with a suitable buffer to remove any unbound 30C complexes.

[00185] Some examples of the kit may also include fluid(s) to be used with the 10D reader system during single molecule reading. In this example, the fluid(s) includes any example of the carrier fluid disclosed herein, labeled nucleotides 12 or 12A-12C and secondary substrate 34 (which, in these examples, is a single molecule relative to labeled nucleotides 12 or 12A-12C).

[00186] The 18E tag that attaches to labeled nucleotides 12 or 12A-12C may be a ligand for the metal of the 50E metal coordination complex, wherein the ligand changes the catalytic property of the 50E metal coordination complex. The ligand can be selected to improve the kinetics of the 50E metal coordination complex or slow down the kinetics of the 50E metal coordination complex. In these examples, label 18E is one example of a pH change component that attaches to labeled nucleotide 12 or 12A-12C. This example system 10D therefore includes two different examples of a pH change component (eg, a 50E metal coordination complex and an 18E tag).

[00187] Other examples of the 18E tag can function as a separate catalyst (in addition to the 50E metal coordination complex) for consuming secondary substrate 34. Examples of 18E tags that function as separate catalysts include non-metal organocatalysts such as diazabicycloundecene, N -heterocyclic carbenes, tetramethylguanidine, etc. In these examples, label 18E is one example of a pH change component that attaches to labeled nucleotide 12 or 12A-12C. This example system 10D therefore includes two different examples of a pH change component (eg, a 50E metal coordination complex and an 18E tag).

[00188] Still other examples of the 18E label slow down the kinetics of the 50E metal coordination complex and therefore also slow the kinetics of the acid or base formation reaction comprising the 50E metal coordination complex and the secondary substrate 34. Examples of 18E labels that slow the kinetics of the metal coordination complex 50E can be any ligand that inhibits the catalytic activity of the metal, or any ligand that changes the electronic state of the metal to change the catalytic activity of the metal. In these examples, label 18E is one example of a pH change component that attaches to labeled nucleotide 12 or 12A-12C. This example system 10D therefore includes two different examples of a pH change component (eg, a 50E metal coordination complex and an 18E tag).

[00189] In an example, corresponding 18E labels that attach to different nucleotides 12 or 12A-12C (eg, A, T, C, G) can be selected to increase or decrease acid or base formation at different rates. In this example, when one of the labeled nucleotide 12 or 12A-12C is turned on by polymerase 28, the 18E label moves to a position adjacent to the 50E metal coordination complex and secondary substrate 34 and enhances or slows down the response at a unique label-dependent rate. In turn, the pH in the conductive channel 26 changes. The change in pH correlates with increased or decreased acid or base formation and can therefore be used to identify the included labeled 12 or 12A-12C nucleotide.

[00190] Since labeled nucleotides 12 or 12A-12C do not include secondary substrate 34, the fluid(s) provided in the example of FIG. 5 also includes secondary substrate 34. Secondary substrate 34 can be any secondary substrate 34 that can react with the 50E metal coordination complex to form an acid or base.

[00191] The following description relates to an exemplary method that includes labeled 12 nucleotides and a 10D reading system. In this example, template polynucleotide strand 44 may be introduced into the 10D readout system in a fluid such as a biologically stable solution, together with or separately from labeled nucleotides 12 and secondary substrate 34. Polymerase 28 binds to template polynucleotide strand 44 such that strand 44 is held in place. The complementary base of one of the labeled nucleotides 12 must be incorporated into the nascent strand 46 (which is formed along the template polynucleotide strand 44) by polymerase 28. During the insertion event, the label 18E of labeled nucleotide 12 appears in the vicinity of channel 26, the 50E metal coordination complex, and any secondary substrate 34 in a fluid that is also in the vicinity of metal coordination complex 50E. In some examples, the acid or base formation reaction with secondary substrate 34 may be enhanced, retarded, or inhibited depending on the 18E ligand tag and its effect on the reaction involving the 50E metal coordination complex. In other examples, the 18E tag may participate in the consumption of secondary substrate 34 along with the 50E metal coordination complex, thereby generating more acid or base. All of these examples change the pH of the fluid and the charge on the surface of the channel 26. In these exemplary methods, after incorporation of a nucleotide base into the resulting strand 46, the bond between alpha-phosphate and binding molecule 16 or between alpha-phosphate and beta-phosphate is naturally cleaved. In these examples, it may be desirable if the binding of the 18E ligand tag is sufficiently weak such that it rapidly weakens when the phosphate backbone is naturally cleaved. This natural cleavage allows the 18E label (and binding molecule 16) to dissociate from the incorporated nucleotide base and diffuse away from the 10D reading system. Polymerase 28 can then accept another labeled nucleotide 12, which includes a nucleotide base that is complementary to the next nucleotide in the 44 template polynucleotide strand.

[00192] The following description relates to an exemplary method that includes labeled nucleotides 12A-12C and a 10D reading system. In this example, template polynucleotide strand 44 may be introduced into the 10D readout system in a fluid such as a biologically stable solution, together with or separately from labeled nucleotides 12A-12C and secondary substrate 34. Polymerase 28 binds to template polynucleotide strand 44 such that chain 44 is held in place. The complementary base of one of the labeled nucleotides 12A-12C must be incorporated into the nascent strand 46 (which is formed along the template polynucleotide strand 44) by polymerase 28. In some examples, the acid or base formation reaction with the secondary substrate 34 may be enhanced, retarded, or inhibited depending on 18E ligand label and its effect on the reaction involving the 50E metal coordination complex. In other examples, the 18E tag may participate in the consumption of secondary substrate 34 along with the 50E metal coordination complex, thereby generating more acid or base. All of these examples change the pH of the fluid and the charge on the surface of the channel 26. In this example, the labeled nucleotide 12A-12C includes a reversible terminator. In this regard, the inclusion of the labeled nucleotide 12A-12C serves as a polymerization terminator. This provides improved, delayed or inhibited acid or base formation and charge detection similar to those described above. In this exemplary method, washing and exposure to a deblocking agent can be used to prepare the 10D reader system for another sequencing run.

[00193] In any of the examples described with reference to FIG. 5, it should be understood that the distance between polymerase 28 and the 50E metal coordination complex and the length of the binding molecule 16, 16' can be chosen such that the 18E tag moves to a position near the 50E metal coordination complex when its labeled nucleotide 12 or 12A-12C is switched on by polymerase 28. This provides predictable effects on the acid or base formation reaction.

[00194] Reader System 10E

[00195] Referring specifically to FIG. 6A and FIG. 6B, the reading system 10D includes a pH sensor 20 and a complex 30D connected to a conductive channel 26 of the pH sensor 20.

[00196] Complex 30D includes polymerase 28 associated with enzyme 50A. In this example, complex 30D further includes a nucleic acid hairpin/enzyme inhibitor conjugate 58 attached to enzyme 50A.

[00197] Polymerase 28 of complex 30D may be any of the examples of the polymerase described with reference to FIG. 2.

[00198] Enzyme 50A can be any example of the enzyme described with reference to FIG. 3A and FIG. 3b.

[00199] Nucleic acid hairpin/enzyme inhibitor conjugate 58 includes a single stranded DNA or RNA having two regions that are complementary and that undergo intramolecular base pairing to form a helical double helix that terminates in an unpaired loop. One end of the unpaired loop is attached to enzyme 50A, and the other end of the unpaired loop includes an enzyme inhibitor 60. In the contour configuration, as shown in FIG. 6A, the enzyme inhibitor 60 at least reduces the activity of the enzyme.

[00200] In complex 30D, enzyme 50A and nucleic acid hairpin/enzyme inhibitor conjugate 58 are conjugated to each other, and enzyme 50A is also conjugated to polymerase 28.

[00201] Complex 30D may be connected to conduction channel 26 via tie-in 32, examples of which are described with reference to FIGS. 2. Alternatively, the 30D complex can be directly attached via binding between the pH change component 50A and the groups on the surface of the conduction channel 26.

[00202] Although not shown, the readout system 10E may also include a detector to detect voltage and/or current changes that correspond to a change in pH occurring at/near the surface of the pH readout channel 26.

[00203] Also, although not shown, the reader system 10E may be positioned on a support, such as a silicon chip. The electrodes 22, 24 can be connected to an electronic circuit that makes them work (for example, after being connected to a detector and a power source).

[00204] In some examples, sensor 10E may come pre-assembled.

[00205] In other examples, the sensor components may be part of a kit, and the kit components may be used to assemble the sensor 10E. An exemplary kit includes a pH sensor 20 on a support and a fluid that is used to inject the 30D complex into the pH sensor 20. The fluid includes a carrier fluid and the 30D complex. In some examples, the 30D fluid complex is attached to tether 32. In other examples, tether 32 is attached to pH readout channel 26 as part of the 10E readout system. In still other examples where there is a direct link created between the pH change component 50A and the surface groups of channel 26, it should be understood that the 30D complex in fluid does not attach to tether 32 and tether 32 does not attach to pH readout channel 26 In an example, the carrier fluid is water or an example of a low buffer fluid such as saline citrate at millimolar concentrations.

[00206] When using these kit examples, the user can deposit fluid on the support and allow the fluid to remain on the support for a suitable time to attach, by tying 32, the complex 30D to the conduit 26, to attach, via the complex 30D, to binding 32 on channel 26, or to bind, via pH change component 50A, to the surface groups of channel 26. The support may be washed with a suitable buffer to remove any unbound 30D complexes.

[00207] Some examples of the kit may also include fluid(s) to be used with the 10E reader system during single molecule readings. In this example, the fluid(s) includes any example of the carrier fluid disclosed herein, labeled nucleotides 12 or 12A-12C and secondary substrate 34 (which, in these examples, is a single molecule relative to labeled nucleotides 12 or 12A-12C).

[00208] The 18F tag that attaches to labeled nucleotides 12 or 12A-12C is a chain of nucleotides that includes bases that are complementary to at least some of the nucleotide bases of the 58 nucleic acid hairpin/enzyme inhibitor conjugate. The 18F label allows initiation of a hairpin opening reaction and then base pairing with a portion of one DNA or RNA strand of a 58 nucleic acid hairpin/enzyme inhibitor conjugate. As shown in FIG. 6B, the introduction and reaction of the 18F label moves the enzyme inhibitor 60 away from the enzyme 50A, thereby providing its enzymatic activity. Because the 18F label can affect enzymatic activity that results in acid or base formation, it is also one example of a pH change component. An acid or base forming reaction with the secondary substrate 34 can occur when the 18F label opens the nucleic acid hairpin/enzyme inhibitor conjugate 58, resulting in a pH change at the surface of the conduction channel 26. The opening and closing of the hairpin can be a rapid process resulting in a moderate level of polymerase inhibition. 28. Thus, different levels of activity or inhibition can be achieved using different numbers of complementary bases, thereby allowing base discrimination.

[00209] Since labeled nucleotides 12 or 12A-12C do not include secondary substrate 34, the fluid(s) provided in the example of FIG. 6A and 6B also includes secondary substrate 34. Secondary substrate 34 can be any secondary substrate 34 that can react with enzyme 50A to form an acid or base.

[00210] The following description relates to an exemplary method that includes labeled 12 nucleotides and a 10E reading system. In this example, template polynucleotide strand 44 may be introduced into the 10E reader system in a fluid such as a biologically stable solution, together with or separately from labeled nucleotides 12 and secondary substrate 34. Polymerase 28 binds to template polynucleotide strand 44 such that strand 44 is held in place. The complementary base of one of the labeled nucleotides 12 must be incorporated into the nascent strand 46 (which is formed along the template polynucleotide strand 44) by polymerase 28. any secondary substrate 34 in a fluid that is also in the vicinity of the nucleic acid hairpin/enzyme inhibitor conjugate 58. The 18F label opens the nucleic acid hairpin/enzyme inhibitor conjugate 58, which allows the enzyme 50A to react with the secondary substrate 34 to form an acid or base. This changes the pH of the fluid and the charge on the surface of the channel 26. In this exemplary method, after incorporation of a nucleotide base into the resulting strand 46, the bond between alpha phosphate and binding molecule 16 or between alpha phosphate and beta phosphate is naturally cleaved. In these examples, it may be desirable if the binding of the 18F ligand tag is sufficiently weak such that it rapidly weakens when the phosphate backbone is naturally cleaved. This natural cleavage allows the 18F label (and binding molecule 16) to dissociate from the incorporated nucleotide base and diffuse away from the 10E reader system. The nucleic acid hairpin/enzyme inhibitor conjugate 58 returns to the origin of the loop, thereby again at least partially inhibiting enzyme 50A activity. Polymerase 28 can then accept another labeled nucleotide 12, which includes a nucleotide base that is complementary to the next nucleotide in the 44 template polynucleotide strand.

[00211] The following description relates to an exemplary method that includes labeled nucleotides 12A-12C and a 10E reading system. In this example, template polynucleotide strand 44 may be introduced into the 10C readout system in a fluid such as a biologically stable solution, together with or separately from labeled nucleotides 12A-12C and secondary substrate 34. Polymerase 28 binds to template polynucleotide strand 44 such that chain 44 is held in place. The complementary base of one of the labeled nucleotides 12A-12C must be incorporated into the nascent strand 46 (which is formed along the template polynucleotide strand 44) by polymerase 28. The 18F tag opens the nucleic acid hairpin/enzyme inhibitor conjugate 58, allowing enzyme 50A to react with secondary substrate 34 for to form an acid or base. This changes the pH of the fluid and the charge on the surface of the channel 26. In this example, the labeled nucleotide 12A-12C includes a reversible terminator. In this regard, the inclusion of the labeled nucleotide 12A-12C serves as a polymerization terminator. This allows acid or base formation and charge detection to be carried out in a manner similar to that described above. In this exemplary method, washing and exposure to a deblocking agent can be used to prepare the 10E reader system for another sequencing run.

[00212] In any of the examples described with reference to FIG. 6A and 6B, it should be understood that the distance between polymerase 28 and the NA hairpin/enzyme inhibitor conjugate 58 and the length of the binding molecule 16, 16' can be chosen such that the label 18F moves to a position in the vicinity of the NA hairpin/enzyme inhibitor conjugate 58 when its labeled nucleotide 12 or 12A-12C is switched on by polymerase 28. This provides predictable effects on the acid or base formation reaction.

[00213] Reading system arrays

[00214] Any example of single molecule reading systems 10A-10E may be included in an array of such reading systems. In some examples, the array may include multiple reader systems 10A-10E, each positioned on a support and electronically configured to be individually addressable and readable. In an example, each array reader system 10A-10E may be positioned on a support in a separate recess. The recesses physically separate each of the readout systems 10A-10E, which at least reduces cross-contamination of pH gradients with adjacent readout systems 10A-10E. The array of readout systems 10A-10E may be part of a flow cell 62, an example of which is illustrated in FIG. 7A. In this example, flow cell 62 includes flow channels 64. Although multiple flow channels 64 are shown, it should be understood that any number of channels 64 may be included in flow cell 62 (e.g., one channel 64, four channels 64, etc. ). Each flow channel 64 is a zone defined between two associated components (eg, a substrate and a lid, or two substrates) that may have fluids (eg, the fluids described herein) introduced into and removed from them. Each flow channel 64 may be isolated from the other flow channel 64 such that fluid introduced into any particular flow channel 64 does not leak into any adjacent flow channel 64. Some examples of fluids introduced into flow channels 64 may introduce components reactions (eg, complexes 30A-30D, labeled nucleotides 12, 12A-12C, 12', etc.), wash solutions, deblocking agents, etc.

[00215] An example architecture in the flow channels 64 of the flow cell 62 is shown in FIG. 7B.

[00216] In the example shown in FIG. 7B, flow cell 64 includes a support 66 and patterned material 68 positioned on support 66. Patterned material 68 defines recesses 70 separated by intermediate regions 72. In this example, the surface of support 66 is exposed in each of recesses 70, and the reader system 10A-10E is positioned on the surface.

[00217] Support 66 in FIG. 7B provides support for the other components of the flow cell 62. Support 66 is generally rigid and insoluble in aqueous liquid. Examples of suitable supports 66 include epoxy, glass, modified glass, plastics, nylon, ceramic/ceramic oxides, silicon dioxide (silicon oxide (SiO ₂ )), fused silica, silica based materials, aluminum silicate, silicon, modified silicon (for example, boron-doped p+-silicon), silicon nitride (Si ₃ N ₄ ), tantalum pentoxide (TaO ₅ ) or other tantalum oxide(s) (TaO _x ), hafnium oxide (HfO ₂ ), inorganic glasses, etc. . Some examples of suitable plastics for substrate 14 include acrylic polymers, polystyrene, copolymers of styrene and other materials, polypropylene, polyethylene, polybutylene, polyurethanes, polytetrafluoroethylene (such as Teflon® from Chemours), cyclic olefins/cycloolefin polymers (COPs) (such as zeonor® by Zeon), polyimides, etc. The support may also be glass or silicon with a coating layer of tantalum oxide or other ceramic oxide on the surface.

[00218] The shape of support 66 may be a laminated plate, panel, rectangular sheet, die, or any other suitable configuration. In an example, support 66 may be a circular laminated plate or panel having a diameter ranging from about 2 mm to about 300 mm. As a more specific example, support 66 is a sandwich plate having a diameter ranging from about 200 mm to about 300 mm. In another example, support 66 may be a rectangular sheet or panel having the largest dimension up to about 10 feet (~3 meters). As a specific example, support 66 is a die having a width ranging from about 0.1 mm to about 10 mm. While exemplary dimensions are provided, it should be understood that support 66 may be used in any suitable dimension.

[00219] In the example shown in FIG. 7B, patterned material 68 is positioned on support 66. It should be understood that any material that can be selectively deposited or deposited and patterned to form depressions 70 and intermediate regions 72 can be used for patterned material 68.

[00220] As one example, the inorganic oxide can be selectively applied to support 66 via vapor deposition, aerosol printing, or inkjet printing. Examples of suitable inorganic oxides include tantalum oxide (eg Ta ₂ O ₅ ), alumina (eg Al ₂ O ₃ ), silicon oxide (eg SiO ₂ ), hafnium oxide (eg HfO ₂ ), etc. .

[00221] As another example, resin can be applied to support 66 and then patterned. Suitable deposition technologies include chemical vapor deposition, dip coating, dipping coating, spin coating, spray coating, pot pouring, ultrasonic spray coating, knife coating, spray printing, screen printing, micro contact printing, etc. Suitable patterning technologies include photolithography, nanoprinting lithography (NIL), stamping technologies, embossing technologies, molding technologies, microetching technologies, printing technologies, etc. Some examples of suitable resins include polyhedral oligomeric silsesquioxane resin (POSS), non-POSS epoxy, poly(ethylene glycol) resin, polyester resin (e.g., ring-opening epoxies), acrylic, acrylate, methacrylate, amorphous fluoropolymer resin (e.g., Cytop® from Bellex), and combinations of the above.

[00222] As used herein, the term "polyhedral oligomeric silsesquioxane (POSS)" means a chemical compound that is a hybrid intermediate (eg, RSiO _1.5 ) between silica (SiO ₂ ) and silicone (R ₂ SiO). An example of a POSS may be the POSS described in Kehagias et al., "Microelectronic Engineering 86" (2009), pp. 776-778, which is incorporated by reference in its entirety. In an example, the composition is an organosilicon compound with the chemical formula [RSiO _3/2 ] _n in which the R groups may be identical or different. Exemplary R groups for POSS include epoxide, azide/azido, thiol, poly(ethylene glycol), norbornene, tetrazine, acrylates and/or methacrylates, or additionally, for example, alkyl, aryl, alkoxy and/or haloalkyl groups. The resin composition disclosed herein may contain one or more different cellular or core structures as monomeric units.

[00223] As another example, the patterned material 68 may be a pH buffer material. The pH buffer material has a high pH buffer capacity which allows the use of a fluid with a low buffer solution concentration. An example of a high buffering pH capacity can be in the range of about 0.01 meq/g to about 2 meq/g. An example of a pH buffer material is SiCOH, which has a large surface area and a high buffer capacity. In instances where the patterned material 68 is not a pH buffer material, the sidewalls of the recesses 70 may be coated with a pH buffer material to provide a high pH buffer capacity for each of the recesses 70.

[00224] As shown in FIG. 7B, patterned material 68 includes recesses 70 defined therein and intermediate regions 72 separating adjacent recesses 70. Many different patterns of recesses 70 can be provided, including regular, repeating, and irregular patterns. In an example, recesses 70 are arranged in a hexagonal pattern for tight packing and increased density. Other layouts may include, for example, straight (rectangular) layouts, triangular layouts, and so on. In some examples, the layout or pattern may be the x-y format of the recesses 70 that are in rows and columns. In some other examples, the pattern or pattern may be a repeating arrangement of recesses 70 and/or intermediate regions 72. In yet other examples, the pattern or pattern may be a random arrangement of recesses 70 and/or intermediate regions 72. The pattern may include dots, pillows, holes, columns, stripes, swirls, lines, triangles, rectangles, circles, arcs, ticks, cells, diagonals, arrows, squares and / or hatching.

[00225] The pattern or pattern of recesses 70 may be characterized with respect to the density of recesses 70 (number of recesses 70) in a given area. For example, recesses 70 may be present at a density of approximately 2 million per mm ² . The density can be adjusted as various densities, including, for example, a density of approximately 100 per mm ² , approximately 1000 per mm ² , approximately 0.1 million per mm ² , approximately 1 million per mm ² , approximately 2 million per mm ² , approximately 5 million per mm ² , approximately 10 million per mm ² , approximately 50 million per mm ² or more or less. Additionally, it should be understood that the density of the recesses 70 in the patterned material 68 may be between one of the lower values and one of the upper values selected from the above ranges. As examples, a high density grating can be characterized as having pits 70 separated by less than about 100 nm, a medium density grating can be characterized as having pits 20 separated by about 400 nm to about 1 µm, and a low density grating can be characterized as having depressions 70 separated by more than about 1 micron. While exemplary densities are provided, it should be understood that any suitable densities may be used. The density of the recesses 70 may depend, in part, on the depth of the recesses 70 and the diffusivity of the formed acid or base. In some cases, it may be desirable if the spacing between the recesses is even greater than in the examples listed herein.

[00226] The pattern or pattern of recesses 70 can also or alternatively be characterized in terms of average pitch or spacing from the center of recess 70 to the center of adjacent recess 70 (center-to-center spacing), or from the edge of one recess 70 to the edge of adjacent recess 70 (inter-edge spacing). The pattern may be regular such that the coefficient of variation around the average pitch is small, or the pattern may be irregular, in which case the coefficient of variation may be relatively large. In any case, the average pitch, for example, may be about 50 nm, about 0.1 µm, about 0.5 µm, about 1 µm, about 5 µm, about 10 µm, about 100 µm, or more or less. The average pitch for a particular recess pattern 20 may be between one of the lower values and one of the upper values selected from the above ranges. In the example, the recesses 20 have a pitch (center-to-center spacing) of approximately 1.5 µm. While exemplary average pitch values are provided, it should be understood that other average pitch values may be used.

[00227] The size of each recess 70 may be characterized by its volume, hole area, depth, and/or diameter.

[00228] Each recess 70 may have any volume that allows fluid restriction. The minimum volume or maximum volume may be selected, for example, to accommodate throughput (e.g., multiplexability), resolution, labeled nucleotides 12, 12A-12C, or 12', secondary substrates 34, or analyte reactivity expected for downstream uses of the flow cell 62. For example, the volume may 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 more. Alternatively or additionally, the volume may 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. The area occupied by each recess opening may be selected based on criteria similar to those set out above for volume. For example, the area for each recess hole may be at least about 1*10 ^-3 µm ² , at least about 1*10 ^-2 µm ² , at least about 0.1 µm ² , at least , about 1 µm ² , at least about 10 µm ² , at least about 100 µm ² or more. Alternatively or additionally, the area may be at most about 1*10 ³ µm ² , at most about 100 µm ² , at most about 10 µm ² , at most about 1 µm ² , at most about 0.1 µm ² , at most about 1*10 ^-2 µm ² or less. The area occupied by each opening of the recess may be greater, less, or between the values indicated above.

[00229] The depth of each recess 70 may be large enough to accommodate one reader system 10A-10E. In an example, the depth may be at least about 1 µm, at least about 10 µm, at least about 100 µm, or greater. Alternatively or additionally, the depth may be at most about 1*10 ³ µm, at most about 100 µm, at most about 10 µm or less. The depth of each recess 70 may be greater than, less than, or between the values indicated above.

[00230] In some cases, the diameter or length and width of each recess 70 may be at least about 50 nm, at least about 0.1 microns, at least about 0.5 microns, at least about 1 µm, at least about 10 µm, at least about 100 µm or more. Alternatively or additionally, the diameter or length and width may be at most about 1* ¹⁰³ µm, at most about 100 µm, at most about 10 µm, at most about 1 µm, at most about 0.5 µm, at most about 0 .1 µm or less (eg, approximately 50 nm). The diameter or length and width of each recess 70 may be greater than, less than, or between the values indicated above.

[00231] As illustrated in FIG. 7B, each of the recesses 70 in the array includes a respective sensor 10A-10E. Desirably, each sensor 10A-10E in each recess 70 has one polymerase 28, either alone or as part of a complex 30A-30D attached thereto. In some examples, each sensor 10A-10E in each recess 70 has one polymerase 28 or complex 30A-30D attached to it. In other examples, some sensors 10A-10E in some recesses 70 have one polymerase 28 or complex 30A-30D attached to them; other sensors 10A-10E in other recesses 70 have more than one polymerase 28 or complex 30A-30D attached to them; and still other sensors 10A-10E in other recesses 70 do not have polymerase 28 or complex 30A-30D attached to them. In these examples, the number of polymerases 28 or complexes 30A-30D that become attached to any given reading system 10A-10E may be random and determined by a Poisson distribution.

[00232] To attach polymerases 28 or complexes 30A-30D, the method includes introducing a fluid into a sensor array including a plurality of separately addressable conductive channels 26, thereby attaching polymerase 28 or complex 30A-30D to at least to some of a plurality of separately addressable pathways 26, a complex 30A-30D including polymerase 28, and other examples of a pH change component 50A-50E associated with polymerase 28, wherein the pH change component 50A-50E is selected from the group consisting of enzyme 50A , which should catalyze the consumption of secondary substrate 34 in solution, which should be available to the sensor array, the 50E metal coordination complex, which should catalyze the consumption of secondary substrate 34 in solution, which should be available to the sensor array, and cofactor 50B or catalytic label activator 50C attached to the labeled nucleotide to be inserted into the sensor lattice ov. The fluid may be allowed to incubate for the required time and at the required temperature to allow polymerases 28 or complexes 30A-30D to attach.

[00233] In any of the exemplary methods disclosed herein for reading single molecules, one of the labeled nucleotides 12 or 12A-12C or 12' is incorporated, via the appropriate polymerase 28, into the resulting strand 46, which is formed in the reading system 10A-10E in each of the recesses 70. In other words, in each template polynucleotide strand 44 in the flow cell 62, the respective polymerases 28 expand the resulting strand 46 with one of the nucleotides 12, 12A-12C, or 12' present in the injected fluid.

[00234] Each of the sensors 10A-10E is separately electrically addressable and readable. In this regard, the charge signals resulting from the pH changes occurring in each recess 70 in response to the consumption of the secondary substrate 34 can be separately detected and analyzed to identify the included labeled nucleotide 12, 12A-12C or 12'. The acid or base forming reaction that occurs in each well will depend on the combination of labels 18A-18F and polymerases 28 or complexes 30A-30D that is used, examples of which are described with reference to FIGS. 3A and FIG. 3B, FIG. 4, fig. 5 and FIG. 6A and FIG. 6b.

[00235] Ensemble detection

[00236] Other examples of reader systems 40A and 40B are shown in FIG. 7C and FIG. 7D and these reading systems 40A and 40B can be used in reading ensembles. In these examples, readout systems 40A and 40B may be positioned in each of the recesses 70 of the flow cell 62. The flow cell 62 includes channels 64, support 66, patterned material 68, and patterned material 68 as described herein.

[00237] In the example shown in FIG. 7C, the reading system 40A includes a pH sensor 20 and primers 74 attached to a conductive channel 26 of the pH sensor 20 and/or to a support surface 66 accessible in each recess 70.

[00238] The surface of the support 66 accessible in the recesses 70 can be functionalized such that the primers 74 can be attached to the surface rather than to the intermediate regions 72 of the patterned material 68. In some examples, functionalizing the surface of the substrate includes silanizing the surface available in recesses 70 and forming a polymer layer on the silanized surface. The silanization can be carried out using any silane or silane derivative. A polymer (not shown) can then be applied to the silanized surface. The polymer may be a semi-rigid polymeric material that is permeable to liquids and gases. An exemplary polymer includes an acrylamide copolymer such as poly-(N-(5-azidoacetamidylpentyl)-acrylamide-co-acrylamide, PAZAM, or other suitable polymeric hydrogel.

[00239] Primers 74 can be any forward amplification primer or reverse amplification primer that includes a functional group that can be attached to the surface of the conduction channel 26 and/or to the surface of the support 66. Examples of suitable functional group-terminal primers include an alkyne-terminal primer, a tetrazine-terminal primer, an azido-terminal primer, an amino-terminal primer, an epoxy or glycidyl-terminal primer, a thiophosphate-terminal primer, a thiol-terminal primer, an aldehyde-terminal primer, a hydrazine-terminal primer, and a triazolinedione-terminal primer. A mixture of primers can also be used. Specific examples of suitable primers include the P5 and/or P7 primers that are used on the surface of some commercial flow cells sold by Illumina Inc.

[00240] In an example, primers 74 may be attached using an attachment process such as flow through settling (e.g., when flow cell 62 has a lid associated therewith), dipping, spray coating, pouring from a bath, or other a suitable method that would attach the primer(s) 74. Each of these exemplary techniques may use a primer solution or mixture that may include primer(s), water, a buffer (eg, saline), and a catalyst.

[00241] In the example shown in FIG. 7D, reader system 40B includes a planar pathway 26' and primers 74 attached to planar pathway 26'. The planar conduction channel 26' can be any material that allows charged ions to be read, or that includes surface groups that can be protonated or deprotonated by the charged ions formed during the acid or base formation reactions disclosed herein. The planar conductive channel 26' is connected to electrodes 22, 24 (not shown in FIG. 7D) which can be integrated into the support 66 and connected to the electronic circuit that makes them work. Any of the primers 74 may be used and attached as described herein. In addition, if the planar pathway 26' has surface groups that can attach primers 74, additional functionalization of the planar pathway 26' may not be performed.

[00242] These examples of flow cell 62 can be used for ensemble sequencing. In ensemble sequencing, the 44 template polynucleotide strand (not shown in Figure 7C and FIG. 7D) to be sequenced can be formed on the surface of the flow cell using primers 74. At the start of template polynucleotide strand formation, library templates can be prepared from any sample. nucleic acid (for example, a DNA sample or an RNA sample). The nucleic acid sample can be assembled into single-stranded DNA or RNA components of a similar size (eg, <1000 bp). During preparation, adapters can be added to the ends of these components. Through short cycle amplification, various motifs can be introduced in adapters such as sequencing binding sites, indices, and regions that are complementary to primers 74 in recesses 70. The final library templates include a DNA or RNA component and adapters at both ends. In some examples, components from the same nucleic acid sample have identical adapters added to them.

[00243] Multiple library templates can be introduced into flow cell 62. Because flow cell 62 includes an array of recesses 70, multiple library templates hybridize to, for example, one of two types of primers 74 immobilized therein.

[00244] Clustering can then be performed. In one example of cluster formation, library templates are copied from 74 hybridized primers via 3' extension using high fidelity DNA polymerase. The original library templates are denatured leaving copies immobilized in recesses 70. Isothermal bridge amplification can be used to amplify the immobilized copies. For example, the copied templates form a circuit in order to hybridize into the adjacent complementary primer 74, and the polymerase copies the copied templates in order to form double-stranded bridges, which are denatured so that they form two single-stranded strands. These two strands form a loop and hybridize into adjacent 74 complementary primers and extend again so that they form two new double strand loops. The process is repeated for each copy of the template through cycles of isothermal denaturation and amplification to create dense clonal clusters. Each cluster of double-stranded bridges is denatured. In an example, the reverse strand is removed by cleavage of a specific base, leaving forward template polynucleotide strands. Clustering results in the formation of multiple template polynucleotide strands 44 in each recess 70. This clustering example is bridged amplification and is one example of an amplification that can be performed. It should be understood that other amplification technologies may be used, such as the exclusion amplification workflow (Examp) (Illumina Inc.).

[00245] To initiate sequencing, the inclusion mix may be added to the 62 flow cell.

[00246] In one example, the inclusion mixture includes a carrier fluid, any example of the 30A-30D complex disclosed herein, and any example of a labeled nucleotide 12 or 12A-12C. Complex 30A-30D includes polymerase 28 and a pH altering component 50A-50E coupled to polymerase 28, wherein the pH altering component 50A-50E is selected from the group consisting of an enzyme 50A which is to catalyze the consumption of a secondary substrate 34, a metal coordination complex 50D , which is supposed to catalyze consumption of secondary substrate 34, and cofactor 50B or activator 50C, which is supposed to catalyze consumption of secondary substrate 34. Labeled nucleotide 12 includes nucleotide 14; binding molecule 16 attached to the terminal phosphate group of nucleotide 14; and a label 18 attached to a binding molecule 16, the label must participate in a pH change reaction involving a secondary substrate 34. The labeled nucleotide 12A-12C includes nucleotide 14' having a blocking group 3'-OH; a cleavable binding molecule 16' attached to the base or sugar of nucleotide 14'; and a label 18 attached to a binding molecule 16', while the label 18 should participate in the reaction of changing the pH, containing the secondary substrate 34.

[00247] Label 18 of labeled nucleotide 12 or 12A-12C should depend on the 30A-30D complex being used and combinations, and coupled acid or base formation reactions may be used as described with reference to FIG. 3A and FIG. 3B, FIG. 4, fig. 5 and FIG. 6A and FIG. 6b. As one example, when the pH changing component of the 30A complex is the 50A enzyme, label 18B is used and may be selected from the group consisting of a first group that improves the kinetics of the 50A enzyme and a second group that slows down the kinetics of the 50A enzyme. As another example, the pH change component is the 50E metal coordination complex, and the 18E label can be used. As explained herein with reference to FIG. 5, tag 18E is a ligand for the metal of the 50E metal coordination complex, wherein the ligand changes the catalytic property of the 50E metal coordination complex. As yet another example, the pH change component is a 50B cofactor or 50C activator, and the 18C label is a catalytic label that is activated by a 50B cofactor or 50C activator. As yet another example, the pH changing component is the 50A enzyme and the 30D complex includes a 58 nucleic acid hairpin/enzyme inhibitor conjugate. The 18F tag is an oligonucleotide sequence that is the complementary portion of the 58 nucleic acid hairpin/enzyme inhibitor conjugate.

[00248] The inclusion mix introduces the 30A-30D complex and labeled nucleotide 12 or 12A-12C into the flow cell 62. The inclusion mix can also include a sequencing primer that hybridizes to a complementary sequence on the template polynucleotide strand 44. This primer for sequencing ensures that the template polynucleotide chain 44 is ready for sequencing.

[00249] Another fluid may be used to introduce secondary substrate 34 into flow cell 62. The inclusion mix and fluid including secondary substrate 34 may be part of a kit. Any example of a flow cell 62 (shown in Fig. 7C and Fig. 7D) may be included in the kit.

[00250] When the inclusion mix includes labeled nucleotide 12 (label 18 of which is naturally cleaved after incorporation), it should be understood that the inclusion mix and secondary substrate fluid 34 may be introduced into the flow cell 62 simultaneously or directly one after the other. such that secondary substrate 34 is present in flow cell 62 at the time of the on event. Because the label 18 cleaves naturally after being turned on, it is desirable if the secondary substrate 34 is present while the label 18 is held in the vicinity of the conductive channel 26 or 26'. In contrast, when the inclusion mix includes a labeled 12A-12C nucleotide (which includes a blocking group and therefore its tag 18 is not naturally cleaved), it should be understood that the inclusion mix and secondary substrate fluid 34 may injected into the flow cell 62 at the same time directly one after the other, or the secondary substrate fluid 34 may be injected after the inclusion is carried out. Because the label 18 remains attached until the deblocking agent is injected, the secondary substrate 34 can be injected at any time during or after the trigger event.

[00251] When the inclusion mix and the secondary substrate fluid 34 are introduced into the flow cell 62, the fluids enter the recesses 70 (in which the template polynucleotide strands 44 are present). One of the labeled nucleotides 12 or 12A-12C is incorporated, via the respective polymerase 28 of the respective complex 30A-30D, into the resulting strand 46, which extends the sequencing primer and which is complementary to template polynucleotide strand 44. In other words, at least some template polynucleotide strands 44 in the flow cell 62, the respective polymerases 28 extend the hybridized sequencing primer with one of the labeled nucleotides 12 or 12A-12C in solution.

[00252] In these examples, since polymerase 28 is part of a complex 30A-30D that includes a pH change component 50A-50E, and since polymerase 28 is involved in nucleotide incorporation, the pH change component 50A-50E moves to a position near the conduction channel 26 or 26' during the enable event. Label 18 of the included nucleotide 12 or 12A-12C also appears in the vicinity of the pH change component 50A-50E and the conduction channel 26, 26'. This allows the pH change component 50A-50E and the label 18 to participate in an acid or base formation reaction with the secondary substrate 34 near the conduction channel 26, 26'. The various reactions and effects of specific combinations of components 50A-50E and labels 18 are identical to those described with reference to FIG. 3A and FIG. 3B, FIG. 4, fig. 5 and FIG. 6A and FIG. 6b. The change in pH as a result of the acid or base forming reaction changes the charge in the conductive channel 26, 26' and the charge change is detected. The change in charge and/or the rate of charge change can be used to identify incorporated nucleotides. Because multiple turn-on events occur on multiple primers 74 in a single well, charge signals can be strong and easily detectable in each individual conductive channel 26, 26'.

[00253] In examples of sequencing assemblies including labeled nucleotide 12, label 18 and binding molecule 16 are naturally cleaved after incorporation.

[00254] In examples of sequencing assemblies including the labeled nucleotide 12A-12C, the label 18 and the binding molecule 16' remain on until a deblocking agent is added and washed through the flow cell 62.

[00255] In some examples of ensemble sequencing, polymerase 28 of complex 30A-30D may be highly processive and thus may not necessarily be introduced in every sequencing run. In other examples, novel polymerase 28 (eg, as part of complex 30A-30D) may be introduced in each sequencing run.

[00256] In another example of ensemble sequencing using flow cell 62, as described with reference to FIG. 7C and FIG. 7D, the inclusion mix may not include the complex example 30A-30D, but may instead include polymerase 28 directly. This example is similar to the example described in FIG. 2, except that polymerase 28 is present in the inclusion mixture and does not bind to the surface of the conduction channel 26, 26'. This exemplary inclusion mix may include carrier fluid, polymerase 28, and labeled nucleotides 12 or 12A-12C, including label 18A; which, as described with reference to FIG. 2 is a catalyst that initiates or accelerates an acid or base formation reaction comprising a secondary substrate 34. A second fluid including a carrier liquid and a secondary substrate 34 may be used in this example of an incorporation mixture.

[00257] When this example of the inclusion mixture and secondary substrate fluid 34 is introduced into the example flow cell 62 shown in FIG. 7B and FIG. 7C, fluids enter recesses 70 (in which template polynucleotide strands 44 are present). One of the labeled nucleotides 12 or 12A-12C is incorporated, via the appropriate polymerase 28, into the resulting strand 46, which extends the sequencing primer and which is complementary to template polynucleotide strand 44. In other words, at least some of the template polynucleotide strands 44 in the flow cuvette 62, the respective polymerases 28 extend the hybridized sequencing primer with one of the labeled nucleotides 12 or 12A-12C in solution.

[00258] In these examples, the 18A catalytic label of the included nucleotide 12 or 12A-12C appears in the vicinity of the conduction channel 26, 26'. This allows the catalytic label 18A to participate in an acid or base formation reaction with the secondary substrate 34 near the conduction channel 26.26'. The various reactions and effects of catalytic tag 18A are identical to those described with reference to FIG. 2. The change in pH as a result of the acid or base forming reaction changes the charge in the conductive channel 26, 26' and the charge change is detected. The change in charge and/or the rate of charge change can be used to identify incorporated nucleotides. Because multiple turn-on events occur on any number of primers 74 in a single well, charge signals can be strong and easily detectable in each individual conductive channel 26, 26'.

[00259] Conclusion

[00260] Although single molecule detection and ensemble detection are described in detail, it should be understood that the acid or base formation reactions disclosed herein may be used with a different sequencing protocol, for genotyping, or in other chemical and/or biological applications.

[00261] It should be appreciated that all combinations of the above principles and additional principles explained in more detail below (unless such principles are mutually inconsistent) are considered part of the inventive subject matter disclosed herein. In particular, all combinations of the claimed subject matter listed at the end of this disclosure are considered part of the inventive subject matter disclosed herein. It should also be appreciated that terms explicitly used in this document, which may also be indicated in any entity disclosure contained by reference, should correspond to the meaning that is most consistent with the specific principles disclosed in this document.

[00262] Reference throughout the detailed description to "one example", "another example", "example", etc. means that a particular element (eg, feature, structure, and/or characteristic) described in connection with an 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 the described elements for any example can be combined in any suitable way in various examples, unless the context clearly dictates otherwise.

[00263] The terms "substantially" and "approximately" as used throughout this disclosure, which includes the claims, are used to describe and account for small fluctuations, for example, due to processing variations. For example, they may mean 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%.

[00264] In addition, it should be understood that the ranges provided herein include the specified range and any value or subrange within the specified range, as if expressly set forth. For example, a range presented as being from 1 nm to less than 1 µm should be interpreted as including not only the explicitly stated limits from 1 nm to less than 1 µm, but also as including individual values, for example, approximately 5 nm , 222.5 nm, 275 nm, etc. and subbands, for example, from about 150 nm to about 800 nm, and so on.

[00265] Although several examples are described in detail above, it should be understood that the disclosed examples may be modified. Therefore, the above description should be considered non-limiting.

Claims (109)

1. A system that reads the change in pH, containing: - pH sensor, including: - two electrodes; And - a conductive channel functionally connected to two electrodes; And - a complex with a pH-changing component attached to the conductive channel of the pH sensor, and the complex includes a polymerase associated with at least one pH-changing component, which should participate in the formation of a pH change in the vicinity of the conductive channel due to the consumption of a secondary substrate in a fluid that is in contact with the pH sensor, wherein at least one pH-altering component is an enzyme, wherein the enzyme generates an acid or base in reaction with a secondary substrate. 2. The reading system of claim 1, wherein the enzyme is selected from the group consisting of hydrolases and oxidases. 3. The reading system of claim 1, wherein the kinetics of the at least one pH-changing component is at least 10 times faster than the kinetics of the polymerase. 4. The reading system of claim 1, wherein the complex further comprises a nucleic acid hairpin/enzyme inhibitor conjugate attached to the enzyme. 5. The reading system of claim 1, wherein the complex further includes a second enzyme attached to the polymerase. 6. The reading system of claim 1, wherein the complex is a fusion protein or protein chimera. 7. The reading system of claim 1, wherein the conductive channel of the pH sensor is selected from the group consisting of a semiconductive nanostructure, a graphene nanostructure, a metal nanostructure, and a conductive polymer nanostructure. 8. Reading system according to claim 1, further comprising: a substrate including a plurality of recesses separated by intermediate regions, wherein at least the conductive channel of the pH sensor is at the bottom of one of the plurality of recesses; And - a plurality of additional pH sensors, wherein at least the conductive channel of each of the plurality of additional pH sensors is at the bottom of the corresponding one of the plurality of recesses. 9. The reading system of claim 8, wherein each of the plurality of recesses includes sidewalls, wherein the sidewalls include a pH buffer material. 10. A labeled nucleotide for use in reading pH change, comprising: - nucleotide; - a linker molecule attached to the terminal phosphate group of the nucleotide; And a catalytic label attached to a linker molecule, wherein the catalytic label should create a pH change by consuming a secondary substrate in the labeled nucleotide fluid, where the catalytic label is selected from the group consisting of hydrolases and oxidases; moreover, the complex with the pH-altering component is involved in creating the pH change. 11. A labeled nucleotide according to claim 10, wherein the linker molecule is cleavable. 12. A kit for reading the change in pH created as a result of the consumption of a secondary substrate, containing: - a nucleotide containing: - a linker molecule attached to the terminal phosphate group of the nucleotide; And - a cofactor or inhibitor attached to the linker molecule; And - reading system, including: - two electrodes; - a conductive channel functionally connected to two electrodes; And - a complex with a pH-changing component attached to the conduction channel, and the complex includes a polymerase conjugated to the pH-changing component, where the pH changing component is an enzyme, and where the cofactor or activator changes the kinetics of the enzyme. 13. The kit of claim 12 wherein the linker molecule is cleavable. 14. A method for detecting a change in pH in a reaction involving a secondary substrate, which includes the steps of: - introducing a template polynucleotide chain into a sensor having a polymerase bound to a conductive channel, where the sensor polymerase is part of a complex with an enzyme catalyst; - a fluid is introduced into the sensor, which includes a secondary substrate and labeled nucleotides, due to which the nucleotide of one of the labeled nucleotides binds to the polymerase, and the label of this one of the labeled nucleotides participates in a pH change reaction in which the secondary substrate that is nearby participates from a conductive channel, wherein the pH-changing reaction is carried out using a complex with a pH-changing component; And - detecting the response of the conductive channel. 15. The method according to claim 14, in which: - the label is a group that improves or slows down the kinetics of the enzyme catalyst; And - the method further includes the step of detecting a change in charge compared to the base charge. 16. The method according to claim 14, in which: - the label is a secondary substrate; And - the method further includes the step of detecting a change in charge compared to the base charge. 17. A method for detecting a change in pH in a reaction involving a secondary substrate, which includes the steps of: - enter the matrix polynucleotide chain in the sensor having a polymerase attached to the conductive channel; - a fluid is introduced into the sensor, which includes a secondary substrate and labeled nucleotides, due to which the nucleotide of one of the labeled nucleotides binds to the polymerase, and the label of this one of the labeled nucleotides participates in a pH change reaction in which the secondary substrate that is nearby participates from a conductive channel, wherein the pH-changing reaction is carried out using a complex with a pH-changing component; And - detecting the response of the conducting channel, and - the sensor polymerase is part of a complex with a metal coordination complex; - the label is a ligand for the metal of the metal coordination complex; And - the method further includes the step of detecting a change in charge compared to the base charge. 18. A method for detecting a change in pH in a reaction involving a secondary substrate, which includes the steps of: - enter the matrix polynucleotide chain in the sensor having a polymerase attached to the conductive channel; - a fluid is introduced into the sensor, which includes a secondary substrate and labeled nucleotides, due to which the nucleotide of one of the labeled nucleotides binds to the polymerase, and the label of this one of the labeled nucleotides participates in a pH change reaction in which the secondary substrate that is nearby participates from a conductive channel, wherein the pH-changing reaction is carried out using a complex with a pH-changing component; And - detecting the response of the conducting channel, and - the sensor polymerase is part of a complex with an enzyme catalyst; - the nucleic acid hairpin/enzyme inhibitor conjugate is attached to the enzyme catalyst; the label is an oligonucleotide sequence that is complementary to the nucleic acid hairpin/enzyme inhibitor conjugate portion; And - the method further includes the step of detecting a change in charge compared to the base charge. 19. The method according to any one of paragraphs. 15-18, further comprising identifying a polymerase-bound nucleotide based on charge change and/or charge change rate. 20. The method of claim 14 wherein the labeled nucleotides have different individual accumulation rates, the method further comprising identifying the associated labeled nucleotide by its individual accumulation rate. 21. An inclusion mix to be included in the pH change reading kit of claim 12, comprising: - carrier fluid; - a complex with a pH-changing component, including: - polymerase; And - a pH changing component associated with the polymerase, and the pH changing component is selected from the group consisting of an enzyme, which should catalyze the consumption of the secondary substrate, a metal coordination complex, which should catalyze the consumption of the secondary substrate, and a cofactor or activator, which should catalyze the consumption of the secondary substrate ; And - labeled nucleotide, including: - nucleotide; - a linker molecule attached to the terminal phosphate group of the nucleotide; And - a label attached to the linker molecule, while the label must participate in the pH change reaction, in which the secondary substrate participates. 22. A mixture for inclusion according to claim 21, in which: the pH-changing component is an enzyme, and the label is selected from the group consisting of a first group that improves the kinetics of the enzyme and a second group that slows down the kinetics of the enzyme; the pH-changing component is a metal coordination complex, and the label is a ligand for the metal of the metal coordination complex, wherein the ligand changes the catalytic property of the metal coordination complex; or the pH-changing component is a cofactor or activator, and the label is a catalytic label that is activated by the cofactor or activator. 23. A mixture for inclusion according to claim 21, in which: the pH-changing component is an enzyme; the complex further includes a nucleic acid hairpin/enzyme inhibitor conjugate attached to the enzyme; And the label is an oligonucleotide sequence that is complementary to the nucleic acid hairpin/enzyme inhibitor conjugate portion. 24. A kit for reading the change in pH in a reaction involving a secondary substrate, containing: - a mixture for inclusion according to claim 21; And - a mixture with a secondary substrate, including a second carrier liquid and a secondary substrate. 25. The kit according to claim 24, additionally containing: - a flow cell, including: a substrate including a plurality of depressions separated by intermediate regions; - a conductive channel at the bottom of each of the plurality of recesses; And - at least one primer attached in each of the recesses. 26. A mixture for inclusion in a system for reading the change in pH, containing: - a carrier fluid comprising a buffer; - a complex with a pH-changing component, including: - polymerase; And - a pH changing component associated with the polymerase, and the pH changing component is selected from the group consisting of an enzyme, which should catalyze the consumption of the secondary substrate, a metal coordination complex, which should catalyze the consumption of the secondary substrate, and a cofactor or activator, which should catalyze the consumption of the secondary substrate ; And - labeled nucleotide, including: - a nucleotide having a blocking group 3'-OH; - a cleavable linker molecule attached to the base or sugar of the nucleotide; And - a label attached to the linker molecule, while the label must participate in the pH change reaction, in which the secondary substrate participates, while in the specified mixture: the pH-changing component is an enzyme, and the label is selected from the group consisting of a first group that improves the kinetics of the enzyme, a second group that slows down the kinetics of the enzyme, and a secondary substrate; or the pH-changing component is a metal coordination complex, and the label is a ligand for the metal of the metal coordination complex, wherein the ligand changes the catalytic property of the metal coordination complex; or the pH-changing component is a cofactor or activator, and the label is a catalytic label that is activated by the cofactor or activator. 27. The mixture for inclusion according to claim 26, in which: the pH-changing component is an enzyme; the complex further includes a nucleic acid hairpin/enzyme inhibitor conjugate attached to the enzyme; And the label is an oligonucleotide sequence that is complementary to the nucleic acid hairpin/enzyme inhibitor conjugate portion. 28. A kit for reading the change in pH in a reaction involving a secondary substrate, containing: - a mixture for inclusion according to claim 26; And - a mixture with a secondary substrate, including a second carrier liquid and a secondary substrate. 29. The kit according to claim 28, further comprising: - a flow cell, including: a substrate including a plurality of depressions separated by intermediate regions; - a conductive channel at the bottom of each of the plurality of recesses; And - a primer attached in each of the recesses. 30. The kit according to claim 28, additionally containing a solution of a deblocking agent.

Images