Selectively functionalized nanoscale wells

Abstract

Methods, substrates, devices and systems related to substrates having localized regions with a single molecule of interest or with groups of molecules of interest attached and other regions having few or no molecules of interest attached are provided. In particular, the invention relates to nanoscale wells and arrays of nanoscale wells in which the nanoscale wells have a bottom surface comprising a group 4-5 transition metal oxide and sidewalls comprising silicon dioxide or aluminum oxide. The nanoscale wells of the invention can be selectively functionalized with surface modifying agents for the selective attachment of molecules of interest. The invention also includes substrates with selectively functionalized regions comprising a plurality of active regions that each comprise a group 4-5 transition metal oxide that are separated by surrounding regions comprising silicon dioxide or aluminum oxide.

Description

SELECTIVELY FUNCTIONALIZED NANOSCALE WELLSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit of U.S. Provisional Application No. 63 / 650,135 filed on May 21, 2024, the disclosure of which is incorporated herein by reference in its entirety.BACKGROUND OF THE INVENTION

[0002] There are a wide range of analytical operations that can benefit from the ability to analyze the reaction of individual molecules or groups of molecules such as clonal populations that are localized to specific regions.

[0003] One approach to such localization is the use of substrates having different surface regions, each surface region made up of a different material, and each different material having different chemical reactivities, allowing for selective functionalization of one material over the other. This approach can be particularly useful for substrates comprising nanoscale wells, where it is desirable that the molecule or molecules of interest are localized on the bottoms of the nanoscale wells.

[0004] While some such selective functionalization approaches are known, there is a need for improved selectively functionalized substrates and methods to create them.BRIEF SUMMARY OF THE INVENTION

[0005] In some aspects, the invention provides a substrate comprising a plurality of nanoscale wells disposed through a top surface of the substrate, wherein each nanoscale well comprises a sidewall surface that comprises silicon dioxide or aluminum oxide and a bottom surface that comprises a group 4-5 transition metal oxide. In some cases the group 4-5 transition metal oxide is hafnium oxide. In some cases the group 4-5 transition metal oxide is selected from the group consisting of titanium oxide, zirconium oxide, niobium oxide, and tantalum oxide. In some cases there is a bottom surface modifying agent that is selectively associated with the bottom surface and that comprises a coupling group. In some cases there is a sidewall modifying agent that is associated with the sidewall surface and that passivates the sidewall. In some cases the bottom surface comprises a bottom surface modifying agent comprising one or more phosphonic acid groups, one or more catechol groups, one or more phosphate groups, or a combination thereof. In some cases the bottom surface comprises a bottom surfacemodifying agent comprising one or more phosphonic acid groups and a coupling group. In some cases the bottom surface modifying agent comprises a biotin-PEG-phosphonic acid. In some cases the bottom surface comprises a bottom surface modifying agent comprising one or more catechol groups and a coupling group. In some cases the bottom surface modifying agent is a biotin-PEG-catechol. In some cases the bottom surface comprises a first bottom surface modifying agent that comprises a coupling group and a second bottom surface modifying agent that lacks the coupling group. In some cases the sidewall surface comprises silicon dioxide, further comprising a silane on the silicon dioxide. In some cases the sidewall surface comprises silicon dioxide, further comprising a PEG-silane on the silicon dioxide. In some cases the bottom surface comprises a coupling group bound thereto, wherein the coupling group is selected from the group consisting of biotin, a biotin-binding protein, an antibody, an antibody fragment, a lectin, and a nucleic acid.

[0006] In some cases the bottom surface comprises a coupling group bound thereto, wherein the coupling group is selected from the group consisting of an amine group, a carboxyl group, a hydroxyl group, a sulfhydryl group, an azido group, an alkyne group, a cyclooctyne group, a dibenzocyclooctyne (DBCO) group, a trans-cyclooctene (TCO) group, a norbornene group, a tetrazine group, an epoxy group, a glycidyl group, an aldehyde group, an alkenyl group, an acrylate group , a methacrylate group, an anhydride group, an activated carboxylic acid group, and an n-hydroxysuccinimide (NHS) ester group. In some cases the bottom surface comprises a coupling group bound thereto, wherein density of the coupling group on the bottom surface is 100 times or more greater than density of the coupling group on the sidewall surface or the top surface. In some cases the bottom surface comprises a coupling group bound thereto, wherein density of the coupling group on the bottom surface is 1000 times or more greater than density of the coupling group on the sidewall surface or the top surface. In some cases the bottom surface comprises an immobilized molecule or molecules of interest each bound to a coupling group, wherein density of the molecules of interest on the bottom surface is 100 times or more greater than density of the molecules of interest on the sidewall surface and the top surface. In some cases the molecule or molecules of interest comprise a polymerase- template complexes. In some cases the bottom surface comprises an immobilized molecule or molecules of interest, wherein density of the molecules of interest on the bottom surface is 1000 times or more greater than density of the molecules of interest on the sidewall surface or the top surface. In some cases molecule or molecules of interest comprise polymerase-template complexes. In some cases the substrate comprises a layer of silicon dioxide overlying a layer of group 4-5 transition metal oxide, wherein the nanoscale wells are nanoscale apertures that penetrate through the layer of silicon dioxide to the layer of group 4-5 transition metal oxide, whereby the base of the nanoscale well comprises the group 4-5 transition metal oxide. In some cases the top surface comprises silicon dioxide. In some cases the top surface comprises aluminum oxide. In some cases the bottom surface of each well has an area less than 196,000 nm2. In some cases the bottom surface of each well has an area less than 70,700 nm2. In some cases the bottom surface of the well is the top surface of an optical waveguide comprising a group 4-5 transition metal oxide.

[0007] In some aspects, the invention provides an analytic device comprising: the substrate recited above; and one or more optical waveguides disposed within the substrate below the plurality of nanoscale wells, each optical waveguide comprising a core; wherein the bottom surface of each nanoscale well is disposed sufficiently proximal to a core of at least one of the one or more optical waveguides to be illuminated by an evanescent field emanating from the core when optical energy is passed through the core. In some cases the bottom surface of the each nanoscale well comprises a single optically resolvable immobilized molecule of interest. In some cases the immobilized molecule of interest comprises a polymerase-template complex. In some cases the immobilized molecule of interest comprises a protein or a peptide. In some cases there is an integrated optical detector disposed below the one or more optical waveguides. In some cases there is one or more integrated optical element below the one or more optical waveguides and above the optical detector. In some cases the one or more integrated optical element comprises a diffractive optical element, lens, filter, or aperture.

[0008] In some aspects, the invention provides an analytic device comprising: a substrate comprising i) at least one optical waveguide and ii) at least one nanoscale well that is disposed through a top surface of the substrate and that penetrates into a top of the substrate and extends toward an optical waveguide core of the optical waveguide, wherein the nanoscale well comprises a sidewall surface that comprises silicon dioxide or aluminum oxide and a bottom surface that comprises a group 4-5 transition metal oxide; and a detector disposed within the substrate below the at least one nanoscale well to receive photons emitted from within the nanoscale well. In some cases a single optically resolvable molecule of interest is bound to the bottom surface of the at least one nanoscale well and is sufficiently proximal to the optical waveguide core to be illuminated by an evanescent field emanating from theoptical waveguide core when optical energy is passed through the optical waveguide. In some cases the at least one nanoscale well comprises a plurality of nanoscale wells. In some cases there are one or more integrated optical elements below the one or more optical waveguides and above the detector. In some cases the one or more integrated optical element comprises a diffractive optical element, lens, filter, or aperture. In some cases the group 4-5 transition metal oxide is hafnium oxide. In some cases the group 4-5 transition metal oxide is selected from the group consisting of titanium oxide, zirconium oxide, niobium oxide, and tantalum oxide. In some cases a bottom surface modifying agent that is selectively associated with the bottom surface and that comprises a coupling group. In some cases a sidewall surface modifying agent that is associated with the sidewall surface. In some cases the bottom surface comprises a bottom surface modifying agent comprising one or more phosphonic acid groups, one or more catechol groups, one or more phosphate groups, or a combination thereof. In some cases the bottom surface comprises a bottom surface modifying agent comprising one or more phosphonic acid groups and a coupling group. In some cases the bottom surface modifying agent comprises a biotin-PEG-phosphonic acid. In some cases the bottom surface comprises a bottom surface modifying agent comprising one or more catechol groups and a coupling group. In some cases the bottom surface modifying agent comprises a biotin-PEG- catechol. In some cases the bottom surface comprises a first bottom surface modifying agent that comprises a coupling group and a second bottom surface modifying agent that lacks the coupling group. In some cases the sidewall surface comprises silicon dioxide, further comprising a silane coating on the silicon dioxide. In some cases the sidewall surface comprises silicon dioxide, further comprising a PEG-silane coating on the silicon dioxide. In some cases the bottom surface comprises a coupling group bound thereto, wherein the coupling group is selected from the group consisting of biotin, a biotin-binding protein, an antibody, an antibody fragment, a lectin, and a nucleic acid.

[0009] In some cases the bottom surface comprises a coupling group bound thereto, wherein the coupling group is selected from the group consisting of an amine group, a carboxyl group, a hydroxyl group, a sulfhydryl group, an azido group, an alkyne group, a cyclooctyne group, a dibenzocyclooctyne (DBCO) group, a trans-cyclooctene (TCO) group, a norbornene group, a tetrazine group, an epoxy group, a glycidyl group, an aldehyde group, an alkenyl group, an acrylate group , a methacrylate group, an anhydride group, an activated carboxylic acid group, and an n-hydroxysuccinimide (NHS) ester group. In some cases the bottom surface comprisesa coupling group bound thereto, wherein density of the coupling group on the bottom surface is 100 times or more greater than density of the coupling group on the sidewall surface and the top surface. In some cases the bottom surface comprises a coupling group bound thereto, wherein density of the coupling group on the bottom surface is 1000 times or more greater than density of the coupling group on the sidewall surface and the top surface. In some cases the bottom surface comprises an immobilized molecule or molecules of interest, wherein density of the molecule or molecules of interest on the bottom surface is 100 times or more greater than density of the molecules of interest on the sidewall surface and the top surface. In some cases the molecule or molecules of interest comprise immobilized polymerase-template complexes. In some cases the molecule or molecules of interest comprise immobilized proteins or peptides. In some cases the bottom surface comprises an immobilized molecule or molecules of interest, wherein density of the molecule or molecules of interest on the bottom surface is 1000 times or more greater than density of the molecules of interest on the sidewall surface and the top surface. In some cases the molecule or molecules of interest comprise immobilized polymerase-template complexes. In some cases the substrate comprises a layer of silicon dioxide overlying a layer of group 4-5 transition metal oxide, wherein the at least one nanoscale well is a nanoscale aperture that penetrates through the layer of silicon dioxide to the layer of group 4-5 transition metal oxide. In some cases the top surface comprises silicon dioxide. In some cases the bottom surface of the at least one nanoscale well has an area less than 196,000 nm2. In some cases the bottom surface of the at least one nanoscale well has an area less than 70,700 nm2. In some cases the optical core comprises a group 4-5 transition metal oxide, and the at least one nanoscale well penetrates to the top of the of the optical core such that the top of the optical core forms the bottom of the nanoscale well.

[0010] In some aspects the invention provides a method of detecting a molecule of interest, comprising: providing an optical waveguide substrate comprising: an optical waveguide core surrounded by a lower refractive index cladding including a optical waveguide upper cladding above the core, and a nanoscale well that penetrates into the optical waveguide upper cladding and extends toward the optical waveguide core, the nanoscale well comprising a molecule of interest on its bottom surface, the bottom surface disposed sufficiently proximal to the optical waveguide core to be illuminated by an evanescent field emanating from the optical waveguide core when optical energy is passed through the optical waveguide core, wherein the bottom surface of the nanoscale well comprises a single optically resolvable immobilizedmolecule of interest, wherein the nanoscale well comprises a sidewall surface that comprises silicon dioxide or aluminum oxide, and a bottom surface that comprises a group 4-5 transition metal oxide; b) exposing the substrate to fluid comprising an analyte comprising a fluorescent or fluorogenic moiety that emits a signal in response to the evanescent field; c) directing optical energy through the optical waveguide core, thereby generating the evanescent field and illuminating the single optically resolvable immobilized molecule of interest; and d) detecting a signal from the analyte upon interaction of the analyte with the single optically resolvable immobilized molecule of interest. In some cases the optical waveguide substrate comprises two or more optical waveguides and a plurality of nanoscale wells, the bottom surface of each nanoscale well disposed sufficiently proximal to an optical waveguide core of one of the two or more optical waveguides to be illuminated by an evanescent field emanating from the optical waveguide core when optical energy is passed through the optical waveguide core. In some cases the detecting a signal from the analyte comprises disposing a detector within the substrate below the optical waveguide cores; and, during the directing optical energy of c), detecting fluorescent or fluorogenic emissions from the analyte. In some cases the single optically resolvable immobilized molecule of interest comprises an enzyme. In some cases the enzyme is a polymerase. In some cases the analyte comprises a nucleotide analog. In some cases the single optically resolvable immobilized molecule of interest comprises a polypeptide and the analyte comprises an amino acid recognition molecule. In some cases the group 4-5 transition metal oxide is hafnium oxide. In some cases the group 4-5 transition metal oxide is selected from the group consisting of titanium oxide, zirconium oxide, niobium oxide, and tantalum oxide. In some cases there is a bottom surface modifying agent that is selectively associated with the bottom surface and that comprises a coupling group. In some cases there is a sidewall surface modifying agent that is associated with the sidewall surface and that passivates the sidewall. In some cases the bottom surface comprises a bottom surface modifying agent comprising one or more phosphonic acid groups, one or more catechol groups, one or more phosphate groups, or a combination thereof. In some cases the bottom surface comprises a bottom surface modifying agent comprising one or more phosphonic acid groups and a coupling group. In some cases the bottom surface modifying agent comprises a biotin-PEG-phosphonic acid. In some cases the bottom surface comprises a bottom surface modifying agent comprising one or more catechol groups and a coupling group. In some cases the bottom surface modifying agent comprises a biotin-PEG-catechol. In some cases thebottom surface comprises a first bottom surface modifying agent that comprises a coupling group and a second bottom surface modifying agent that lacks the coupling group. In some cases the sidewall surface comprises silicon dioxide, further comprising a silane coating on the silicon dioxide. In some cases the sidewall surface comprises silicon dioxide, further comprising a PEG-silane coating on the silicon dioxide. In some cases the bottom surface comprises a coupling group bound thereto, wherein the coupling group is selected from the group consisting of biotin, a biotin-binding protein, an antibody, an antibody fragment, a lectin, and a nucleic acid.

[0011] In some cases the bottom surface comprises a coupling group bound thereto, wherein the coupling group is selected from the group consisting of an amine group, a carboxyl group, a hydroxyl group, a sulfhydryl group, an azido group, an alkyne group, a cyclooctyne group, a dibenzocyclooctyne (DBCO) group, a trans-cyclooctene (TCO) group, a norbornene group, a tetrazine group, an epoxy group, a glycidyl group, an aldehyde group, an alkenyl group, an acrylate group , a methacrylate group, an anhydride group, an activated carboxylic acid group, and an n-hydroxysuccinimide (NHS) ester group. In some cases the substrate comprises a layer of silicon dioxide overlying a layer of group 4-5 transition metal oxide, wherein the nanoscale well is a nanoscale aperture that penetrates through the layer of silicon dioxide to the layer of group 4-5 transition metal oxide. In some cases the top surface comprises silicon dioxide. In some cases the top surface comprises aluminum oxide. In some cases the bottom surface of the well has an area less than 196,000 nm2. In some cases the bottom surface of the well has an area less than 70,700 nm2.

[0012] In some aspects the invention provides a method of performing sequencing-by- synthesis comprising: providing an analytic device comprising: a substrate comprising an optical waveguide and a nanoscale well disposed through a top surface of the substrate and extending toward an optical waveguide core of the optical waveguide, wherein the nanoscale well comprises a sidewall surface that comprises silicon dioxide or aluminum oxide and a bottom surface that comprises a group 4-5 transition metal oxide, and wherein the nanoscale well comprises a polymerase-template complex immobilized to the bottom surface of the well; providing illumination light into the optical waveguide core whereby the polymerase-template complex is illuminated by an evanescent field emanating from the optical waveguide core; providing fluorescently labeled nucleotide analogs; and monitoring stepwise addition of the fluorescently labeled nucleotide analogs as they are added to a nascent strand of the template.In some cases the group 4-5 transition metal oxide is hafnium oxide. In some cases the group 4-5 transition metal oxide is selected from the group consisting of titanium oxide, zirconium oxide, niobium oxide, and tantalum oxide. In some cases a bottom surface modifying agent that is selectively associated with the bottom surface and that comprises a coupling group. In some cases a sidewall surface modifying agent that is associated with the sidewall surface and that passivates the sidewall.

[0013] In some cases the bottom surface comprises a bottom surface modifying agent comprising one or more phosphonic acid groups, one or more catechol groups, one or more phosphate groups, or a combination thereof. In some cases the bottom surface comprises a bottom surface modifying agent comprising one or more phosphonic acid groups and a coupling group. In some cases the bottom surface modifying agent comprises a biotin-PEG- phosphonic acid. In some cases the bottom surface comprises a bottom surface modifying agent comprising one or more catechol groups and a coupling group. In some cases the bottom surface modifying agent comprises a biotin-PEG-catechol. In some cases the bottom surface comprises a first bottom surface modifying agent that comprises a coupling group and a second bottom surface modifying agent that lacks the coupling group. In some cases the sidewall surface comprises silicon dioxide, further comprising a silane coating on the silicon dioxide. In some cases the sidewall surface comprises silicon dioxide, further comprising a PEG-silane coating on the silicon dioxide. In some cases the bottom surface comprises a coupling group bound thereto, wherein the coupling group is selected from the group consisting of biotin, a biotin-binding protein, an antibody, an antibody fragment, a lectin, and a nucleic acid.

[0014] In some cases the bottom surface comprises a coupling group bound thereto, wherein the coupling group is selected from the group consisting of an amine group, a carboxyl group, a hydroxyl group, a sulfhydryl group, an azido group, an alkyne group, a cyclooctyne group, a dibenzocyclooctyne (DBCO) group, a trans-cyclooctene (TCO) group, a norbornene group, a tetrazine group, an epoxy group, a glycidyl group, an aldehyde group, an alkenyl group, an acrylate group , a methacrylate group, an anhydride group, an activated carboxylic acid group, and an n-hydroxysuccinimide (NHS) ester group. In some cases the substrate comprises a layer of silicon dioxide overlying a layer of group 4-5 transition metal oxide, wherein the nanoscale well is a nanoscale aperture that penetrates through the layer of silicon dioxide to the layer of group 4-5 transition metal oxide. In some cases the top surface comprises silicondioxide. In some cases the top surface comprises aluminum oxide. In some cases the bottom surface of the well has an area less than 196,000 nm2. In some cases the bottom surface of the well has an area less than 70,700 nm2.

[0015] In some aspects, the invention provides a method of preparing a substrate for selective immobilization of a molecule or molecules of interest thereon, comprising: providing a substrate comprising a plurality of nanoscale wells disposed through a top surface of the substrate, wherein each nanoscale well comprises a sidewall surface that comprises silicon dioxide or aluminum oxide and a bottom surface that comprises a group 4-5 transition metal oxide; and contacting the substrate with a bottom surface modifying agent that comprises a coupling group and that selectively associates with the group 4-5 transition metal oxide, thereby selectively locating the coupling group on the bottom surface of the nanoscale wells. In some cases the bottom surface modifying agent comprises one or more phosphonic acid groups, one or more catechol groups, one or more phosphate groups, or a combination thereof. In some cases the bottom surface modifying agent comprises a biotin-PEG-phosphonic acid. In some cases the bottom surface modifying agent comprises a biotin-PEG-catechol. In some cases the sidewall comprises silicon dioxide, comprising contacting the substrate with a sidewall surface modifying agent that associates with and passivates the silicon dioxide. In some cases the sidewall comprises silicon dioxide, comprising contacting the substrate with a sidewall surface modifying agent that comprises one or more silane groups and that associates with and passivates the silicon dioxide. In some cases the sidewall surface modifying agent comprises a PEG-silane. In some cases the substrate is contacted with the bottom surface modifying agent and then contacted with the sidewall surface modifying agent.

[0016] In some cases, after contacting the substrate with the sidewall surface modifying agent, the substrate is contacted with a removal compound that preferentially removes the sidewall surface modifying agent from the group 4-5 transition metal oxide. In some cases the substrate is contacted with the sidewall surface modifying agent and then contacted with the bottom surface modifying agent. In some cases coupling the molecule or molecules of interest to the coupling group, thereby selectively immobilizing the molecule or molecules of interest on the bottom surface of the nanoscale wells. In some cases the group 4-5 transition metal oxide is hafnium oxide. In some cases the group 4-5 transition metal oxide is selected from the group consisting of titanium oxide, zirconium oxide, niobium oxide, and tantalum oxide.In some cases the coupling group is selected from the group consisting of biotin, a biotinbinding protein, an antibody, an antibody fragment, a lectin, and a nucleic acid.

[0017] In some cases the coupling group is selected from the group consisting of an amine group, a carboxyl group, a hydroxyl group, a sulfhydryl group, an azido group, an alkyne group, a cyclooctyne group, a dibenzocyclooctyne (DBCO) group, a trans-cyclooctene (TCO) group, a norbomene group, a tetrazine group, an epoxy group, a glycidyl group, an aldehyde group, an alkenyl group, an acrylate group , a methacrylate group, an anhydride group, an activated carboxylic acid group, and an n-hydroxysuccinimide (NHS) ester group. In some cases the density of the coupling group on the bottom surface is 100 times or more greater than density of the coupling group on the sidewall surface and the top surface. In some cases the density of the coupling group on the bottom surface is 1000 times or more greater than density of the coupling group on the sidewall surface and the top surface. In some cases the substrate comprises a layer of silicon dioxide overlying a layer of metal oxide, wherein the nanoscale wells are nanoscale apertures that penetrate through the layer of silicon dioxide to the layer of group 4-5 transition metal oxide. In some cases the top surface comprises silicon dioxide. In some cases the top surface comprises aluminum oxide. In some cases the bottom surface of each well has an area less than 196,000 nm2. In some cases the bottom surface of each well has an area less than 70,700 nm2.

[0018] In some aspects the invention provides a method for producing an array of analytic devices on a substrate, comprising: providing a substrate comprising an optical waveguide core layer that comprises one or more optical waveguide cores; depositing an optical waveguide upper cladding layer comprising silicon dioxide on the optical waveguide core layer; depositing a group 4-5 transition metal oxide on the optical waveguide upper cladding layer to form a group 4-5 transition metal oxide layer; depositing a silicon dioxide layer on the group 4-5 transition metal oxide layer to form a well forming layer; and patterning and etching to form an array of nanoscale apertures penetrating through the well forming layer to the group 4-5 transition metal oxide layer, thereby forming an array of nanoscale wells that each comprise a silicon dioxide sidewall surface and a group 4-5 transition metal oxide bottom surface. In some cases the group 4-5 transition metal oxide layer is an etch-stop layer to control the depth of the apertures. In some cases the optical waveguide core layer overlies a detector layer. In some cases the group 4-5 transition metal oxide is hafnium oxide. In some cases the group 4-5 transition metal oxide is selected from the group consisting of titaniumoxide, zirconium oxide, niobium oxide, and tantalum oxide. In some cases the bottom surface of each well has an area less than 196,000 nm2. In some cases the bottom surface of each well has an area less than 70,700 nm2. In some cases reflective layer is deposited on the well forming layer prior to patterning and etching the array of nanoscale apertures.

[0019] In some aspects the invention provides a substrate with selectively functionalized regions, the substrate comprising: a plurality of active regions that each comprise a group 4-5 transition metal oxide, each active region having a molecule or molecules of interest attached thereto; and surrounding regions around the plurality of active regions, the surrounding regions comprising silicon dioxide or aluminum oxide. In some cases the molecule or molecule or molecules of interest are attached to the plurality of active regions through a coupling group on an active surface region surface modifying agent. In some cases the surrounding regions comprise a surrounding region surface modifying agents that passivates the surrounding regions.BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figs 1A-1G show embodiments of nanoscale wells of the invention having bottoms comprising group 4-5 transition metal oxides. Fig. 1G illustrates how in any of these embodiments, the substrate can comprise a waveguide below the nanoscale well.

[0021] Figs 2A-2F show embodiments of nanoscale wells of the invention having well bottoms that comprise waveguide cores that comprise group 4-5 transition metal oxides.

[0022] Figs 3A-3F illustrate how surface modifying agents can be used for selective functionalization of the nanoscale wells of the invention for selectively attaching a molecule or a plurality of molecules of interest to the bottom of the nanoscale well.

[0023] Fig. 4 provides an embodiment of a method of producing a selectively functionalized nanoscale well of the invention.

[0024] Fig. 5 provides another embodiment of a method of producing a selectively functionalized nanoscale well of the invention.

[0025] Fig. 6 provides another embodiment of a method of producing a selectively functionalized nanoscale well of the invention.

[0026] Figs 7A and 7B show examples of phosphonate-linker-coupling group compounds useful in the invention.

[0027] Fig. 8 provides an exemplary method of producing a shallow well of the invention having well bottom comprising group 4-5 transition metal oxide.

[0028] Fig. 9A shows a cross section of a substrate comprising an active region comprising a group 4-5 transition metal oxide with surrounding regions comprising silicon dioxide or aluminum oxide. Fig. 9B shows how this substrate can be treated with active region surface modifying agents and surrounding region surface modifying agents as described herein to selectively functionalize the regions. Fig. 9C shows an array of active regions separated by surrounding regions comprising silicon dioxide or aluminum oxide of the invention.

[0029] Fig. 10 shows a cross-section of a unit cell of a chip comprising an array of integrated analytical devices, in which each unit cell has a nanoscale well and the components required for illuminating the nanoscale well and detecting light emitted from it.

[0030] Fig. 11 A illustrates an embodiment of a nanoscale well of the invention in a chip having an optical waveguide below the nanoscale well for providing excitation illumination. Fig. 1 IB illustrates an embodiment of a nanoscale well of the invention in a chip having an optical waveguide below the nanoscale well for providing excitation illumination.

[0031] Throughout the drawings, reference numbers may be re-used to indicate correspondence between referenced elements. The drawings are provided to illustrate example embodiments described herein and are not intended to limit the scope of the disclosure. Schematic figures are not necessarily to scale.DETAILED DESCRIPTION OF THE INVENTIONGeneral

[0032] The present invention is generally directed to substrates, analytic devices, methods, and processes for providing desired molecules in preselected locations, areas, or regions on a substrate, such as on the bottoms of nanoscale wells. The inventors have found that having a group 4 or group 5 transition metal oxide on the base or bottom of nanoscale wells provides advantages over other approaches. The group 4-5 transition metal oxides provide robust coatings in aqueous environments. The surfaces of the group 4-5 transition metal oxides can be selectively treated with surface modifying agents having coupling groups allowing for the attachment of molecules of interest to the bottom of the nanoscale wells.

[0033] The inventors have also found that silicon dioxide or aluminum oxide on the sidewalls of the nanoscale wells allows for orthogonal chemistry to be employed such that molecules of interest are attached highly selectively to the bases but not to the sidewalls of the wells. Sidewall surface modifying agents, e.g., passivating agents, can be applied the sidewalls to further minimize binding of molecules of interest or other reaction components to the sidewalls or other regions of the surface than the bottoms of the wells. In some cases, a removal compound can be applied to selectively remove surface modifying agents from regions where the presence of that surface modifying agent is not desired. In some cases, silicon dioxide or aluminum oxide is on the tops of the substrates as well as on the sidewalls. In some cases the selectively functionalized regions are not nanoscale wells, but are active regions of group 4-5 transition metal oxide separated by surrounding regions of silicon dioxide or aluminum oxide.

[0034] The nanoscale wells constructed as described herein can be used for analysis and detection of molecules of interest. In some cases, the analysis or detection of a single molecule of interest within the nanoscale well is carried out. In some cases, multiple molecules of interest, e.g. clonal populations, within the nanoscale well can be analyzed or detected. In some cases, the molecule or molecules of interest are detected directly. In some cases the molecule or molecules of interest are detected by observing labeled analytes that interact with the molecules of interest. In some aspects, the invention relates to how molecules (molecules of interest) are selectively attached to preferred regions of substrates and the substrates and analytic devices that are produced. In other aspects, the invention relates to analytic methods that use these selectively functionalized surfaces to detect molecules (molecules of interest). It will be understood by those of skill in the art that a molecule that is detected, e.g. a labeled molecule, may not itself be the “molecule of interest”, but will provide detection or identification of the molecule of interest indirectly.

[0035] The molecules of interest can be biomolecules, for example, nucleotides, nucleic acids, amino acids, peptides, or proteins. A preferred use of the selectively functionalized nanoscale wells is in single-molecule real-time nucleic acid sequencing.

[0036] Substrates with the selectively functionalized nanoscale wells of the invention will typically have a plurality of nanoscale wells. In some cases, a substrate will have arrays of nanoscale wells of greater than a thousand wells, greater than 100,000 wells, greater than a million wells, or greater than ten million wells. Substrates with these numbers of nanoscalewells can be produced by semiconductor processing methods, for example, carried out by building layers of materials on top of a silicon wafer. The group 4-5 transition metal oxides can be deposited with known semiconductor processing approaches.

[0037] The nanoscale wells of the invention can be incorporated into analytic chips having optical and / or electronic functionality. For example, the nanoscale wells can be produced in substrates which also include optical waveguides, lenses, apertures, filters, and detectors.

[0038] The analytic devices or chips of the invention can be used as part of analytic systems or instruments. The analytic chip typically has thousands to millions of nanoscale wells. The analytic chips are contacted with a solution having analytic reagents. The system or instrument is then used to monitor chemical reactions or interactions that provide an analytic signal to identify or characterize molecules of interest within the nanoscale wells.

[0039] An analytic instrument can provide excitation light and or input electronic signals to the analytic chips, and the instrument can receive emission light or output electronic signals from the analytic chips. The received emission light or output electronic signals can provide data to a computer for use in carrying out the analysis. There can be computers in the analytical instrument or outside the analytical instrument. In some cases, there are computers both inside and outside of the analytical instrument for use in the analysis.

[0040] In a preferred embodiment the analytic instrument is used for nucleic acid sequencing. There are a number of nucleic acid sequencing approaches for which the instant invention can provide a significant advantage. The analytic devices of the invention can be used with either single molecule sequencing approaches or multi-molecule (clonal population) approaches to sequencing. The sequencing methods can be real-time (e.g. observing the incorporation of nucleotides into a growing strand), or the sequencing methods can be sequential methods wherein the sequencing reaction is halted between nucleotide additions for observation. A number of methods of nucleic acid sequencing using multi-molecule or clonal populations on surfaces are known. In some cases, reversibly blocked nucleotides can be employed to control the nucleotide addition. One method of clonal population sequencing for which the invention can be useful is in sequencing employing sequencing by binding or SBB chemistry. Multi-molecule or clonal population sequencing approaches are described, for example in U.S. Patents US8728729, US8637242, US9267173, US9051612, 7635562, EP1957667, US10768173, US11203778, and U.S. Patent Applications US20200199668, US20210318295,US20210223531, US20210292810, US20240035082A1, the full contents of which are incorporated herein by reference for all purposes.

[0041] In some cases, the nucleic acid sequencing chip is an integrated sequencing chip. Such chips can have millions of nanoscale wells of the invention with 4-5 transition metal oxides on the bottoms and typically silicon dioxide on the sidewalls of the nanoscale wells. The sequencing chip can have an array of optical waveguides extending below the nanoscale wells to provide evanescent wave illumination to the nanoscale wells. The chip can have an array of photodetectors arranged to correspond to the spacings of the nanoscale wells and disposed such that light emitted from the nanoscale wells passes down, through the optical waveguides, to the photodetectors, all within the chip. The chip may also include other optical components between the bottom of the nanoscale well and the detector such as lenses, filters, and apertures. The nanoscale wells in the chip are selectively functionalized as described herein with bottom surface modifying agents having coupling groups such as phosphonates or catechols on the bottoms of the nanoscale wells, and sidewall surface modifying agents such as silanes on the sidewalls of the wells. The integrated sequencing chips of the invention can be used for single molecule or for multiple-molecule sequencing approaches.

[0042] Where single molecule sequencing methods are used, nanoscale wells can be produced such that a single polymerase-DNA template complex is attached to the bottom of a significant fraction of the nanoscale wells in the chip through coupling groups on the bottoms of the wells. Analytical reagents, in this case fluorescently labeled nucleotides and components for nucleic acid synthesis, are put into contact with the chip. As labeled nucleotides diffuse into the nanoscale wells and become incorporated into a DNA strand complementary to the template nucleic acid, the fluorescent labels on the nucleotides are excited by the evanescent illumination from the optical waveguides below the nanoscale wells. The fluorescent labels emit fluorescent signals which pass down, through the optical waveguides to the photodetectors. In this way, the synthesis of the DNA strand is monitored in real time, allowing the sequence of that strand, and therefore the template strand, to be determined.

[0043] The inventors have found nanoscale wells of the invention can be produced by using the group 4-5 transition metal oxides as etch stop layers in semiconductor processes. For example, a group 4-5 transition metal oxide layer can be deposited onto a base substrate, followed by the deposition of a well forming layer, e.g. a silicon dioxide layer.Semiconductor photolithography and etching processes can be used to etch a nanoscale well into the silicon dioxide layer, and the group 4-5 transition metal oxide acts as the etch stop. This process produces a nanoscale well having a bottom that is group 4-5 transition metal oxide and sidewalls that are silicon dioxide. These wells can be selectively functionalized as described herein.

[0044] Molecules of interest can be selectively attached to the group 4-5 transition metals on the bottoms of the nanoscale wells, for example, using selective surface modifying agents containing coupling groups. The inventors have found that surface modifying agents containing, for example, phosphate, phosphonate or catechol functionality can provide for robust functionalization of the group 4-5 transition metals on the bottoms of the nanoscale wells. Surface modifying agent based on phosphate, phosphonate, or catechols having coupling groups attached to them can be employed. In some cases the molecules of interest can be attached directly to the surface modifying agents without the use of intermediate coupling groups. In other cases, the molecules of interest can be indirectly attached to the surface modifying agent through one or more intermediate coupling groups. For example, a biotinylated molecule of interest can be attached to a biotinylated bottom surface modifying agent through a biotin binding protein such as avidin or streptavidin. As another example, a molecule of interest can be reacted with a bifunctional crosslinker that is also reacted with a coupling group on the bottom surface modifying agent.

[0045] The inventors have discovered that, while 4-5 transition metal oxides and aluminum oxide react similarly with surface modifying agents such as those containing phosphonates and catechols, removal compounds can be used to selectively remove these surface modifying agents from aluminum oxide while removing little or no bottom surface modifying agents from group 4-5 transition metal oxides. In some cases, this selectivity is based on the difference in etch rate. Such removal compounds have a much higher etch rate for aluminum oxide than for 4-5 transition metal oxides.

[0046] In some cases, removal compounds can be employed to enhance the selectivity of the surface modifying agents. Removal compounds can, for example, selectively remove surface modifying agents from the walls while leaving these surface modifying agents on the bottoms of the nanoscale wells. For example, where the base of a nanoscale well has a 4-5 transition metal oxide, and a phosphonate or catechol bottom surface modifying agent is employed, some of this surface modifying agent can undesirably remain on the walls. Treatment withremoval compounds can selectively remove the unwanted bottom surface modifying agents from the sidewalls. Other removal compounds can be used to remove unwanted sidewall surface modifying agents from the bottoms of the nanoscale wells.

[0047] The substrates, devices, systems, and methods of the invention may be broadly practical in providing individual molecules or groups of molecules within any of a variety of given desired spaces, for example, regions on a substrate. In preferred aspects, the processes are used to selectively deposit or immobilize a desired molecule or group of molecules within the optically accessible portion of nanoscale wells.

[0048] In general, nanoscale wells of the invention are used to provide electromagnetic radiation and to allow emission of radiation only from very small spaces or volumes. In some cases, the nanoscale wells have one or more dimensions such as height or width that are less than 500 nanometers, less than 300 nanometers, less than 200 nanometers, less than 100 nanometers, less than 50 nanometers, or less than 30 nanometers. Nanoscale wells can, in some cases, have a bottom with a somewhat circular area where the circle has a diameter from about 10 nm to about 200 nm. Typically, the nanoscale wells are illuminated from below, for example from the evanescent waves from a waveguide core disposed below the nanoscale well. As a result, the radiation will permeate the core only a very short distance up from the bottom of the well, and consequently illuminate only a very small volume within the nanoscale well. Other illumination methods can be employed and are contemplated, including, e.g., field enhancement by sharp metal tips, nanotube confinement, thin slit confinement, near-field resonant energy transfer confinement, near field aperture confinement, diffraction limited nanoscale well, and stimulated emission depletion confinement, as well as all other confinements described in pending US. Patent No. 7,170,050, 7,056,661, and 6,917,726, each of which is incorporated herein by reference in its entirety for all purposes.

[0049] In accordance with the present invention, the nanoscale well is typically fluid filled. The portion of the nanoscale well near the bottom provides a zone or volume in which a chemical, biochemical, and / or biological reaction may take place that is characterized by having an extremely small volume, and in some cases is sufficient to include only a single molecule or set of molecules. Nanoscale wells can comprise zero mode waveguides (ZMWs), whose fabrication, structure, and use in analytical operations are described in detail in U.S. Patent No. 6,917,726 and Levene, et al., Science 299(5607):609-764 (2003), the fulldisclosures of which are hereby incorporated herein by reference in their entirety for all purposes.

[0050] In the context of chemical or biochemical analyses within nanoscale wells, it is clearly desirable to ensure that the reactions of interest are taking place within the optically interrogated portions of the nanoscale well, at a minimum, and in some cases such that only the reactions of a single molecule are occurring within an interrogated portion of a nanoscale well. A number of methods may generally be used to provide individual molecules within an observation volume. A variety of these are described in U.S. Patents 10,525,438, 8.906.831, 8,802,600, 8,501,406 which are incorporated herein by reference in their entirety for all purposes, which describes, inter alia, modified surfaces that are designed to immobilize individual molecules to the surface at a desired density, such that approximately one, two, three or some other select number of molecules would be expected to fall within a given observation volume. In some case, such methods utilize dilution techniques to provide relatively low densities of coupling groups on a surface, either through dilution of such groups on the surface or dilution of intermediate or final coupling groups that interact with the molecules of interest, or combinations of these.

[0051] In some cases, it may be further desirable that reactions of interest be reduced or even eliminated from regions outside of the observation volume of the nanoscale well, e.g., on sidewalls of the nanoscale well or on the tops of the chip. In some cases, it is also desirable to prevent molecules in a reaction mixture from binding to the portions of the sidewall within or outside of the illumination region. In particular, reaction components that are outside of the range of interrogation may, nonetheless, impact the reaction of interest or the monitoring of that reaction, by affecting reaction kinetics through depletion of reagents, increasing concentration of products, contributing to signal background noise levels, e.g., through the generation of products or consumption of reactants, that may interfere with the interrogated reaction or that provide excessive detectable background product levels that diffuse into and out of the interrogation volume of the nanoscale well.

[0052] Accordingly, selective and preferential deposition and / or immobilization of the reaction components within the observation volume are particular advantages of the invention. In the context of the foregoing, molecules of interest may be described as being preferentially located in a particular region, or localized substantially in a given region. It will be appreciated that use of the term preferentially is meant to indicate that the molecule islocalized in a given location at a concentration or surface density that exceeds that of other locations in which it is not preferentially localized. Thus preferential immobilization of a given molecule in a first region will mean that the molecule is present in such region at a higher density or concentration than in other regions. Density in such regions may be as much as 20% greater, 30% greater, 50% greater, 100% greater, or upwards of 200%, up to 1000% or more of the concentration or density in other regions, and in some cases 100 times greater, 1000 times greater or more. Similar meaning is generally applicable to indications that a given molecule is substantially only located in a given region.

[0053] In the case of, for example, nanoscale wells used for single molecule enzymatic analysis, it may be desirable to provide a single enzyme molecule within the illumination volume of a nanoscale well, and preferably upon the bottom or base surface of the well. As noted above, it may therefore be further desirable to ensure that additional enzyme molecules are not present upon surfaces other than the bottom surface.

[0054] As recited above, a particularly valuable application of the substrates produced by the process of the invention is in processes termed “single molecule sequencing applications.” By way of example, a complex of a template nucleic acid, a primer sequence and a polymerase enzyme may be monitored, on a single molecule basis, to observe incorporation of each additional nucleotide during template dependent synthesis of the nascent strand. By identifying each added base, one can identify the complementary base in the template, and thus read off the sequence information for that template. In the context of nanoscale wells, an individual polymerase / template / primer complex may be provided within the observation volume of the nanoscale well. As each of four labeled (e.g., fluorescent) nucleotides or nucleotide analogs is incorporated into the synthesizing strand, the prolonged presence of the label on such nucleotide or nucleotide analogs will be observable by an associated optical detection system. Suitable nucleotide analogs are described, for example in U.S. Patents 10,676,788, 11,884,862, and 10,781,483 the full disclosures of which are incorporated herein by reference in their entirety for all purposes. Suitable sequencing detection systems are described in U.S. Patents 8,46,569, 9,410,891, 8,467,061, 9,372,308 9,223,084, 9,624,540, 9,606,068, the full disclosures of which are incorporated herein by reference in their entirety for all purposes. Such single molecule sequencing applications are envisioned as being benefited by the methods described herein, through the selected immobilization ofpolymerases, templates or primers or complexes of any or all of these, preferentially within selected regions on a substrate, and / or substantially not on other portions of the substrate.

[0055] Although generally discussed in terms of localization of enzymes or other macromolecular groups, for purposes of the present invention, the molecule or molecules of interest may be any of a variety of different functional molecules for which one desires to provide spatial individuality or enhanced localization. Such groups include active molecules, such as catalytic molecules like enzymes, but also include molecules with other functionality, e.g., non-catalytic groups, such as binding or coupling groups, hydrophobic or hydrophilic groups, structural enhancement groups, e.g., for adhesion promotion, activatable or deactivatable groups, or the like. Catalytically active molecules will typically include any catalytically active molecule for which one desires spatial individuality, e.g., to exploit in single molecule analyses, or the like.

[0056] The substrates, devices, instruments, and systems of the invention have numerous advantages over previously described approaches. The treatment of group 4-5 transition metal oxide surfaces with modified phosphonates, phosphates, or catechols (e.g. a biotin-PEG- phosphonic acid or a biotin-PEG-catechol) does not require “activation” as is often required where the bottom surface comprises silicon dioxide (where, for example, treatment with solvent-based alkoxy silane requires initial hydrolysis or activation). In addition, the treatment of group 4-5 transition metal oxide surfaces with modified phosphonates, phosphates, or catechols can be carried out in aqueous solution. Solutions of surface modifying agents comprising phosphonates, phosphates, or catechols are typically very stable, having long shelf lives. Even neat material does not typically require special storage (such as freeze drying). This enhanced stability can enable efficient processes, because since the solutions are very stable, material quality control is simplified, allowing for use of the solutions without repeated quality control steps. Bottom surface modifying agents comprising phosphonates, phosphates, or catechols can deposit onto group 4-5 transition metal oxide surfaces at fast rates. For example, in some cases, the deposition can be accomplished in a few minutes using processes such as dipping, spraying, or spin casting. For the sidewall surface modification, there are many low-cost SiCh surface modification and passivation reagents in the market (e.g., silane reagents), including several that can be dispensed by vapor phase (i.e. solvent-free “dry process”). A typical CVD process typically requires less than 1g of material for 25 wafers, with negligible chemical waste.Substrates having sel ctively functionalized nanoscale wells

[0057] The substrates having selectively functionalized nanoscale wells of the invention are typically produced on base substrates at least partly via semiconductor processing techniques. The base substrate for this type of processing can be any suitable material. The base substrate can comprise, for example, silicon, germanium, gallium arsenide, gallium nitride, silicon carbide, silicon on insulator (SIN), or a flexible polymer. Typical base substrate materials are silicon and silicon dioxide (SiCh). The base substrate on which the nanoscale wells are formed will often have one or multiple layers on top of a base substrate. For example, the base substrate as described herein may have the layers that will constitute a multilayer device produced on top of a semiconductor wafer having multiple photosensors. An analyitic or sensing device is then produced by adding layers over the photosensors. The nanoscale wells are typically on the top of the substrate to facilitate interaction with analytic samples in contact with nanoscale wells from above.

[0058] As used herein, nanoscale wells are wells that have dimensions, e.g. height and width on the nanometer scale. In some cases, the nanoscale wells have one or more dimensions such as height or width that are between 1 nm and 500 nm. In some cases, the nanoscale wells have one or more dimensions such as height or width that are less than 500 nanometers, less than 300 nanometers, less than 200 nanometers, or less than 100 nanometers. In some cases, the height is between 10 nm and 500 nm. In some cases a width of the base of the well is between 30 nm and 300 nm. In some cases a width of the base of he the well is between 50 nm and 200 nm. In some cases, the nanoscale wells have bottoms with areas between 79 nm2and 196,000 nm2. In some cases, the nanoscale wells have bottoms with areas between 1,900 nm2and 71,000 nm2. In some cases, the nanoscale wells have bottoms with areas less than 196,000 nm2, less than 71,000 nm2, less than 31,000 nm2, or less than 7,900 nm2. The shape of the bottom of the well is often roughly circular, but any suitable shape can be used, for example, roughly oval, square, or rectangular. For applications where single molecules of interest are identified, nanoscale wells having heights between 100 nm and 400 nm and base width dimensions between 30 nm and 200 nm can be used.

[0059] The nanoscale wells of the invention typically extend through a well forming layer to a group 4-5 metal oxide layer. In addition to these layers there can be other layers. In some cases there can be more than one well forming layer.

[0060] Figs. 1A - IF show cross sections of embodiments of selectively functionalized nanoscale wells of the invention. Fig. 1A shows an embodiment of a nanoscale well of the invention. The nanoscale well 100 extends through a well forming layer 130. The well forming layer can comprise silicon dioxide. In some cases, the material of the well forming layer 130 is silicon dioxide. The nanoscale well 100 extends down to a group 4-5 transition metal oxide layer 120, which forms the bottom of the well. In some cases, the group 4-5 transition metal oxide layer comprises hafnium oxide. The nanoscale well 100 has sidewalls comprising silicon dioxide and a bottom comprising a group 4-5 transition metal oxide allowing for selective functionalization of the sidewalls and the bottom as described herein. For example, a sidewall surface modifying agent (not shown) can be present on the sidewalls, and a bottom surface modifying agent (not shown) can be present on the bottom of the well. The bottom surface modifying agent can comprise coupling groups for the attachment of a single molecule of interest 190 or for the attachment of multiple molecules of interest, e.g. clonal populations of molecules, to the base of the well. The nanoscale well is formed on the base substrate 110. In a preferred embodiment, the layer of the base substrate 110 on which the bottom of the nanoscale well resides is a cladding layer (e.g. silicon dioxide) above a waveguide core (not shown).

[0061] Fig. IB shows an embodiment of a nanoscale well of the invention. The nanoscale well 101 extends through a well forming layer 131. The well forming layer can comprise silicon dioxide. In some cases, the material of the well forming layer 131 is silicon dioxide. The nanoscale well 101 extends down to a group 4-5 transition metal oxide layer 121, which forms the bottom of the well. In some cases, the group 4-5 transition metal oxide layer comprises hafnium oxide. On top of the well forming layer 131 there is a reflective layer 141 which can be a metal layer, for example aluminum. The reflective layer can be useful in some types of analytic devices, for example where the well is illuminated from below. On top of the reflective layer 141, there is a surface layer 151. The material forming the surface layer can comprise silicon dioxide and, in some cases, can be silicon dioxide. In some cases, another layer (not shown) such as titanium nitride (TiN) is present between the reflective layer 141 and the surface layer 151.

[0062] The nanoscale well has sidewalls comprising silicon dioxide and a bottom comprising a group 4-5 transition metal oxide allowing for selective functionalization of the sidewalls and the bottom as described herein. For example, a sidewall surface modifying agent (not shown)can be present on the sidewalls, and a bottom surface modifying agent (not shown) can be present on the bottom of the well. The bottom surface modifying agent can comprise coupling groups for the attachment of a single molecule of interest 191 or for the attachment of multiple molecules of interest, e.g. clonal populations of molecules, to the base of the well. The nanoscale well is formed on the base substrate 111. Preferably, the layer of the base substrate 111 on which the bottom of the nanoscale well resides is a cladding layer (e g. silicon dioxide) above a waveguide core (not shown). In this embodiment, the surface layer 151 and the well forming layer 131, which forms the largest portion of the sidewall, both comprise silicon dioxide, such that surface modifying agents can be used to simultaneously treat the sidewalls and the top surface. In this embodiment, a portion of the edge of the reflective layer 141 is exposed on the sidewall. One aspect of the invention is the use of removal compounds.Where the reflective layer 141 comprises aluminum, the exposed portions of the aluminum will typically comprise a native oxide layer of aluminum oxide (alumina). As described herein, removal compounds can be used to remove any bottom surface modifying agents which could have reacted with exposed aluminum oxide. This ensures that the selective functionality, e.g. coupling groups and molecules of interest are located selectively on the bottom of the wells.

[0063] Fig. 1C shows an embodiment of a nanoscale well of the invention. The nanoscale well 102 extends through a well forming layer 132. The well forming layer can comprise silicon dioxide. In some cases, the material of the well forming layer 132 is silicon dioxide. The nanoscale well 102 extends down to a group 4-5 transition metal oxide layer 122, which forms the bottom of the well. In some cases, the group 4-5 transition metal oxide layer comprises hafnium oxide. On top of the well forming layer 132 there is a reflective layer 142 which can be a metal layer, for example aluminum. On top of the reflective layer 142, there is a surface layer 152. In this embodiment, the surface layer 152 extends across the top of the substrate and extends into the nanoscale well 102, coating the sidewalls.

[0064] The material forming the surface layer can comprise silicon dioxide and, in some cases, can be silicon dioxide. In some cases, another layer (not shown) such as titanium nitride is present between the reflective layer 142 and the surface layer 152. The nanoscale well has sidewalls comprising silicon dioxide and a bottom comprising a group 4-5 transition metal oxide allowing for selective functionalization of the sidewalls and the bottom as described herein. For example, a sidewall surface modifying agent (not shown) can be presenton the sidewalls, and a bottom surface modifying agent (not shown) can be present on the bottom of the well. The bottom surface modifying agent can comprise coupling groups for the attachment of a single molecule of interest 192 or for the attachment of multiple molecules of interest, e.g. clonal populations of molecules, to the base of the well. The nanoscale well is formed on the base substrate 112. In a preferred embodiment, the layer of the base substrate 112 on which the bottom of the nanoscale well resides is a cladding layer (e g. silicon dioxide) above a waveguide core (not shown). In this embodiment, the surface layer 152 comprising silicon dioxide forms both the top and the sidewalls, such that surface modifying agents can be used to simultaneously treat the sidewalls and the top surface. In this embodiment, the edge of the reflective layer 142 is not exposed on the sidewall.

[0065] Fig ID shows an embodiment of a nanoscale well of the invention. The nanoscale well 103 extends through a well forming layer 133. The well forming layer comprises silicon dioxide. In some cases, the material of the well forming layer 133 is silicon dioxide. The nanoscale well 103 extends down to a group 4-5 transition metal oxide layer 123, which forms the bottom of the well. In some cases, the group 4-5 transition metal oxide layer comprises hafnium oxide. On top of the well forming layer 133 there is a reflective layer 143 which can be a metal layer, for example aluminum. On top of the reflective layer 143, there is a surface layer 153. In this embodiment the material forming the surface layer comprises aluminum oxide. In some cases, surface layer 153 is a separate layer of aluminum oxide, for example, which has been deposited. Where the reflective layer 143 is aluminum, the aluminum oxide layer 153 can be a native oxide layer formed on the aluminum. In some cases, another layer (not shown) such as titanium nitride is present between the reflective layer 143 and the surface layer 153. The nanoscale well has sidewalls comprising silicon dioxide and a bottom comprising a group 4-5 transition metal oxide allowing for selective functionalization of the sidewalls and the bottom as described herein. For example, a sidewall surface modifying agent (not shown) can be present on the sidewalls, and a bottom surface modifying agent (not shown) can be present on the bottom of the well. The bottom surface modifying agent can comprise coupling groups for the attachment of a single molecule of interest 193 or for the attachment of multiple molecules of interest, e.g. clonal populations of molecules, to the base of the well. The nanoscale well is formed on the base substrate 113. In a preferred embodiment, the layer of the base substrate 113 on which the bottom of the nanoscale well resides is a cladding layer (e.g. silicon dioxide) above a waveguide core (not shown). In thisembodiment, the surface layer 153 comprises aluminum oxide and the well forming layer 133, which forms the largest portion of the sidewall, comprises silicon dioxide. One aspect of the invention is the use of removal compounds. As described herein, removal compounds can be used to remove from the aluminum oxide top surface any bottom surface modifying agents which could have reacted with exposed aluminum oxide. This ensures that the selective functionality, e.g. coupling groups, are located selectively on the bottom of the wells.

[0066] Fig. IE shows an embodiment of a nanoscale well of the invention. The nanoscale well 104 extends through a well forming layer 134. The well forming layer can be any suitable material. The nanoscale well 104 extends down to a group 4-5 transition metal oxide layer 124, which forms the bottom of the well. In some cases, the group 4-5 transition metal oxide layer comprises hafnium oxide. On top of the well forming layer 134 there is a reflective layer 144 which can be a metal layer, for example aluminum. On top of the reflective layer 144, there is a surface layer 154 which comprises aluminum oxide. In this embodiment, the surface layer 154 extends across the top of the substrate and extends into the nanoscale well 140, coating the sidewalls. In some cases, another layer (not shown) such as titanium nitride is present between the reflective layer 144 and the surface layer 154.

[0067] The nanoscale well in this embodiment has sidewalls comprising aluminum oxide and a bottom comprising a group 4-5 transition metal oxide allowing for selective functionalization of the sidewalls and the bottom as described herein. For example, a sidewall surface modifying agent (not shown) can be present on the sidewalls, and a bottom surface modifying agent (not shown) can be present on the bottom of the well. The bottom surface modifying agent can comprise coupling groups for the attachment of a single molecule of interest 194 or for the attachment of multiple molecules of interest, e.g. clonal populations of molecules, to the base of the well. The nanoscale well is formed on the base substrate 114. In a preferred embodiment, the layer of the base substrate 114 on which the bottom of the nanoscale well resides is a cladding layer (e.g. silicon dioxide) above a waveguide core (not shown). In this embodiment, the surface layer 154 comprising aluminum oxide forms both the top and the sidewalls. Removal compounds can be used to selectively remove bottom surface modifying agents from the aluminum oxide on the sidewalls and top surface while leaving the bottom surface modifying agents on the bottoms of the wells in place, allowing for selective functionalization.

[0068] Fig. IF shows an embodiment of a nanoscale well of the invention. The nanoscale well 105 extends through a well forming layer 135. The well forming layer in this embodiment comprises aluminum oxide. In some cases, the material of the well forming layer 135 is aluminum oxide. The nanoscale well 105 extends down to a group 4-5 transition metal oxide layer 125, which forms the bottom of the well. In some cases, the group 4-5 transition metal oxide layer comprises hafnium oxide. The nanoscale well 105 has sidewalls comprising aluminum oxide and a bottom comprising a group 4-5 transition metal oxide allowing for selective functionalization of the sidewalls and the bottom as described herein. For example a bottom surface modifying agent (not shown) can be present on the bottom of the well. The bottom surface modifying agent can comprise coupling groups for the attachment of a single molecule of interest 195 or for the attachment of multiple molecules of interest, e.g. clonal populations of molecules, to the base of the well. The nanoscale well is formed on the base substrate 115. In a preferred embodiment, the layer of the base substrate 115 on which the bottom of the nanoscale well resides is a cladding layer (e.g. silicon dioxide) above a waveguide core (not shown). In this embodiment, both the sidewalls and the top surface comprise aluminum oxide. Removal compounds can be used to selectively remove surface modifying agents from the aluminum oxide on the sidewalls and top surface while leaving the surface modifying agents on the bottoms of the wells in place, allowing for selective functionalization.

[0069] Fig. 1G illustrates a preferred embodiment in which the portion of the substrate just below the bottom of the nanoscale well is the upper cladding above a waveguide core. While this arrangement is shown here for a similar structure to that of Fig. 1C, it is to be understood that this arrangement can also be used with all of the structures of Fig. 1A-F and for other variations of the invention. Here, the nanoscale well 106 extends through a well forming layer 136. The well forming layer can comprise silicon dioxide. In some cases, the material of the well forming layer 136 is silicon dioxide. The nanoscale well 106 extends down to a group 4- 5 transition metal oxide layer 126, which forms the bottom of the well. In some cases, the group 4-5 transition metal oxide layer comprises hafnium oxide. On top of the well forming layer 136 there is a reflective layer 146 which can be a metal layer, for example aluminum. On top of the reflective layer 146, there is a surface layer 156. In this embodiment, the surface layer 156 extends across the top of the substrate and extends into the nanoscale well 106, coating the sidewalls.

[0070] The material forming the surface layer can comprise silicon dioxide and, in some cases, can be silicon dioxide. The nanoscale well has sidewalls comprising silicon dioxide and a bottom comprising a group 4-5 transition metal oxide allowing for selective functionalization of the sidewalls and the bottom as described herein. For example, a sidewall surface modifying agent (not shown) can be present on the sidewalls, and a bottom surface modifying agent (not shown) can be present on the bottom of the well. The bottom surface modifying agent can comprise coupling groups for the attachment of a single molecule of interest 196 or for the attachment of multiple molecules of interest, e.g. clonal populations of molecules, to the base of the well. The nanoscale well is formed on the base substrate 116. In this preferred embodiment, layer 166 is an upper cladding layer, e.g. silicon dioxide, layer 176 is the waveguide core with a relatively high refractive index material, e.g. silicon nitride, and layer 186 is a lower cladding layer, e.g. silicon dioxide. This arrangement allows for light propagating down the waveguide (formed from the waveguide core and upper and lower cladding layers) to illuminate a row of nanoscale wells such as nanoscale well 106 from below.

[0071] In the above examples and throughout the specification it is described that a layer or other element comprises a group 4-5 transition metal. It is to be understood that in some cases these layers or elements comprising the group 4-5 transition metal will be or will consist essentially of a group 4-5 transition metal. Also, in the above examples and throughout the specification it is described that a layer or other element comprises silicon dioxide, aluminum oxide or other specified material. It is to be understood that in some cases these layers or elements comprising silicon dioxide, aluminum oxide, or other specified material will be or will consist essentially of a such materials.

[0072] Those of skill in the art will understand that each of the specific embodiments described are not limiting, and that combinations of the embodiments illustrated here can also be useful.

[0073] The nanoscale well containing substrates of the present invention can be used in analytic devices and systems, for example in analytic chips. In some cases, these analytical devices or chips are illuminated using optical waveguides in the chip, allowing evanescent wave illumination of the nanoscale wells from below. Chips with such integrated optical waveguides are described, for example in U.S. Patents 8,46,569, 9,410,891, 8,467,061,9,372,308 9,223,084, 9,624,540, 9,606,068, the full disclosures of which are incorporated herein by reference in their entirety for all purposes.

[0074] One aspect of the invention is a chip having an array of nanoscale wells where the nanoscale wells are illuminated from below with one or more optical waveguides. The optical waveguides typically have a core of transparent core material surrounded by a cladding of a transparent cladding material. The refractive index of the core material is higher than the surrounding cladding material. Silicon dioxide is commonly used as the cladding material. An optical waveguide in the chip typically illuminates a row of nanoscale wells disposed above the waveguide. The bottom of the nanoscale well is typically close to the waveguide core. For example, the distance between the bottom of the nanoscale well and the top of the waveguide core can be less than 200 nm, less than 100 nm, less than 50 nm, less than 30 nm, less than 20 nm, or less than 10 nm. In some cases, the distance between the bottom of the nanoscale well and top of the waveguide core is greater than 10 nm, greater than 20 nm, greater than 30 nm, greater than 50 nm, or greater than 100 nm. In some cases the distance between the distance between the bottom of the nanoscale well and top of the waveguide core is between 10 nm and 200 nm, or between 20 nm and 100 nm.

[0075] Another aspect of the invention is a chip having an array of nanoscale wells where the nanoscale wells are illuminated from below with one or more optical waveguides where the bottom of the nanoscale well is formed by the top of the optical waveguide core and the waveguide core comprises a group 4-5 transition metal oxide. In some cases, the waveguide core comprises hafnium oxide. The group 4-5 transition metals have relatively high refractive indices relative to silicon dioxide allowing them to be used as waveguide core materials. Silicon dioxide, a typical cladding material, has a refractive index in the visible of about 1.5. Having the top of the waveguide core as the base of the nanoscale wells has processing advantages in that the group 4-5 transition metal oxide materials of the waveguide core can act as the etch stop, providing a straightforward method for forming the wells of the invention. Note that where the bottom of the nanoscale wells is the top of the waveguide core, the volume of the nanoscale well forms part of the cladding. During operation, the nanoscale well will typically be filled with aqueous solution. The aqueous solution typically has a refractive index that is about 1.3 for visible light. This is a relatively low refractive index, allowing for this volume to function as a cladding material and allowing for light propagation through the optical waveguide.

[0076] Fig. 2A shows an embodiment of the invention in which a waveguide core comprising a group 4-5 transition metal forms the bottom of the nanoscale well. The nanoscale well 200 extends into the well forming layer 230. In this embodiment, the well forming layer 230 comprises or consists of silicon dioxide. The optical waveguide core 220 comprises a group 4-5 transition metal and forms the base of the nanoscale well. The figure is a cross-section. The waveguide core 220 is shown in cross section, and extends above and below the drawing. The waveguide core typically extends under a row of nanoscale wells whereby one optical waveguide illuminates multiple nanoscale wells. The waveguide core can illuminate 1,000 or more, 100,000 or more, or a million or more nanoscale wells. The material 210 provides a cladding to the sides and below the waveguide core 220. In some cases, the cladding material surrounding the waveguide core including 210 and 230 comprises or consists of silicon dioxide. Chips having these types of nanoscale wells illuminated by waveguide cores can be useful in devices that monitor a single molecule of interest. Here, a single molecule of interest 290 is attached to the bottom of the nanoscale well (top of the waveguide core). To attach the single molecule of interest, a bottom surface modifying agent (not shown) having coupling groups is selectively applied to the bottoms of the nanoscale wells for attaching the single molecule of interest.

[0077] Fig. 2B shows another embodiment of the invention in which the waveguide core comprising a group 4-5 transition metal forms the bottoms of the nanoscale well. The nanoscale well 201 extends into the well forming layer 231. In this embodiment, the well forming layer 231 comprises or consists of silicon dioxide. On top of the well forming layer 231 is a reflective layer 241. The reflective layer 241 can be an aluminum layer. On top of the reflective layer 241 is a silicon dioxide top surface layer 251. In this embodiment, an edge of the reflective layer 241 is exposed within the nanoscale well. As described herein, removal compounds can be used to reduce or eliminate any unwanted binding of bottom surface modifying agents to this exposed edge. The optical waveguide core 221 comprising a group 4-5 transition metal forms the bottom of the nanoscale well. The material 211 provides a cladding to the sides and below the waveguide core 221. In some cases, the material for the cladding material surrounding the waveguide core including 211 and 231 comprises or consists of silicon dioxide. As shown here, a single molecule of interest 291 is attached to the bottom of the nanoscale well (top of the waveguide core). To attach the single molecule ofinterest, a bottom surface modifying agent (not shown) having coupling groups is selectively applied to the bottom of the nanoscale well for attaching the single molecule of interest.

[0078] Fig. 2C shows another embodiment of the invention in which the waveguide core comprising a group 4-5 transition metal forms the bottom of the nanoscale well. The nanoscale well 202 extends into the well forming layer 232. In this embodiment, the well forming layer 232 comprises or consists of silicon dioxide. On top of the well forming layer232 is a reflective layer 242. The reflective layer 242 can be an aluminum layer. On top of the reflective layer 242 is a silicon dioxide top surface layer 252. The silicon dioxide top surface layer 252 extends over the top of the chip and into the nanoscale wells, covering the sidewalls. The optical waveguide core 222 comprising a group 4-5 transition metal forms the bottom of the nanoscale well. The material 212 provides a cladding to the sides and below the waveguide core 222. In some cases, the material for the cladding material surrounding the waveguide core including 212 and 232 comprises or consists of silicon dioxide. As shown here, a single molecule of interest 292 is attached to the bottom of the nanoscale well (top of the waveguide core). To attach the single molecule of interest, a bottom surface modifying agent (not shown) having coupling groups is selectively applied to the bottom of the nanoscale well for attaching the single molecule of interest.

[0079] Fig. 2D shows another embodiment of the invention in which the waveguide core comprising a group 4-5 transition metal forms the bottom of the nanoscale well. The nanoscale well 203 extends into the well forming layer 233. In this embodiment, the well forming layer 233 comprises or consists of silicon dioxide. On top of the well forming layer233 is a reflective layer 243 that comprises aluminum. The aluminum layer has a native aluminum oxide layer on its surface. The optical waveguide core 223 comprising a group 4-5 transition metal forms the bottom of the nanoscale well. The material 213 provides a cladding to the sides and below the waveguide core 223. In some cases, the material for the cladding material surrounding the waveguide core including 213 and 233 comprise or consist of silicon dioxide. As shown here, a single molecule of interest 293 is attached to the bottom of the nanoscale well (top of the waveguide core). To attach the single molecule of interest, a bottom surface modifying agent (not shown) having coupling groups is selectively applied to the bottom of the nanoscale well for attaching the single molecule of interest. As described herein, removal compounds can be employed to remove unwanted bottom surface modifying agents from aluminum oxide on the top of the reflective layer 243.

[0080] Fig. 2E shows another embodiment of the invention in which the waveguide core comprising a group 4-5 transition metal forms the bottom of the nanoscale well. The nanoscale well 204 extends into the well forming layer 234. In this embodiment, the well forming layer 234 comprises or consists of silicon dioxide. On top of the well forming layer 234 is a reflective layer 244. In some cases, the reflective layer 244 comprises or consists of aluminum. On top of the reflective layer is a top surface layer comprising or consisting of aluminum oxide 254. Here, the top surface layer 254 extends over the top of the chip and extends into the nanoscale wells, coating the sidewalls. The optical waveguide core 224 comprising a group 4-5 transition metal forms the bottom of the nanoscale well. The material 214 provides a cladding to the sides and below the waveguide core 224. In some cases, the material for the cladding material surrounding the waveguide core including 214 and 234 comprises or consists of silicon dioxide. As shown here, a single molecule of interest 294 is attached to the bottom of the nanoscale well (top of the waveguide core). To attach the single molecule of interest, a bottom surface modifying agent (not shown) having coupling groups is selectively applied to the bottoms of the nanoscale wells for attaching the single molecule of interest. As described herein, removal compounds can be employed to remove unwanted bottom surface modifying agents from aluminum oxide on the sidewalls and top of the aluminum oxide top surface layer 254.

[0081] Fig. 2F shows another embodiment of the invention in which the waveguide core comprising a group 4-5 transition metal forms the bottom of the nanoscale well. The nanoscale well 205 extends into the well forming layer 235. In this embodiment, the well forming layer 235 comprises or consists of aluminum oxide. The refractive index of aluminum oxide of about 1.7 is not as low as that of silicon dioxide, but is lower than the refractive indices of the group 4-5 transition metals that make up the waveguide core, allowing the aluminum oxide to be an effective cladding material. The optical waveguide core 225 comprising a group 4-5 transition metal forms the bottom of the nanoscale well. The material 215 provides a cladding to the sides and below the waveguide core 225. As shown here, a single molecule of interest 295 is attached to the bottom of the nanoscale well (top of the waveguide core). To attach the single molecule of interest, a bottom surface modifying agent (not shown) having coupling groups is selectively applied to the bottom of the nanoscale well for attaching the single molecule of interest. As described herein, removal compoundscan be employed to remove unwanted bottom surface modifying agents from aluminum oxide on the sidewalls and top of the well forming layer 235.

[0082] One significant aspect of the instant invention is that the nanoscale wells of the invention are amenable to selective functionalization whereby the base or bottom of the nanoscale well is selectively functionalized with bottom surface modifying agents to allow for coupling of molecules of interest, while the sidewalls have little or no bottom surface modifying agent and molecules of interest attached. In addition, separate sidewall functionalization chemistry can be used on the sidewalls. The sidewall surface modifying agents used for this functionalization can be selective or non-selective. The sidewall functionalization chemistry typically does not have coupling groups for attaching molecules of interest. The sidewall surface modifying agents can have functionality for preventing binding or adhesion of molecules of interest. Where the sidewall surface modifying agents are non- selective, subsequent chemistry to remove unwanted sidewall surface modifying agents from the bottoms of the wells can be applied.

[0083] In some cases, the top surface is also treated with surface modifying agents and / or with removal compounds. In some cases, these are the same agents as the sidewall surface modifying agents. In some cases, separate surface modifying agents are used on the sidewalls and the tops.

[0084] The types of chemistry that are used in this way can be referred to as orthogonal chemistry. The nanoscale wells of the invention have bottoms with group 4-5 transition metal oxide. In some cases, orthogonal chemistry is used for silicon dioxide on the sidewalls of the nanoscale wells and / or the tops of the chips. In some cases, orthogonal chemistry is used for aluminum oxide on the sidewalls of the nanoscale wells and / or the tops of the chips. These chemistries are described in more detail herein.

[0085] Figures 3A-3F illustrate how surface modifying agents can be used for selective functionalization of the nanoscale wells of the invention for selectively attaching a molecule or a plurality of molecules of interest to the bottom of the nanoscale well. Fig. 3 A shows an embodiment of a selectively functionalized nanoscale well of the invention. The nanoscale well 301 extends through a well forming layer 331. The well forming layer comprises silicon dioxide. In some cases, the material of the well forming layer 331 is silicon dioxide. The nanoscale well 301 extends down to a group 4-5 transition metal oxide layer 321, which forms the bottom of the well. In some cases, the group 4-5 transition metal oxide layer compriseshafnium oxide. On top of the well forming layer 331 there is an aluminum reflective layer 341. On top of the aluminum reflective layer 341, there is a surface layer 351. The material forming the surface layer can comprise silicon dioxide and, in some cases, is silicon dioxide. In some cases, another layer (not shown) such as titanium nitride is present between the reflective layer 341 and the surface layer 351. The nanoscale well has sidewalls comprising silicon dioxide and a bottom comprising a group 4-5 transition metal oxide allowing for selective functionalization of the sidewalls and the bottom as described herein. The nanoscale well is formed on the base substrate 311.

[0086] Here in Fig. 3A, a bottom surface modifying agent 381 is present on the bottom of the nanoscale well formed by the group 4-5 transition metal oxide layer 321. The bottom surface modifying agent can be chosen to react specifically with the group 4-5 transition metal oxides on the base of the well. Bottom surface modifying agents useful for the present invention include but are not limited to agents comprising phosphates, phosphonates, or catechols. The bottom surface modifying agents also typically include coupling groups for attachment of molecules of interest.

[0087] In addition, a sidewall surface modifying agent 361, for example a silane, is present on the sidewalls of the nanoscale well. In this embodiment, the sidewall surface modifying agent 361 is also present on the top of the top silicon dioxide layer on the top surface of the chip. This sidewall surface modifying agent can act to prevent the adhesion or binding of unwanted components, such a molecules of interest or analytic reaction components. The sidewall surface modifying agents do not have coupling groups for the attachment of molecules of interest.

[0088] It can be seen in Fig. 3A that the edge of the reflective aluminum layer is exposed. Because the edge of aluminum reflective layer 341 will have a surface layer of aluminum oxide (native oxide), the sidewall surface modifying agents directed to silicon dioxide will not typically coat the surface of the edge of the aluminum reflective layer 341. In addition, the aluminum oxide on the edge may react with the bottom surface modifying agents. The inventors have discovered that this issue can be addressed by treating the chip with removal compounds. Removal compounds can be chosen to specifically remove bottom surface modifying agents from aluminum oxide while leaving the bottom surface modifying agents on the group 4-5 transition metal oxide surface at the bottom of the nanoscale well. In some cases, the removal compounds act selectively by etch rate. That is, removal compounds canbe used that etch aluminum oxide at a relatively high rate as compared to the etch rate for the group 4-5 transition metal oxides.

[0089] Fig. 3B illustrates how the nanoscale well of Fig. 3 A can be used to attach molecules of interest selectively to the bottom of the well. The molecules of interest are described herein as attached, bound, coupled, or immobilized to the surface. The attachment, binding, coupling, immobilizing or reacting of the molecules of interest can be covalent or non- covalent. The bottom surface modifying agent 381 on the bottom of the nanoscale well has coupling groups (alternately referred to as binding groups or reactive groups). These coupling groups are chosen to react or bind with complementary functional groups on the molecule or molecules of interest 391. As shown, in some cases a single molecule of interest is attached. In other cases (not shown), the coupling groups of the bottom surface modifying agent 381 can be used to attach multiple molecules (e.g. clonal populations) to the bottom of the well.

[0090] Fig. 3C shows another embodiment of a selectively functionalized nanoscale well of the invention. The nanoscale well 302 extends through a well forming layer 332. The well forming layer comprises silicon dioxide. In some cases, the material of the well forming layer 332 is silicon dioxide. The nanoscale well 302 extends down to a group 4-5 transition metal oxide layer 322, which forms the bottom of the well. In some cases, the group 4-5 transition metal oxide layer comprises hafnium oxide. On top of the well forming layer 332 there is a reflective layer 342. On top of the reflective layer 342, there is a surface layer 352. The material forming the surface layer can comprise silicon dioxide and, in some cases, is silicon dioxide. In this embodiment, the surface layer 352 covers the top surface and extends into the nanoscale well, coating the sidewalls. In some cases, another layer (not shown) such as titanium nitride is present between the reflective layer 342 and the surface layer 352. The nanoscale well has sidewalls comprising silicon dioxide and a bottom comprising a group 4-5 transition metal oxide allowing for selective functionalization of the sidewalls and the bottom as described herein. The nanoscale well is formed on the base substrate 312.

[0091] Here in Fig. 3C, a bottom surface modifying agent 382 is present on the bottom of the nanoscale well formed by the group 4-5 transition metal oxide layer 322. The bottom surface modifying agent can be chosen to react specifically with the group 4-5 transition metal oxides on the base of the well. Bottom surface modifying agents useful for the present invention include agents comprising phosphates, phosphonates, and catechols. The bottom surface modifying agents also include coupling groups for attachment of molecules of interest.

[0092] In addition, a sidewall surface modifying agent 362, for example a silane, is present on the sidewalls of the nanoscale well. In this embodiment, the sidewall surface modifying agent 362 is also present on the top of the top silicon dioxide layer on the top surface of the chip. This sidewall surface modifying agent can act to prevent the adhesion or binding of unwanted components, such a molecules of interest or analytic reaction components. The sidewall surface modifying agents do not have coupling groups for the attachment of molecules of interest. Removal compounds can be used in some cases to specifically remove bottom surface modifying agents from the sidewall surface modifying agents on the sidewall of the nanoscale well and top of the chip while leaving the surface modifying agents on the group 4- 5 transition metal oxide surface at the bottom of the nanoscale well.

[0093] Fig. 3D illustrates how the nanoscale well of Fig. 3C can be used to attach molecules of interest selectively to the bottom of the well. The bottom surface modifying agent 382 on the bottom of the nanoscale well has coupling groups. These coupling groups are chosen to react with complementary functional groups on the molecule or molecules of interest 392. As shown, in some cases a single molecule of interest is attached. In other cases (not shown), the coupling groups of the bottom surface modifying agent 382 can be used to attach multiple molecules (e.g. clonal populations) to the bottom of the well.

[0094] Fig. 3E shows another embodiment of a selectively functionalized nanoscale well of the invention. The nanoscale well 303 extends through a well forming layer 333. The well forming layer comprises silicon dioxide. In some cases, the material of the well forming layer 333 is silicon dioxide. The nanoscale well 303 extends down to a group 4-5 transition metal oxide layer 323, which forms the bottom of the well. In some cases, the group 4-5 transition metal oxide layer comprises hafnium oxide. On top of the well forming layer 333 there is an aluminum reflective layer 343. In this embodiment, there is no silicon dioxide top surface layer. The nanoscale well has sidewalls comprising silicon dioxide and a bottom comprising a group 4-5 transition metal oxide allowing for selective functionalization of the sidewalls and the bottom as described herein. The nanoscale well is formed on the base substrate 313.

[0095] Here in Fig. 3E, a bottom surface modifying agent 383 is present on the bottom of the nanoscale well formed by the group 4-5 transition metal oxide layer 323. The bottom surface modifying agent can be chosen to react specifically with the group 4-5 transition metal oxides on the base of the well. Bottom surface modifying agents useful for the present inventioninclude agents comprising phosphates, phosphonates, and catechols. The bottom surface modifying agents also include coupling groups for attachment of molecules of interest.

[0096] In addition, a sidewall surface modifying agent 363, for example a silane, is present on the sidewalls of the nanoscale well. This sidewall surface modifying agent can act to prevent the adhesion or binding of unwanted components, such a molecules of interest or analytic reaction components. The sidewall surface modifying agents do not have coupling groups for the attachment of molecules of interest.

[0097] It can be seen in Fig. 3E that the reflective aluminum layer 343 is exposed. Under typical atmospheric conditions, the aluminum reflective layer 343 will have a surface layer of aluminum oxide (native oxide). In some cases, the sidewall surface modifying agent 363 will react specifically with the silicon dioxide of the sidewall. In some cases, the sidewall surface modifying agent 363 will not be sufficiently specific and will also react with the aluminum oxide on the aluminum layer 343. In these cases, a removal compound, e.g. a phosphonate, can be used to selectively remove the unwanted sidewall surface modifying agent from the aluminum layer 343.

[0098] In addition, the aluminum oxide may react with the bottom surface modifying agents. The inventors have discovered that this issue can be addressed by treating the chip with selective removal compounds. Removal compounds can be used in some cases to specifically remove unwanted bottom surface modifying agents from the sidewall surface modifying agents on the sidewall of the nanoscale well and top of the chip while leaving the surface modifying agents on the group 4-5 transition metal oxide surface at the bottom of the nanoscale well. In some cases, the removal compounds act selectively by etch rate. That is, removal compounds can be used that etch aluminum oxide at a relatively high rate as compared to the etch rate for the group 4-5 transition metal oxides.

[0099] Fig. 3F illustrates how the nanoscale well of Fig. 3E can be used to attach molecules of interest selectively to the bottom of the well. The bottom surface modifying agent 383 on the bottom of the nanoscale well has coupling groups. These coupling groups are chosen to react with complementary functional groups on the molecule or molecules of interest 393. As shown, in some cases a single molecule of interest is attached. In other cases (not shown), the coupling groups of the bottom surface modifying agent 383 can be used to attach multiple molecules (e.g. clonal populations) to the bottom of the well.Group 4-5 transition metal oxides

[0100] As used herein the term group 4 transition metals (group IVB elements) are the three elements: titanium, zirconium, and hafnium, and the group 5 transition metals (group VB elements) are the three elements: vanadium, niobium, and tantalum. The term group 4-5 transition metals refers to the six elements: titanium, zirconium, hafnium, vanadium, niobium, and tantalum. For clarity, as used herein, rutherfordium is not included as a group 4 transition metal and dubnium is not included herein as a group 5 transition metal. The group 4-5 transition metal oxides are the oxides of these transition metals.

[0101] The inventors have found that the group 4-5 transition metal oxides are useful for providing orthogonal surface chemistry in conjunction with silicon dioxide and / or aluminum oxide. Table 1 lists the group 4-5 transition metal oxides, their common forms, and their refractive indices.Table 1

[0102] One property that the inventors have identified as particularly useful for many embodiments of the invention is the relatively low etch rates in aqueous solution for these materials, particularly as compared to aluminum oxide. The relatively low etch rates allow for molecules of interest to be attached reliably and robustly to surfaces such as the bottoms of nanoscale wells. Different group 4-5 transition metal oxides are most useful in certain embodiments. For example, in some cases hafnium oxide, with its relatively lower refractive index, is preferred for embodiments in which the hafnium oxide is deposited in a layer above an optical waveguide core. This lower refractive index can result in more efficient light transmission through the waveguide.

[0103] Layers of group 4-5 transition metal oxides can be formed using known semiconductor processing methods. In some cases, the layer of group 4-5 transition metal oxide, e.g hafnium oxides, is formed using chemical vapor deposition (CVD). In some cases, the layer of group 4-5 transition metal oxide, e.g hafnium oxides, is formed using atomic layerdeposition (ALD). The group 4-5 transition metal oxides can be deposited with sputtering or with ion assisted deposition (IAD). The thickness of the layer can be, for example, from 300 nm to 10 microns. The thickness of the layer is typically from 5 nm to 200 nm. In some cases, the thickness of the layer is greater than or equal to 10 nm. In some cases, the thickness of the layer is less than 200 nm.Selective functionalization chemistry

[0104] The selectively functionalized nanoscale wells, chips, instruments, systems and methods of the invention employ selective chemistry for different surface regions on a substrate. This involves: 1) producing substrates having the desired materials in desired regions, and 2) treating these substrates with agents to selectively modify these surface regions. In some embodiments, nanoscale wells are produced such that the bottom surface of the nanoscale well comprises a group 4-5 transition metal oxide such as hafnium oxide. These bottom surfaces are treated with selective surface modifying agents which allow for the attachment of molecules of interest to the bottoms of the nanoscale wells. In some cases, surface modifying agents comprising phosphates, phosphonates, or catechols are used.

[0105] In addition, sidewall surface modifying agents can be applied to be selectively present on the sidewalls of the nanoscale wells. In the nanoscale wells of the invention, the sidewalls comprise silicon dioxide or aluminum oxide. The sidewall surface modifying agents are different than the bottom surface modifying agents. The sidewall surface modifying agents do not have coupling groups for the molecules of interest. In addition, the sidewall surface modifying agents can functionalize the sidewall surface to discourage or prevent the adhesion of molecules of interest or other reaction components. This type of functionalization is sometimes referred to as passivation. That is, this functionalization makes the sidewall surface passive, or inert to unwanted reactions. In some cases, in addition to the sidewall, the top surfaces of the chip comprise silicon dioxide or aluminum oxide. In these cases, the sidewall surface modifying agents can also functionalize the top surfaces of the chip to prevent unwanted reactivity.

[0106] Another aspect of the selective functionalization chemistry of the invention is the use of selective removal compounds to remove unwanted agents from a surface region. Even where the surface modifying agents provide good selectivity, removal compounds can be used to further increase the selective functionalization. Where the sidewalls comprise silicondioxide, preferential removal of sidewall surface modifying agents from a group 4-5 transition metal oxide surface can be accomplished using an acidic compound, such as a polymer comprising one or more of carboxylic acid, sulfonic acid, or phosphonic acid moieties. For example, these compounds can selectively remove a sidewall surface modifying agent, such as a silane, from a group 4-5 transition metal oxide surface.

[0107] In the case of aluminum oxide on the sidewalls or top surfaces, removal compounds can provide the desired orthogonality of chemistry. In many cases, aluminum oxide will have similar reactivity to group 4-5 metal oxides to surface modifying agents, making it difficult to obtain selective functionalization. The inventors have found that removal compounds that etch aluminum oxide at higher rates than they etch group 4-5 metal oxides can be used to provide the desired highly selectively functionalized surfaces.Bottom surface modifying agents

[0108] The bottom surface modifying agents are agents that bind strongly and often specifically with group 4-5 transition metal oxides, for example on the bottoms of the nanoscale wells. The bottom surface modifying agents typically include coupling groups for coupling the molecule of interest selectively to the bottom of the nanoscale well. In some cases, a single bottom surface modifying agent is used. In some cases, mixtures of bottom surface modifying agents are used. In some cases, a mixture of bottom surface modifying agents is used in which one bottom surface modifying agent comprises a coupling group and a second bottom surface modifying agent does not comprise a coupling group. Varying the relative amounts of these agents can be used to adjust the density of coupling groups on the bottom of the nanoscale well.

[0109] Exemplary bottom surface modifying agents include phosphate, phosphonate or catechol groups which will react with group 4-5 transition metal oxide surfaces. As used herein, the terms phosphonate and phosphonic acid are used interchangeably. The terms catechol and catecholate are also used interchangeably, and the terms phosphate and phosphoric acid are used interchangeably. It is understood by those of skill in the art that the free acid and salt forms of these compounds typically readily exchange in aqueous solution, and that the relative proportion in the salt or free acid form will vary with the pH of the solution.

[0110] Exemplary useful bottom surface modifying agents have the components phosphate- linker-coupling group, phosphonate-linker-coupling group or catechol -linker-coupling group. In some cases polymeric surface modifying agents are used which have multiple catechol or phosphonate groups and / or multiple coupling groups.

[0111] Thus, selective coating of a bottom surface modifying agent to a group 4-5 transition metal oxide surface as opposed to a silicon dioxide surface means that the compound is present on the surface of a group 4-5 transition metal oxide layer at a higher density or concentration than on a silicon dioxide surface, for example on the sidewall or top surface of the chip. Density in such regions may be as much as 20% greater, 30% greater, 50% greater, 100% greater, or upwards of 200%, up to 1000% or more than the concentration or density on the surface of a silica-based or transparent layer, and in some cases 100 times greater, 1000 times greater or more.

[0112] In some cases, the group 4-5 transition metal oxide layer tends to be positively charged in aqueous solution, and negatively charged surface modifying agents can be employed that bind to the group 4-5 transition metal oxide surface.

[0113] Some preferred bottom surface modifying agents contain phosphorous. These compounds will generally comprise P=O and / or P-OH functionality. In particular, compounds comprising phosphate or phosphonate groups are used. Such phosphate or phosphonate compounds can selectively react with group 4-5 transition metal oxide surfaces, while having low reactivity to other surfaces, for example silicon dioxide surfaces. Preferred bottom surface modifying agents include phosphorous containing polymeric materials. Suitable phosphorous containing polymeric materials include derivatives of poly(vinylphosphonic acid), Albritect™ CP-30, Albritect™ CP- 10, Albritect™ CP-90, Aquarite® ESL, and Aquarite® EC4020. Albritect™ and Aquarite® compounds are commercially available from Rhodia, Inc. Derivatives will typically have one or multiple coupling groups attached, on average, per molecule. Phosphate or phosphonate moieties can bind strongly to metal oxides but do not generally bind strongly to silicon dioxide. Thus, compounds that comprise at least one phosphate group (-OP(O)(OH)2, whether protonated, partially or completely deprotonated, and / or partially or completely neutralized) or phosphonic acid group (- P(O)(OH)2, whether protonated, partially or completely deprotonated, and / or partially or completely neutralized) can be used to selectively modify the group 4-5 transition metal oxide surfaces at the bottoms of the nanoscale wells.

[0114] For example, a group 4-5 transition metal oxide surface can be modified with an alkyl phosphate or an alkyl phosphonate. It is understood that a phosphonic acid will generally have hydrogens associated with two of the phosphonic acid oxygens, and that a phosphonate will generally have other counterions associated with these oxygens. In aqueous solution, hydrogen ions and counterions can exchange rapidly. Thus generally either phosphonic acid and phosphonate compounds can be useful in the invention.

[0115] Exemplary alkyl phosphates and alkyl phosphonates include, but are not limited to, an alkyl phosphate or alkyl phosphonate which contains an alkyl group that is a straight chain unsubstituted alkyl group (e.g., a straight chain alkyl group having from 1 to 26 carbons, e.g., from 8 to 20 carbons, e.g., from 12 to 18 carbons). Additional exemplary alkyl phosphates and alkyl phosphonates include functionalized or substituted alkyl phosphonates and alkyl phosphates, for example, functionalized X-alkyl-phosphonates and X-alkyl-phosphates where X comprises a coupling group. X can be a terminal group comprising any suitable coupling group or consisting of a vinyl (CEE), methyl (CEE), amine (NEE), alcohol (CH2OH), epoxide, acrylate, methacrylate, thiol, carboxylate, active ester (NHS-ester), melamine, halide, phosphonate, or phosphate group, or an ethylene glycol (EG) oligomer (EG4, EG6, EG8) or polyethylene glycol (PEG), photo-initiator (e.g., photo-iniferters such as dithiocarbamates (DTC)), photocaged group, or photoreactive group (e.g., psoralen). The alkyl chain spacer in the X-alkyl-phosphonate or X-alkyl-phosphate molecule is a hydrophobic tether that optionally has 1 to 26 methylene (CH2) repeat units, preferably from 8 to 20, and more preferably from 12 to 18. The alkyl chain may contain one or more (up to all) fluorinated groups and / or can instead be a hydrocarbon chain with one or more double or triple bonds along the chain.

[0116] The X-alkyl-phosphate or X-alkyl-phosphonate layer can furthermore be used as a substrate to anchor other ligands or components of a surface stack, such as a polyelectrolyte multilayer or chemisorbed multilayer. The alkyl phosphates / phosphonates can form a stable, solvent resistant self-assembled monolayer that can protect the underlying metal oxide from corrosion etc.; the role of the alkyl tether in the above structures is to enhance the lateral stability of the chemisorbed monolayer in aqueous environments. In embodiments in which the phosphonate or phosphate compound includes an unsaturated hydrocarbon chain, the double or triple bond(s) can serve as lateral crosslinking moieties to stabilize a self-assembled monolayer comprising the compound.

[0117] Modification of metal oxides with phosphates and phosphonates has been described, e.g., in Langmuir (2001) 17:3428, Chem. Mater. (2004) 16:5670; J. Phys. Chem. B (2005) 109: 1441, Langmuir (2006) 22:6469, Langmuir (2006) 22:9254, Langmuir (2006) 22:3988, J. Phys. Chem. B (2003) 107: 11726, J. Phys. Chem. B (2003) 107:5877, Langmuir (2001) 17:462, J. Phys. Chem. B (2006) 110:25603, Langmuir (2002) 18:3957, Langmuir (2002) 18:3537, and Langmuir (2001) 17:4014, which are incorporated herein by reference for all purposes.

[0118] Group 4-5 transition metal oxide surfaces can be modified with polyphosphates or polyphosphonates. These materials can form a chemical complex with the substrate. Such interaction can be stronger and less reversible to salt exchange than are simple electrostatic interactions. Examples include, but are not limited to, PEG-phosphonates such as those described in Zoulalian et al. (2006) “Functionalization of titanium oxide surfaces by means of poly(alkyl-phosphonates)” J. Phys. Chem. B 110(51):25603-25605 or PEG- polyvinyl(phosphonate) copolymers. In general, copolymers including chemisorbing moieties plus PEG or other anti-fouling moieties are contemplated herein.

[0119] Other suitable phosphonates include derivatives of high molecular weight polymeric phosphonates such as polyvinylphosphonic acid (PVPA):

[0120] wherein n can be from about 1 to about 1000 or from about 10 to about 100.

[0121] Derivatives of phosphonate end-capped polymers of polymers having acidic functional groups such as carboxylic acids, sulfonic acids and mixtures thereof can also be used.

[0122] Exemplary copolymers copolymer include derivatives of the copolymers:, or

[0123] such as vinyl phosphonic acid-acrylic acid copolymers (commercially available from Rhodia as Albritect™ CP30). The values for n and m can range from about 1 to about 1000. In some cases, m is between about 10 and about 100, and n is between about 100 and 300. In some cases, m is between about 50 and about 70, and n is between about 80 and 120. In some cases, m is about 60 and n is about 200.

[0124] Suitable phosphonates also include derivatives of low molecular weight phosphonates such as 2-carboxyethyl phosphonic acid (also known as 3-phosphonopropionic acid; commercially available from Rhodia as Albritect™ PM2).

[0125] Suitable polyphosphates or polyphosphonates include a coupling group and can include PEG or other antifouling moieties. Phosphonate compounds can be supplied as salts (e g., sodium, potassium, lithium, or ammonium salts) or as free acids.

[0126] Suitable polymers for use as bottom surface modifying agents include polymers produced from the following monomers. Particularly useful polymers comprise polymers with these monomers and also comprising one or more phosphate or phosphonate groups, for example copolymers comprising vinyl(phosphonic acid) (VPA) and at least one other of the monomers listed below.

[0127] For PEG-MA and PEG-MA-ME, n is generally chosen such that the molecular weight is between about 100 and 10,000 or about 200, 400, or 1000. For example n can be from about 1 to about 1000 or from about 10 to about 100

[0128] In some cases, monomeric compounds such as bisphosphonic acids, alkyl phosphonic acids, or other phosphonic acids can be used.

[0129] One exemplary approach involves reacting a group 4-5 transition metal oxide region with a compound having a phosphate, phosphonate, or catechol group directly attached to the coupling group or attached to the coupling group through a linker. Exemplary compounds include compounds phosphate-linker-coupling group, phosphonate-linker-coupling group, andcatechol-linker-coupling group. Any suitable linker group can be used. In general, a “linker” of the instant disclosure should be considered broadly to include any chemical moiety that provides a suitable covalent connection between two or more components within a given compound. A linker can be hydrophilic (e.g., tetraethylene glycol, hexaethylene glycol, polyethylene glycol) or it can be hydrophobic (e.g., hexane, decane, etc.). Exemplary linkers include substituted or unsubstituted C6-C30 alkyl groups, polyols (e.g., glycerol), polyethers (e.g., poly(ethylen eglycol)), poly amines, amino acids (e.g., polyaminoacids), peptides, saccharides (e.g., polysaccharides) and combinations thereof. Such linkers typically comprise linear or branched chains, wherein the chain can be substituted at any suitable position, as desired, and wherein any carbon atom can be replaced by any suitable heteroatom. A linker can comprise one or more alkyl, heteroalkyl, cycloalkyl, cycloheteroalkyl, aryl, or heteroaryl groups, if so desired. In some cases the linker group comprises polyethylene glycol (PEG).

[0130] For example, a phosphonate-poly ethylene glycol -biotin compound can be used to produce a surface having selective binding groups, e.g. biotin, bound to the group 4-5 transition metal oxide region. This method provides a one step process for obtaining a group 4-5 transition metal oxide surface having a coupling group such as biotin attached thereto.

[0131] Fig. 7A and 7B show examples of phosphonate-linker-coupling group compounds useful in the invention. In Fig. 7A, a biotin-PEG-NHS ester compound is coupled with a amine-phosphonate compound to form a biotin-PEG-phosphonate bottom surface modifying agent. In Fig. 7B, a biotin-PEG-NHS ester compound is coupled with a amine-bis- phosphonate compound to form a biotin-PEG-phosphonate bottom surface modifying agent having two phosphonate groups per biotin.

[0132] Table 2 provides exemplary compounds for use in the invention.TABLE 2

[0133] These compounds can be used to produce bottom surface modifying agents with coupling groups or can be used, in some cases as bottom surface modifying agents. The ethyl ester groups can be hydrolyzed to produce desired phosphonate functionality. The bromine functionality of compounds 1 and 2 can be used to connect to other molecules. For example, a tertiary amine compound having a coupling group can react with compound 1 or compound 2 to form a corresponding quaternary ammonium salt. Compound 3 can be used, for example, as a hydrophilic cross-linking agent, for example to form a gel. Compound 4 has an azide group which can be used to carry out “Click Chemistry”, for example reacting with alkyne functionality to form a corresponding 1,2, 3 -triazole via 1, 3 -dipolar cycloaddition with or without a copper catalyst. Compound 5 has a methacrylate group that can be used to prepare polymerizable surfaces including gels.

[0134] Other preferred bottom surface modifying agents include catechol or catecholate groups which can react with the group 4-5 transition metal oxide surfaces. Catechol is also known as pyrocatechol or 1,2 dihydroxy benzene, The use of catechol for functionalizing surfaces is described, for example, in "The Chemistry Behind Catechol -based Adhesion" Volume 58, Issue3 January 14, 2019, Pages 696-714, which is incorporated by reference herein for all purposes.

[0135] In some cases these bottom surface modifying agents will include a dopamine moiety. In some cases, a nitrocatechol or nitrodopamine moity is used. An exemplary compound bottom surface modifying agent is a biotin-PEG-catechol compound, e.g. biotin- PEG4-nitrodopamine (see structure below).

[0136] In some embodiments, polymeric molecules having multiple catechol groups per molecule are used. Multiple chatechol groups per molecule can be desirable because of the added stability by "avidity", or multiplicity of attachment points.

[0137] In some cases, a gel is formed by the bottom surface modifying agent. Gels are typically formed by using cross-linking, either to link previously formed polymeric molecules or by having a crosslinking agent present while polymerizing the bottom surface modifying agent on the surface.

[0138] Functionalization with surface modifying agents is generally carried out by exposing the substrate surface to a solution comprising the relevant agent mixed with a solvent. The solvent used is typically one that substantially dissolves the agent. The solvents will generally comprise polar solvents. Typically, aqueous solvent are preferred. While not being bound by theory, it is believed that the use of an aqueous solvent assists in providing specificity of the reaction with the group 4-5 transition metal oxides. In some cases, the aqueous solvent comprises a mixture of water and an alcohol, for example, water / ethanol or water / methanol. The pH of the solution can affect the level of specificity which is obtained. In some cases a pH between about 6 and 12 used with catechols. In some cases a pH between about 7 and 11 is used with catechols. Where phosphonates or phosphonic acids are used, the pH can be between 1 and 8, or in some cases between 2 and 7. The pH ranges that can be used to enhance adhesion and specificity are described, for example in “Mapping out the Aqueous Surface Chemistry of Metal Oxide Nanocrystals: Carboxylate, Phosphonate, and Catecholate Ligands”, JACS Au 2022, 2, 711-722 which is incorporated herein by reference in its entirety for all purposes.

[0139] The bottom surface modifying agent can be provided in an aqueous solution at any suitable concentration. The solution can have from about 5% of the surface modifying agent, to about 95% (w / w or w / v) of surface modifying agent. The reaction can be carried out at a temperature and for a time that will allow the reaction to occur. In some cases, temperatures from 20° C to 100° C are used. In some cases, temperatures from 60° C to 90° C are used. The reaction times generally range from minutes to hours.

[0140] In some cases, the addition of salts can improve the reaction. In some cases, salts having sodium (Na+) are present. In some cases, the addition of bivalent salts such as Ca can be beneficial.

[0141] It will be understood that the bottom surface modification step will often be preceded by one or more washing steps to remove contaminants from the surfaces. Pretreatment steps can also be used, for example to put the chip into the state desired for subsequent reaction.

[0142] The level of selective functionalization can be evaluated by a variety of techniques that are known in the art of surface characterization. For example, techniques such as X-ray photoelectron spectroscopy (XPS), contact angle, or ellipsometry can be used to characterize the level of selectivity of the coating.Coupling groups

[0143] The bottom surface modifying agents can have coupling groups, binding groups, or reactive groups for attaching the molecule or molecules of interest to the bottom of the nanoscale well. The terms coupling group, binding group, or reactive group are used interchangeably. In some cases the coupling is covalent, in other cases the coupling is non- covalent. One of skill in the art will understand from the context where such distinctions are made.

[0144] Any suitable coupling group can be used. Binding or coupling groups may include small molecule coupling groups or they may include macromolecular coupling groups, e.g., antibodies, antibody fragments, specific binding pairs, such as avidin / biotin, binding peptides, lectins, complementary nucleic acids, or any of a variety of other binding groups. The term avidin as used herein refers to protein molecules or moieties that bind strongly to biotin including avidin, streptavidin, neutravidin, traptavidin, and other variants.

[0145] In some cases, the coupling groups are activatable or deactivatable coupling groups. A variety of different activatable or deactivatable coupling groups may be used in conjunction with this aspect of the invention. Typically, such groups include coupling groups that are capped or blocked with a selectively removable group. These include groups that are thermally altered, e.g., thermolabile protecting groups, chemically altered groups, e.g., acid or base labile protecting groups, and photo alterable groups, e.g., photo-cleavable or removable protecting groups. Suitable activatable and deactivatable coupling groups are provided, for example, in U.S. Patent Application 11 / 394,352.

[0146] A variety of different coupling groups may be used in this context, depending upon the nature of the molecule of interest to be subsequently deposited upon and coupled to the substrate. For example, the coupling groups may include functional chemical moieties, such as amine groups, carboxyl groups, hydroxyl groups, sulfhydryl groups, metals, chelators, and the like. Alternatively or additionally, they may include specific binding elements, such as biotin, avidin, streptavidin, neutravidin, lectins or SNAP -tags™ and their substrates (Covalys Biosciences AG; the SNAP-tag™ is a polypeptide based on mammalian O6-alkylguanine- DNA-alkyltransferase, and SNAP-tag substrates are derivates of benzyl purines and pyrimidines), associative or binding peptides or proteins, antibodies or antibody fragments, nucleic acids or nucleic acid analogs, or the like. Click chemistry including the Azide-Alkyne Huisgen Cycloaddition catalyzed, for example, by copper can also be used. Click chemistry reagents are available commercially, for example from Conju-Probe, Thermo-Fisher Scientific, Vector Labs, BroadPharm, APB Biosciences and others.

[0147] In some cases, the coupling group is a biotin, a biotin-binding protein, an antibody, an antibody fragment, a lectin, or a nucleic acid. The coupling group can be an amine group, a carboxyl group, a hydroxyl group, a sulfhydryl group, an azido group, an alkyne group, a cyclooctyne group, a dibenzocyclooctyne (DBCO) group, a trans-cyclooctene (TCO) group, a norbomene group, a tetrazine group, an epoxy group, a glycidyl group, an aldehyde group, an alkenyl group, an acrylate group , a methacrylate group, an anhydride group, an activated carboxylic acid group, or an n-hydroxysuccinimide (NHS) ester group

[0148] Additionally, or alternatively, the coupling group may be used to couple an additional group that is used to couple or bind with the molecule of interest, which may, in some cases include both chemical functional groups and specific binding elements. A preferred set of embodiments utilizes biotin to attach a molecule of interest to the group 4-5 transition metal oxide layer or region. The attachment of biotin or another selective binding group to the surface can be accomplished in a number of ways.

[0149] In some cases, the compound having the selective binding group is diluted with a surface modifying agent that does not contain the selective binding group, e.g. phosphonate- polyethylene glycol in order to control the density of selective binding groups on the group 4- 5 transition metal oxide surface. That is, in some cases, it is desired to provide a surface with a selected, relatively low density, of coupling groups, for example, coupling groups on a surface. For some applications, the desired result is a surface that includes a relatively lowdensity of the selected reactive molecule surrounded by an otherwise non-reactive surface. Although discussed in terms of a molecule or type of molecule of interest, it will be appreciated that mixed functionality surfaces are also encompassed within the scope of the invention, including, e.g., two, three, four, or more different molecules or types of molecules of interest.

[0150] Provision of a wide range of reactive coupling groups on surfaces is readily understood in the art, and includes, for example, ionic functional groups, polyionic groups, epoxides, amides, thiols, hydrophobic groups, e.g., aliphatic groups, mono or polycyclic groups, and the like, e.g., as generally used in reverse phase and / or hydrophobic interaction chromatography (HIC), staudinger ligation groups (see, e.g., Lin et al., J. Am. Chem. Soc. (2005), 127:2686-95), Click chemistry coupling using chemoselective azide-acetylene linkages (See, Deveraj et al., JACS 2005, 127:8600-8601; Lummerstorfer et al., J. Phys. Chem. B (2004) 108:3963-3966, and Collman et al., Langmuir (2004) 20: 1051-1053, each of which is incorporated herein by reference in its entirety for all purposes) and other groups that associate or are capable of being coupled with other groups in a non-specific fashion.Preferred coupling groups include specific binding groups on surfaces, e.g., groups that specifically recognize a complementary binding partner, including, e.g., complementary nucleic acid pairs, antibody-epitope pairs, binding peptides that recognize specific macromolecular structures, e.g., recognition sequences in proteins, peptides or nucleic acids, lectins, chelators, biotin-avidin pairs, and the like. Dilution of coupling groups is described, for example, in US 8,137,942 which is incorporated herein by reference in its entirety for all purposes.

[0151] Another exemplary approach involves first reacting a group 4-5 transition metal oxide surface with a surface modifying agent comprising a coupling group, and reacting the coupling group on the surface with an attaching agent that has both functionality for reacting with the coupling group, and functionality for attaching the desired molecule (e.g. a selective binding group such as biotin). For example, the group 4-5 transition metal oxide surface is reacted with an amino-phosphonate or thiol-phosphonate under conditions where the amino- phosphonate or thiol-phosphonate becomes bound to the substrate. The amino-phosphonate or thiol-phosphonate surface is subsequently reacted with an attaching agent, for example having an activated ester coupled to biotin to link the biotin to the amino-phosphonate surface, or a maleimide group coupled to biotin to link to the thiol- phosphonate surface. The attachingagent can be diluted as described herein with molecules that react, for example, with the amino-phosphonate or thiol-phosphonate, but do not have selective binding groups. This process incorporating an attaching group results in coupling of the selective binding group to the surface in two steps. While this approach uses two steps rather than the one step described above, it can have some advantages in development, processing, and quality control.

[0152] In some cases, the compound comprising the selective binding group is diluted with another agent such as a capping agent that can bind to the surface, but does not have selective binding group. In accordance with the invention, the low density of the coupling group on a surface is designed to provide a single reactive moiety within a relatively large area for use in certain applications, e g., single molecule analyses, while the remainder of the area is substantially non-reactive. As such, coupling groups can be diluted to provide a low density of coupling groups that are typically present on a substrate surface at a density of coupling groups of greater than 1 / 1X106nm2of surface area, but less than about 1 / 100 nm2. In more preferred aspects, the density of coupling groups on the surface will be greater than 1 / 100,000 nm2, 1 / 50,000 nm2, 1 / 20,000 nm2and 1 / 10,000 nm2, and will be less than about 1 / 100 nm2, 1 / 1000 nm2, and 1 / 10,000 nm2. For certain preferred applications, the density will often fall between about 1 / 2500 nm2and about 1 / 300 nm2, and in some cases up to about 1 / 150 nm2.

[0153] The conditions for the attachment of the molecule of interest can be controlled such that, for example, only one molecule of interest or one active molecule of interest is delivered to one or more nanoscale wells on a surface. In some cases, the conditions for the attachment of the molecule of interest are controlled such that 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more of the nanoscale wells have only one molecule of interest or one active molecule of interest. Approaches for obtaining a high fraction of nanoscale wells having one molecule of interest is described in U.S. Patents 10,525,438, 8.906.831, 8,802,600, 8,501,406 which are incorporated herein by reference in their entirety for all purposes.Sidewall surface modifying agents

[0154] Sidewall surface modifying agents can be used to enhance performance of the nanoscale wells, in particular, by preventing the adhesion or binding of molecules of interest or other components to the sidewalls of the nanoscale wells or to the top of the chip.

[0155] Where the sidewall comprises silicon dioxide, surface modifying agents that have an affinity for silicon dioxide can be used. In some cases, silanes are used.

[0156] Some aspects of the invention include the selective passivation of the sidewalls of nanoscale wells or the top surface of the chip. Passivation of a surface generally means treating a surface to change the properties of the surface to make the surface less reactive toward some compounds. In some cases, passivation can involve preventing the surface from participating in a chemical or binding reaction in which the surface would have participated if not passivated. For example, passivation can involve minimizing the surfaces' interactions with the environment (e.g., minimizing or eliminating nonspecific binding to the surfaces). In particular, passivation can involve minimizing the binding of biomolecules or other reagents used in carrying out a reaction with biomolecules, such as proteins, nucleic acids, or nucleotides. In some cases, passivation can be minimizing a surface’s tendency to undergo corrosion. In the context of arrays of nanoscale wells described herein, it can be desirable that there is passivation with respect to the adsorption or binding of compounds in a solution which is observed within the nanoscale well.

[0157] Passivation can, in some cases, be accomplished by treating the surface with a surface modifying agent that is a passivating compound. In some cases, the passivating compound can be bound covalently to the surface. In some cases, the passivating compound can be deposited onto the surface without the formation of a covalent bond, being held in place, for example by van der Waals, hydrogen bonding, or dipolar forces. In one aspect of the invention, the surface is treated with a passivating compound that selectively reacts with, and deposits onto the materials on sidewalls of the nanoscale wells (silicon dioxide or aluminum oxide), while having little deposition on group 4-5 transition metal oxide surfaces.

[0158] The methods of the invention can include a step in which a sidewall comprising silicon dioxide is reacted with a sidewall surface modifying agent. This is carried out using a surface modifying agent which reacts with the silicon dioxide surface either selectively or non-selectively. As used herein, such a surface modifying agent is generally a compound that binds to the silicon dioxide and also has other components that, for example, provide passivation.

[0159] Modifying the sidewall of the nanoscale wells may be carried out by any of a variety of methods known in the art. For example, in the context of sidewalls comprising silicon dioxide, e.g., glass, quartz, fused silica, silicon, or the like, well characterized silane chemistries may be used. Silanes (e.g., methoxy-, or ethoxy-, silane reagents) can form stable bonds with silica surfaces via Si-O-Si bond formation, and can be less reactive to metal ormetal oxide surfaces such as group 4-5 transition metal oxide or aluminum oxide surfaces under appropriately selected reaction conditions (e.g., vapor phase or solution-based treatments). Suitable silanes for passivation include silanes with ether, alkyl, anionic, carbohydrate, peptide, glycol, or zwitterionic moieties. Suitable anionic moieties include sulfonates and carbonates. Zwitterionic moieties can include sulfobetaine, carboxybetaine, phosphorylcholine, dimethylamine oxide, or dimethyl sulfoniopropionate building blocks. Suitable zwitterionic moieties are described, for example in “Interactions between Biomolecules and Zwitterionic Moieties: A Review”, Biomacromolecules 2020, 21, 2557-2573, which is incorporated herein by reference for all purposes. Carboxylate moieties can include sugars such as dextran. Suitable ester moieties include PEG, for example having 2 to 1000 PEG units, or 4 to 100 PEG units. PEG silanes for passivation are described, for example in “Efficient One-Step PEG-Silane Passivation of Glass Surfaces for SingleMolecule Fluorescence Studies”, ACS Appl. Mater. Interfaces 2018, 10, 46 which is incorporated by reference herein for all purposes.

[0160] As another example, negatively charged surfaces can be selectively modified by adsorption of copolymers containing positive polyelectrolyte blocks and PEG-ylated (or similar anti-fouling) blocks. Many silica-based surfaces can be rendered negatively charged under the appropriate conditions in order to facilitate this approach. The polycationic blocks bind to regions of the device that are electronegative, and the PEG components provide a nonreactive surface to preclude nonspecific binding. Exemplary polyelectrolyte-PEG copolymers include PLL-PEG (poly(L-lysine)-poly(ethylene glycol)). Other polycationic blocks can include, for example, PEI (poly(ethylenimine), PDDA (poly(diallyldimethyl ammonium chloride), and PAH (poly(allylamine hydrochloride). In some cases, poly(methyl methacrylate) (PMMA) and copolymers thereof can be used to modify the properties of the surfaces comprising silicon dioxide.

[0161] Phospholipid chemistries can also be used to functionalize the surface of the silicon dioxide-based portions of the substrate. Chemistries using phospholipid compositions, have shown the ability, in the presence and absence of calcium, to form different levels of supported phospholipid bilayers on silicon dioxide surfaces. One can utilize a phospholipid that has high binding selectivity for the underlying silicon dioxide substrate. Examples of these selective phospholipid compositions are described in, e.g., Rossetti, et al., Langmuir. 2005; 21(14):6443-50, which is incorporated herein by reference in its entirety for allpurposes. In some cases, the application of phospholipids is preceded by a step in which the silicon dioxide portions of the substrate are treated with a hydrophobic reagent such as a Silwet material.

[0162] In some cases, multiple layers are applied to the silicon dioxide surfaces in order to provide passivation.

[0163] The treatment of the silicon dioxide-based surface can be performed in a selective manner, such that relatively more of the sidewall surface modifying agent is bound to the silicon dioxide-based surface than is bound to the group 4-5 transition metal oxide surface. The treatment can even be carried out in a highly selective manner, whereby significantly more sidewall surface modifying agent is bound to the silicon dioxide-based surface than is bound to the group 4-5 transition metal oxide surface. However, we have found that for some applications, even a highly selective treatment will result in more sidewall surface modifying agent bound to the group 4-5 transition metal oxide surface than is desired. Where this is the case, a subsequent selective removal step can be employed to remove the unwanted groups.Some methods useful in the invention for passivation of desired surface regions is provided in “A Survey of Structure-Property Relationships of Surfaces that Resist the Adsorption of Protein” Langmuir 2001, 17, 5605-5620, “Recent Developments and Practical Feasibility of Polymer-Based Antifouling Coatings” Adv. Funct. Mater. 2020, 30, 2000936, and “Machine Learning-Enabled Design and Prediction of Protein Resistance on Self-Assembled Monolayers and Beyond” ACS Appl. Mater. Interfaces 2021, 13, 11306-11319, which are incorporated by reference herein in their entirety for all purposes.Removal compounds

[0164] As we have described, in some cases, selective surface modification chemistry can provide the selectively functionalized surfaces desired. In addition, we have found that surfaces having desired low levels of unwanted bottom surface modifying agent bound to the sidewalls or tops of the chips and / or desired low levels of unwanted sidewall surface modifying agent on the bottoms of the nanoscale wells can be obtained by following a surface modifying step with a step involving the selective removal of unwanted surface modifying agent. The use of a selective removal step can, in some cases, allow for the use of less- specific surface modification chemistry.

[0165] The selective removal compounds of the invention will generally remove or deactivate a significant portion of the bottom surface modifying agent on the sidewalls or tops of the chips and / or sidewall surface modifying agent on the bottoms of the nanoscale wells. In some embodiments, the selective removal agent will remove or deactivate substantially all of the bottom surface modifying agent on the sidewalls or tops of the chips and / or sidewall surface modifying agent on to the bottoms of the nanoscale wells. The deactivation of the functionalizing agent can occur in various ways.

[0166] Deactivation of the relevant surface modifying agent can include chemical reaction with the functionalizing agent and can include covering or sequestering the agent. In some embodiments the selective removal agent will remove or deactivate greater than 99.9%, greater than 99%, greater than 98%, greater than 95%, greater than 90%, greater than 80%, greater than 75%, greater than 70% or greater than 60% of the undesired surface modifying agent.

[0167] A suitable selective removal agent will leave enough functionalizing agent on the group 4-5 transition metal oxide surface to allow for the substrate to function, e.g. for molecular analysis. In some embodiments, the selective removal agent will remove little or no functionalizing agent or coupling groups from the group 4-5 transition metal oxide surfaces. In some embodiments, the selective removal agent will remove substantially no functionalizing agent or coupling groups from the group 4-5 transition metal oxide surfaces. In some embodiments the functionalizing agent will remove less than 0.1%, less than 1%, less than 2%, less than 5%, less than 10%, less than 15%, less than 20%, less than 25%, less than 30%, or less than 50% of the functionalizing agent or coupling groups from the group 4-5 transition metal oxide surfaces.

[0168] Suitable selective removal agents include acidic compounds, for example, compounds having one or more functional groups with a pKa of less than about 6, less than about 5, less than about 4, less than about 2, less than about 1 or lower than 1. We have found that the acidic selective removal agents are particularly useful.

[0169] The selective removal agents typically include compounds having, for example carboxylic acid (-CO2H), sulfonic acid (-SO3H), phosphonate (-PO3H2), or phosphate (- OPO3H2) functional groups or combinations thereof.

[0170] Particularly useful selective removal agents comprise polymers having acidic functionality. These polymers include, for example, carboxylic acid groups, e.g. acrylates,including poly(acrylic acid) (PAA), and poly(methacrylic acid), sulfonic acid groups, e.g. poly(vinylsulfonic acid) (PVSA), phosphonic acid groups, e g. poly(vinylphosphonic acid) (PVPA), or phosphoric acid groups or copolymers having two or more of these groups.

[0171] The number average molecular weight (Mn) of the polymeric selective removal agents can be from about 1,000 to about 100,000 or from about 5,000 to about 50,000.

[0172] Polymeric selective removal agents can comprise poly(vinylsulfonic acid) PVSA, having the structure:Poly(vinylsulfonic acid) (PVSA) wherein n can be about 1 to about 1000, or about 10 to about 100. In some cases, n is selected to provide a suitable molecular weight for acting as a selective removal agent. In some embodiments, the average molecular weight of the PVSA is from about 1,000 to about 100,000 or from about 1,000 to about 50,000. In some embodiments, the PVSA has a number average molecular weight of Mn from about 4,000 to about 9,000. In some embodiments the PVSA has a poly dispersity from about 1.2 to about 1.6.

[0173] Selective removal agents of the invention can also comprise poly (styrenesulfonic acid)(PSSA), and poly(styrenesulfonic acid-co-maleic acid)(PSSA-MA) with the structures shown below.Poly(styrenesulfonic acid) (PSSA)Poly(styrenesulfonic acid-co-maleic acid) (PSSA-MA)wherein n and m are selected to provide a suitable molecular weight for acting as a selective removal agent. In some cases n and m are from 1 to about 1000. In some cases, m and n are from about 10 to about 500.

[0174] Suitable copolymers include compounds comprising PAA-PVSA, PAA-PVPA, PVSA-PVPA. In some cases, the polymer or copolymer selective removal agents have attached to them polyethylene glycol, creating PEG-ylated polymers.

[0175] One class of selective removal agents comprise compounds having the structure below:wherein n and m are selected to provide a suitable molecular weight for acting as a selective removal agent. In some embodiments, n is from about 1 to about 1000, and m is from about 1 to about 1000. In some embodiments, n is from about 10 to about 100, and m is from about 10 to about 100. In some embodiments n is about 50 and m is about 50. One preferred embodiment comprises Aquarite® ESL, available from Rhodia, Inc.

[0176] Other exemplary copolymers which comprise selective removal agents of the invention include the copolymers below:Poly(styrenesulfonic acid-co-phosphonic acid) (PSSA / PVPA)Poly(sulfonic acid-co-phosphonic acid) (PVSA / PVPA)Poly(sulfonic acid-co-acrylic acid)(PVSA / PAA) and(PVPA / PAA) wherein n and m are selected to provide a suitable molecular weight for acting as a selective removal agent. In some embodiments, n is from about 1 to about 1000, and m is from about 1 to about 1000. In some embodiments, n is from about 10 to about 100, and m is from about 10 to about 100.

[0177] For copolymers described herein having two polymeric regions, the ratio of the regions can be, e.g., about 10:90, 20:80, 30:70, 40:60, 50:50, 60:40, 70:30, 80:20, or 90: 10 (molar or weight ratio).

[0178] Suitable materials for use as a selective removal agent include Dequest® compounds available from ThermPhos Trading GmbH; including, for example, Dequest® P9000 a homopolymer of maleic acid, Dequest® P9020, a modified polyacrylic acid, sodium salt, and Dequest® P9030, a sulphonated polyacrylic acid copolymer.

[0179] The substrates can be treated with the selective removal agents using methods described herein and methods known in the art. The selective removal agents are typically delivered to the substrates in solution, but other methods of delivery, such as delivery of the selective removal agents in a gaseous form, can be used. The solutions of the selective removal agent for treatment of the substrate will generally utilize solvents in which the selective removal agent is substantially soluble. In some cases, aqueous solutions are used.

[0180] An exemplary method of treating the substrate with selective removal agents comprises putting a solution containing the selective removal agent into contact with the substrate and bringing the solution to a temperature for a period of time. In some cases agitation or stirring of the solution is carried out during such time. The solution is thenremoved, after which the substrate may be rinsed, for example, with pure solvent, e.g. pure water, and dried.

[0181] The concentration of the selective removal agent will typically be between 0.01% and 20% (weight by volume, e.g. g / mL). In many cases the concentration of the selective removal agent will be less than 1%, for example between 0.1% and 0.8% or between 0.2% and 0.6%.

[0182] The treatment with selective removal agent is generally carried out under acidic conditions, e.g. at a pH of less than 6. In some cases it is carried out at a pH of less than 5, less than 4, less than 3, less than 2, less than 1, or lower. The treatment with selective removal agent can be carried out at between pH 6 and pH 0, between pH 5 and pH 1, or between pH 4 and pH 2. We have found that in some cases, the selective removal reaction tends to proceed more rapidly at lower pH. In some cases, if the pH is too low, it is difficult to control the level of corrosion. The reaction can be carried out at any effective temperatures. For example, temperatures from 20° C to 100° C can be employed. In some cases temperatures between 40° C and 95° C are used. In some cases, temperatures between 80° C and 90° C are used. The time for the selective removal reaction is generally between 1 minute and 1 day. The optimal time may vary depending, for example, on the pH and temperature employed. Times between about 10 min. to about 120 min., or between about 20 min. and about 60 min. can be used.

[0183] In some cases, the addition of salts can improve the selective removal reaction. In some cases, salts having sodium (Na+) are present. In some cases, the addition of bivalent salts such as Ca++can be beneficial with respect to selective removal.Molecule or molecules of interest

[0184] The methods described herein can be used, for example, for the selective attachment of one or molecules of interest to bottoms of the nanoscale wells where they can be observed and used for analysis.

[0185] The terms molecule of interest and molecules of interest as used herein describe molecules that are bound or coupled to the surface that are utilized in an analysis. The molecules of interest can be detected or identified either directly through labels bound to the molecule or molecules of interest, or indirectly though labeled molecules that interact strongly or more weakly with the molecule or molecules of interest. These labeled molecules aresometimes referred to herein as analytes, which can, in some cases, be provided in a solution in contact with the molecules of interest.

[0186] The analysis typically involves the use of a label. This label can be used to detect or identify a molecule of interest. In some cases, the label is directly bound or coupled to the molecule of interest, for example covalently bound or bound by a strong non-covalent interaction, e.g. biotin-streptavidin. In other cases the label is bound to a molecule that more weakly or transiently binds to the molecule of interest. The weak, or transient binding can be, for example, enzyme-substrate binding. The analysis can be, for example, a sandwich assay such as an ELISA assay. Sandwich assays are decribeed, for example in Anal Chim Acta Volume 758, 3 January 2013, Pages 1-18, which is incorporated herein by reference for all purposes.

[0187] In single-molecule real-time nucleic acid sequencing, the molecule of interest can be a polymerase or a nucleic acid, e.g. within a polymerase-template complex. This polymerase- template complex can transiently bind labeled nucleotide analogs while they are interacting with the polymerase to make a nascent strand complementary to the template in the polymerase template complex. For multi-molecule (clonal population) nucleic acid sequencing the molecules of interest can be a clonal population of nucleic acids having the same sequence. A step-wise process can be used to identify each nucleotide added in forming a nascent strand on the clonal population of nucleic acids. In some cases, the step-wise process involves an intermediate with a label covalently bound to the nucleic acid template (molecules of interest). In some cases, the step-wise process involves the non-covalent binding of labeled nucleotide analogs to the template nucleic acids (molecules of interest) in a sequencing-by-binding (SBB) process.

[0188] For protein or peptide identification or sequencing applications, the molecule or molecules of interest can be proteins or peptides bound or coupled to the surface, These protein molecules of interest can be detected or identified using labels that directly bind to the protein or using labeled molecules that interact either weakly or strongly with the bound or coupled proteins, for example, labeled antibodies or labeled aptomers. Labeled amino acid recognition molecules such as those described in U.S Patent Application 2020 / 0209256 can be employed to discriminate between amino acids, e.g. as residues are successively removed from a polypeptide to expose the next residue during protein sequencing.

[0189] The molecules of interest are generally attached to the coupling groups selectively placed onto the group 4-5 transition metal oxide on the bottom of a nanoscale well as described herein. A variety of chemistries are available for specifically attaching a molecule of interest to the coupling groups bound to the surface.

[0190] For example, where biotin is bound to the group 4-5 transition metal oxide regions of the surface, this surface can be used to attach the molecule of interest using a binding agent such as streptavidin, which has a very high affinity for biotin. In one approach, the molecule of interest has a biotin tag which can then be attached to the surface using an intermediate binding agent (e.g., streptavidin, avidin, or another biotin-binding protein) which acts to bind to both the surface and the molecule of interest. In another approach, streptavidin, avidin, or another biotin-binding protein is attached directly to the molecule of interest.

[0191] A wide variety of nucleic acids can be of interest in the methods herein. These include cloned nucleic acids (DNA or RNA), expressed nucleic acids, genomic nucleic acids, amplified nucleic acids cDNAs, and the like. Details regarding nucleic acids, including isolation, cloning and amplification can be found, e.g., in Berger and Kimmel, Guide to Molecular Cloning Techniques, Methods in Enzymology volume 152 Academic Press, Inc., San Diego, CA (Berger); Sambrook et al., Molecular Cloning - A Laboratory Manual (3rd Ed.), Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 2000 (“Sambrook”); Current Protocols in Molecular Biology. F.M. Ausubel et al., eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc; Kaufman et al. (2003) Handbook of Molecular and Cellular Methods in Biology and Medicine Second Edition Ceske (ed) CRC Press (Kaufman); and The Nucleic Acid Protocols Handbook Ralph Rapley (ed) (2000) Cold Spring Harbor, Humana Press Inc (Rapley).Suitable templates are also described in U.S. Patents 8,003,330, 10,768,173, 11,203,778 and U.S. Patent Applications 20210318295 and 20240035082 and U.S. Patents 8,236,499 8,153,375 which are incorporated by reference herein in their entirety for all purposes.

[0192] Similarly, a wide variety of proteins, e.g., enzymes, can also be delivered using the methods herein. A variety of protein isolation and detection methods are known and can be used to isolate enzymes such as polymerases, e.g., from recombinant cultures of cells expressing the recombinant polymerases of the invention. A variety of protein isolation and detection methods are well known in the art, including, e.g., those set forth in R. Scopes, Protein Purification, Springer-Verlag, N.Y. (1982) and Handbook of Bioseparations,Academic Press (2000). Sambrook, Ausubel, Kaufman, and Rapley supply additional useful details.

[0193] For a description of polymerases and other enzymes that are active when bound to surfaces, which is useful in single molecule sequencing reactions in which the enzyme is fixed to a surface (e.g., to the bottom of a nanoscale well the bottom of a nanoscale well), see Hanzel et al. ACTIVE SURFACE COUPLED POLYMERASES, WO 2007 / 075987 and Hanzel et al. PROTEIN ENGINEERING STRATEGIES TO OPTIMIZE ACTIVITY OF SURFACE ATTACHED PROTEINS, WO 2007 / 075873). Suitable polymerases are described, for example, in U.S. Patent application publications 2007-0196846, 2008-0108082, 2010-0075332, 2010-0093555, 2010-0112645, 2011-0189659, 2012-0034602, 2013-0217007, 2014-0094374, and 2014-0094375, each of which is incorporated herein by reference in its entirety for all purposes. For further descriptions of single molecule sequencing applications utilizing ZMWs, see Levene et al. (2003) “Zero Mode Waveguides for single Molecule Analysis at High Concentrations,” Science 299:682-686; Eid et al. (2008) “Real-Time DNA Sequencing from Single Polymerase Molecules” Science DOI:10.1126 / science.322.5905.1263b; Korlach et al. (2008) “Selective aluminum passivation for targeted immobilization of single DNA polymerase molecules in zero-mode waveguide nanostructures” Proceedings of the National Academy of Sciences U.S.A. 105(4): 1176- 1181 ; Foquet et al. (2008) “Improved fabrication of zero-mode waveguides for single-molecule detection” Journal of Applied Physics 103, 034301; “Zero-Mode Waveguides for SingleMolecule Analysis at High Concentrations” USP 7,033,764, USP 7,052,847, USP 7,056,661, and USP 7,056,676, the full disclosures of which are incorporated herein by reference in their entirety for all purposes.

[0194] In order to attach a polypeptide or protein such as an enzyme to the surface, binding elements can be added (recombinantly or, e.g., chemically) including, e.g. biotin, avidin, GST sequences, modified GST sequences, e.g., that are less likely to form dimers, biotin ligase recognition (BiTag) sequences, S tags, SNAP -tags, antibodies or antibody domains, antibody fragments, antigens, receptors, receptor domains, receptor fragments, ligands, or combinations thereof. In some cases, the enzyme can be covalently attached to the substrate through functional groups on the enzyme such as amine, carboxylate, or thiol groups, for example with NHS or maleimide linking chemistry.

[0195] Multiple surface binding domains can be added to orient the polypeptide relative to a surface and / or to increase binding of the polymerase to the surface. By binding a surface at two or more sites, through two or more separate tags, the polymerase is held in a relatively fixed orientation with respect to the surface. Further details on attaching tags is available in the art. See, e.g., U.S. patents 5,723,584 and 5,874,239 for additional information on attaching biotinylation peptides to recombinant proteins.Substrates and chips

[0196] The substrates of the invention are generally rigid, and often planar, but need not be either. Where the substrate comprises an array of nanoscale wells, the substrate will generally be of a size and shape that can interface with optical instrumentation to allow for the illumination of and for the measurement of light from the nanoscale wells. Typically, the substrate will also be configured to be held in contact with liquid media, for instance containing reagents and enzyme substrates and / or labeled components for optical measurements.

[0197] The substrates and chips of the invention often comprises one or more silicon dioxide-based layers and regions. As used herein, the terms silicon dioxide, silica, and SiCh are used interchangeably. The silicon dioxide comprising materials are typically transparent. The term “transparent”, as used herein, refers to a layer or material that will at least partially transmit electromagnetic energy or light of the wavelength appropriate for the use of the substrate. For analytic devices and chips, the wavelength is generally from the infrared to the ultraviolet. In many cases, for example in using fluorescent dyes, it is desirable that the substrate transmit visible light, for example, between 400 nm and 800 nm. The silicon dioxide materials may comprise any of a number of transparent solid materials, depending upon other components of the substrate. Such materials include inorganic materials, such as glass, quartz, fused silica, and the like. In some cases, a silicon dioxide comprising material such as fused silica can be processed using semiconductor processing techniques. In some cases the silicon dioxide material or layer can be an oxide layer, e.g. a native oxide layer. Such a silicon dioxide layer can be provided, for example, by the oxidation of silicon or silicon nitride.

[0198] Where the substrates comprise arrays of nanoscale wells, the arrays may comprise a single row or a plurality of rows of nanoscale wells on the surface of a substrate, where when a plurality of row are present, the number of rows will usually be more than 10, and morecommonly more than 100 or more than 1000. The subject array of nanoscale wells may align horizontally or diagonally along the x-axis or the y-axis of the substrate. The individual wells can be arrayed in any format across or over the surface of the substrate, such as in rows and columns so as to form a grid, or to form a circular, elliptical, oval, conical, rectangular, triangular, or polyhedral pattern. To minimize the nearest-neighbor distance between adjacent nanoscale wells, a hexagonal array is sometimes preferred.

[0199] The array of nanoscale wells may be incorporated into a structure that provides for ease of analysis, high throughput, or other advantages, such as in a microtiter plate and the like. Such setup is also referred to herein as an "array of arrays." For example, the subject arrays can be incorporated into another array such as microtiter plate wherein each micro well of the plate contains a subject array of nanoscale wells.

[0200] In accordance with the invention, arrays of nanoscale wells are provided in arrays of more than 100, more than 1000, more than 10,000, more than 100,000, or more than 1,000,000 separate nanoscale wells on a single substrate. In addition, the arrays typically comprise a relatively high density of nanoscale wells on the surface of the substrate. Such high density typically includes nanoscale wells present at a density of greater than 10 nanoscale wells per mm2, preferably, greater than 100 nanoscale wells per mm2of substrate surface area, and more preferably, greater than 500 or even 1000 nanoscale wells per mm2and in many cases up to or greater than 100,000 nanoscale wells per mm2. Although in many cases, the nanoscale wells in the array are spaced in a regular pattern, e.g., in 2, 5, 10, 25, 50, 100, or 1000 or more rows and / or columns of regularly spaced nanoscale wells in a given array, in certain cases, there are advantages to providing the organization of waveguides in an array deviating from a standard row and / or column format. In preferred aspects, the substrates include nanoscale wells to define the discrete reaction regions on the substrate.

[0201] The surfaces of the substrate are modified according to the methods described herein. The surface modifications are generally carried out by laboratory and manufacturing techniques that would be known to those of skill in the art. In some cases, the compounds used for surface modification and surface functionalization are brought into contact with the surface in liquid form. For example, the surfaces are treated with or exposed to a solution of the surface modification compound. In other cases, the compounds are brought into contact with the surface in gaseous form. In general, the whole surface of the substrate is exposed toor treated with the compounds or functionalizing agents. In other cases, regions of the surface can be masked from exposure to one or more of the compounds or functionalizing agents.

[0202] In some embodiments, the substrates include a base substrate comprising a semiconductor material that is not generally transparent. Suitable semiconductor materials include doped silicon, germanium, or gallium arsenide.

[0203] The arrays of nanoscale wells of the invention can be disposed on substrates or chips that are useful for carrying out chemical or biochemical analyses. In some cases, the chips comprise optical or electronic components for carrying out the analysis. For example, the chips can include optical waveguides, lenses, filters, apertures, and detectors. In a preferred embodiment, the chips are constructed as layers that are deposited on a sensor array. The array of nanoscale wells is on the top of the chip where the wells can be in contact with reagents in solution, and the detectors are on the bottom of the chip. Light emitted from the nanoscale wells travels down through any desired optical elements to be detected by the detectors on the bottom of the chip. The arrangement of the detectors in the sensor array corresponds to the arrangement of nanoscale wells, for example, with one nanoscale well corresponding to and sending light to one, two, three, or four detectors (pixels) in the sensor array.Substrates having high levels of bias

[0204] The methods of the invention can be used to produce substrate surfaces having high levels of bias. The term bias as used herein is generally used to refer a measure of a difference in surface properties of two different surfaces or two different portions of a surface. The measured bias can represent the selective attachment of an agent to one portion of the surface over another portion of the surface. The bias can be represented as a ratio of a property associated with the agent on one portion of the surface to that measured on a different portion of the surface. It will be understood that there may be multiple methods for measuring bias, and that the bias measured by one method may not provide the same level of bias as another method. Bias can be measured, for example, using optical methods (including fluorescence), X-ray photoelectron spectroscopy (XPS), ellipsometry, or contact angle.

[0205] The methods of the invention provide for producing substrates having surfaces with high bias. In particular, the substrates of the invention can have a group 4-5 transition metal oxide portion and a silicon dioxide or aluminum oxide portion, wherein the bias for agentsbound to one portion as compared to the other portion is high. Substrates having these characteristics can comprise, for example, nanoscale wells. In some cases, the level of bias can be measured on an array of nanoscale wells. In some cases, it is not practical to measure the bias on the array of nanoscale wells. In such cases, the bias that is achievable by a surface preparation method can be determined on a surrogate surface. In some cases, fused silica coupons having regions of group 4-5 transition metal oxide, e.g. hafnium oxide, on portions of their top surfaces while in other portions the silicon dioxide remains exposed can be used to determine the bias that is obtained by a method of the invention.

[0206] In some cases, the bias represents the relative levels of a coupling group on the group 4-5 transition metal oxide portions of the substrate as compared to the levels of the coupling group on the silicon dioxide or aluminum oxide portions of the substrate. In these cases, bias can be determined by measuring the amount of attachment of a compound that reacts with the coupling groups. Examples of chemistries that can be used for coupling groups and compounds that react with the coupling groups described herein. Other chemistries are well known in the art. In some cases, the coupling groups can comprise a selective binding group.

[0207] In some cases, the bias represents the relative levels of a selective binding group on the group 4-5 transition metal oxide portions of the substrate as compared to the levels of selective binding group on the silicon dioxide or aluminum oxide portions of the substrate.For example, where the selective binding group is biotin, bias can be determined by measuring the relative amount of a labeled avidin, streptavidin, or neutravidin bound to each surface region. In some cases, for example where fluorescent labels are used, a metal or metal oxide surface can tend to quench the fluorescence of the labeled avidin, streptavidin, or neutravidin. In these cases, the labeled avidin, streptavidin, or neutravidin can be bound to beads, allowing for fluorescence in the bound state.

[0208] The bias for a substrate treated with a selective binding group or other coupling group can also be determined using a molecule of interest, e.g. an enzyme of interest that is labeled. The enzyme of interest can be labeled covalently, or the enzyme could be labeled by having the enzyme bound to a molecule that is labeled. For example, the enzyme could be bound to a labeled substrate molecule. Where the enzyme of interest is a polymerase, the polymerase can be bound to a fluorescently labeled template nucleic acid. For example, a surface having been selectively functionalized with a biotin containing selective binding group as described herein can be reacted first with neutravidin, and then with an enzyme bound to afluorescently bound template where the enzyme comprises a biotin group. The relative fluorescence measured on the group 4-5 transition metal oxide portions of the surface relative to that on the silicon dioxide or aluminum oxide portions provides a measure of the bias.

[0209] In one aspect, the invention provides an array of nanoscale wells having bottom surfaces comprising group 4-5 transition metal oxide and sidewalls comprising silicon dioxide or aluminum oxide, wherein the array has been treated with a sidewall surface modifying agent and with a bottom surface modifying agent comprising a coupling group, wherein when a coupon having portions with the same type of group 4-5 transition metal oxide as the group 4-5 transition metal oxide in the array and the portions with the same type of silicon dioxide or aluminum oxide as the silicon dioxide or aluminum oxide of the array is treated in the same manner as the array, the coupon exhibits a fluorescent intensity bias of greater than about 5, greater than about 8, greater than about 10, greater than about 20, greater than about 30, greater than about 40, greater than about 50, greater than about 60, greater than about 70, greater than about 80, greater than about 90, or greater than about 100 in a fluorescent bias assay such as a neutravidin labeled bead assay or labeled enzyme assay. The coupling group can comprise a selective binding group such as biotin.

[0210] In one aspect, the invention provides a substrate with regions comprising group 4-5 transition metal oxide and regions comprising silicon dioxide or aluminum oxide, wherein the substrate has been treated with a sidewall surface modifying agent and with a bottom surface modifying agent comprising a coupling group, wherein when a coupon having portions with the same type of group 4-5 transition metal oxide as the group 4-5 transition metal oxide regions of the substrate and portions with the same type of silicon dioxide or aluminum oxide as the silicon dioxide or aluminum oxide regions of the substrate is treated in the same manner as the substrate, the coupon exhibits a fluorescent intensity bias of greater than about 5, greater than about 8, greater than about 10, greater than about 20, greater than about 30, greater than about 40, greater than about 50, greater than about 60, greater than about 70, greater than about 80, greater than about 90, or greater than about 100 in a labeled enzyme assay or neutravidin labeled bead assay. The coupling group can comprise a selective binding group such as biotin.Substrates with shallow nanoscale wells

[0211] While many embodiments of the invention utilize relatively deep nanoscale wells, for example where the depth (or height) of the well is greater than the diameter (or other cross-sectional dimension) of the well, the invention can also be used with wells that are relatively shallow. Such a shallow well of the invention, and an exemplary method of producing such a well is shown in Fig. 8. The well formed in this embodiment (see e.g. the structure after step III) has a bottom comprising a group 4-5 transition metal oxide and sidewalls formed by a layer of silicon dioxide. A similar structure can be formed by using an aluminum oxide layer in place of the silicon dioxide layer (not shown). The dimensions of the bottoms of the wells, the number of wells on a chip, and the shapes of the wells can be the same as those described herein.

[0212] In the embodiment shown in Fig 8., a base substrate 810, made, for example, of silicon or silicon dioxide is provided. In some cases, the base substrate comprises multiple layers. In step I, a group 4-5 transition metal oxide layer 820 is deposited onto the base substrate 810. In step II a layer of silicon dioxide 830 is deposited. In step III, lithography and etching is used to etch through the silicon dioxide layer 830 to produce a shallow well 800 with group 4-5 transition metal oxide 820 on the bottom. In step IV, a sidewall surface modifying agent 840 as described herein is applied, typically in solution or by vapor deposition. The sidewall surface modifying agent covers the sidewalls and the tops of the silicon dioxide layer 830. The sidewall surface modifying agent 840 can be selective as shown here or non-selective such that some of the sidewall surface modifying agent is on the bottom of the shallow well (not shown). In step V, a bottom surface modifying agent 850 having coupling groups is deposited, typically in solution or through vapor deposition. The bottom surface modifying agent 850 can be very specific. To the extent that there is unwanted sidewall surface modifying agent on the bottom of the well, the treatment with bottom surface modifying agent 850 can in many cases remove it. In some cases, removal compounds (not shown) are used to ensure highly selective functionalization. In step VI, a molecule or groups of molecules of interest 890 are attached to the bottom of the well through coupling groups on the bottom surface modifying agents 850. The groups of molecules of interest can be, for example, clonal populations of molecules used for analysis.

[0213] While it is not always the case, single molecule applications tend to utilize relatively deep wells, and clonal population applications such as short read sequencing that have sequential extend, wash, detect, and wash steps, e.g., 3’-reversible terminator basedsequencing, tend to use the shallower wells. Applications such as microarrays for DNA hybridization can also utilize the shallow wells of the invention.Substrates with active regions of group 4-5 transition metal oxidesWhile many of the embodiments described herein utilize the group 4-5 transition metal oxides for coupling molecules of interest to the bottoms of wells, the invention can also be used for coupling molecules of interest to specific regions on a chip that are not wells. To describe these embodiments, we use the term “active regions” to refer to the regions comprising group 4-5 transition metal oxides to which molecules of interest can be attached. It is to be understood that for these embodiments, the chemistry and characteristics of “active regions” correspond directly to those described throughout this specification for the bottoms of nanoscale wells. For example, the “active region surface modifying agents” used herein can be any of the “bottom surface modifying agents” described throughout specification. For example, the active regions are treated to couple molecules of interest to the group 4-5 transition metal oxide active regions.

[0214] In these embodiments, the active regions of 4-5 transition metal oxide are surrounded by silicon dioxide and / or aluminum oxide regions (“surrounding regions”). The chemistry and characteristics of the surrounding regions correspond directly to those described throughout the specification for nanoscale well sidewalls. For example, “surrounding region surface modifying agents” used herein can be any of the “sidewall surface modifying agents” described throughout the specification.

[0215] The active regions of group 4-5 transition metal oxide are discrete regions separated by silicon dioxide and / or aluminum oxide surrounding regions. Often there are a million or more active regions on a substrate. In some cases, there are more than a million active regions, more than 10 million active regions, more than 100 million active regions, or more than 500 million active regions on a substrate or chip. The active regions can be raised above the substrate, can be flush with the top of the substrate, or can be recessed below the top of the substrate. The active regions can have one or more dimensions of 10 micrometer or less, one or more dimensions of 1 micrometer or less, one or more dimensions of 500 nanometers or less, one or more dimensions of 200 nanometers or less, one or more dimensions of 100 nanometers or less, one or more dimensions of 50 nanometers or less, or one or more dimensions of 10 nanometers or less. The 2-dimensional shape of the active regions can beany suitable shape. For example, the shape of the active regions can be roughly circular, square, oval, or rectangular.

[0216] Fig. 9A shows a cross section of an an active region comprising a group 4-5 transition metal oxide with surrounding regions comprising silicon dioxide or aluminum oxide. On the substrate 910 is an active region 920 comprising group 4-5 transition metal oxide, e.g. hafnium oxide. The active region 920 is flanked by surrounding regions 930 comprising either silicon dioxide or aluminum oxide. Fig. 9B shows how this substrate can be treated with active region surface modifying agents and surrounding region surface modifying agents as described herein to selectively functionalize the regions. The active region 920 is modified with a acive region surface modifying agent 950 , e.g. a phosphonate. The active region surface modifying agent 950 has a coupling group, e.g. biotin, which is used to attach molecules of interest 990 selectively to the active region 920. The molecules of interest, e.g. DNA, protein, or peptide, can be a clonal population. In some cases (not shown), a single molecule can be attached to the active region 920. The surrounding regions 930, e,g, silicon dioxide, flanking the active region 920 are modified with a surrounding region surface modifying agent 940. The surrounding region surface modifying agent 940, e.g. a silane, can be used to prevent attachment of molecules of interest to these regions.

[0217] The active regions are often provided as an array of active regions. The array can be any suitable distribution of regions. Often the array is provided as a grid having columns and rows. Fig. 9C shows an array of active regions separated by surrounding regions comprising silicon dioxide or aluminum oxide of the invention. Here, an array of roughly square active regions comprising group 4-5 transition metal oxide 921, e.g. hafnium oxide, are present on a planar substrate. The active regions 921 are separated by surrounding regions comprising silicon dioxide and / or aluminum oxide 931. The regions 921 and 931 can be treated with surface modifying agents as described herein to produce arrays of regions having molecules of interest selectively bound to the active regions. Removal compounds are used to further improve the selectivity of the functionalization.Methods of making selectively functionalized substrates

[0218] The selectively functionalized nanoscale wells of the invention can be made using semiconductor processing chemistry. In some cases, this is followed by specific surface deposition of surface modifying agents using solution phase or vapor phase methods. Eitherthe bottom surface modifying agent or the sidewall surface modifying agents or both can be applied in this manner. Vapor phase methods can include chemical vapor deposition (CVD), plasma enhanced chemical vapor deposition (PECVD), and physical vapor deposition (PVD) including evaporative deposition, ion plating, pulsed laser deposition, and sputter deposition. Solution phase methods include dip coating, spray coating, spin coating, slot dye, bar and blade coating.

[0219] The nanoscale wells are typically formed on the top of a substrate or chip to allow for them to be in contact with a fluid above the chip for analysis. The substrate is typically made up of a base substrate onto which layers are deposited, patterned, and etched, as is typical for semiconductor processing. The base of the substrate can be any suitable material. The base substrate can be, for example, silicon, germanium, gallium arsenide, gallium nitride, silicon carbide, silicon on insulator (SIN), or a flexible polymer. Typical base substrate materials are silicon and silicon dioxide (SiCh). The substrate as described herein may be produced by depositing multiple layers on top of a semiconductor wafer that includes multiple photosensors. An analytic device is then produced by adding layers over the photosensors. The nanoscale wells are typically formed on the top of the substrate whereby the locations of the nanoscale wells correspond to the sensing elements (e.g. pixels) in the photosensors. Each photosensor on the wafer can have millions of pixels. In some cases there is a one-to-one correspondence of the nanoscale wells with a pixel. In other cases, there can be 2, 3, 4 or more pixels that correspond to each nanoscale well. Approaches to producing such substrates are described, for example in 8,46,569, 9,410,891, 8,467,061, 9,372,308 9,223,084, 9,624,540, 9,606,068, the full disclosures of which are incorporated herein by reference in their entirety for all purposes which are incorporated by reference herein in their entirety for all purposes.

[0220] Fig. 4 provides an embodiment of a method of producing a selectively functionalized nanoscale well of the invention. The nanoscale well is formed onto the substrate 410. In some cases, the layer of the base substrate 410 can be a silicon dioxide layer, and can be, for example, the top cladding layer for an optical waveguide below (not shown). In step I, a group 4-5 transition metal oxide layer 420, e.g. hafnium oxide, is deposited. In step II, a silicon dioxide well-forming layer 430, for example comprising silicon dioxide, is deposited. In step III, a reflective layer 440, for example, aluminum, is deposited. In step IV, a surface layer 450 of silicon dioxide is deposited. In step V, a nanoscale well 400 is produced by etching through layers 450, 440, and 430 to expose the group 4-5 transition metal oxide layer420. The group 4-5 transition metal oxide layer 420 can act as an etch stop layer in this process allowing for enhanced control of the etching process. The nanoscale well 400 that is formed comprises silicon dioxide on its sidewalls and a group 4-5 transition metal oxide on the bottom. In step VI, the device is treated with a sidewall surface modifying agent 460, for example a silane. In this embodiment, the sidewall surface modifying agent 460 is somewhat selective. It covers the silicon dioxide sidewall and the silicon dioxide surface layer. Some sidewall surface modifying agent is on the bottom group 4-5 transition metal oxide layer 420. In step VII, a highly selective bottom surface modifying agent 470 (e.g. comprising a phosphate, phosphonate, or catechol) is applied. In some cases, the bottom surface modifying agent will selectively remove the unwanted sidewall surface modifying agent from the bottom group 4-5 transition metal oxide layer 420. Where the reflective material 440 comprises aluminum, which will have an aluminum oxide surface, the bottom surface modifying agent 470 can coat the edges of the aluminum layer 440 as shown. Where this is the case, step VIII, treatment with selective removal compounds as described herein, can be employed. The selective removal compound will remove the bottom surface modifying agent 470 from the aluminum oxide edge, while leaving the bottom surface modifying agent 470 on the group 4-5 transition metal oxide layer 420. In step IX, a molecule of interest 490 or group of molecules of interest (not shown) is selectively attached to the bottom of the nanoscale well through a coupling group on the bottom surface modifying agent 470.

[0221] Fig. 5 provides an embodiment of a method of producing a selectively functionalized nanoscale well of the invention. The nanoscale well is formed onto the base substrate 510. In some cases, the layer of the base substrate 510 can be a silicon dioxide layer, and can be, for example, the top cladding layer for an optical waveguide below (not shown). In step I, a group 4-5 transition metal oxide layer 520, e.g. hafnium oxide, is deposited. In step II, a silicon dioxide well-forming layer 530, for example comprising silicon dioxide, is deposited. In step III, a reflective layer 540, for example, aluminum, is deposited. In step IV, a nanoscale well 500 is produced by etching through layers 540 and 530 to expose the group 4-5 transition metal oxide layer 520. The group 4-5 transition metal oxide layer 520 can act as an etch stop layer in this process allowing for enhanced control of the etching process. In step V, a silicon dioxide surface layer 540 is deposited such that it coats the top of the chip and also extends down into the nanoscale well to coat the bottom and sidewalls of the nanoscale well 500. In step VI, etching is performed to remove the silicon dioxide surface layer 540 from the bottomof the well. This exposes the group 4-5 transition metal oxide on the bottom. The nanoscale well 500 that is formed comprises silicon dioxide on its sidewalls and a group 4-5 transition metal oxide on the bottom. In step VII, the device is treated with a sidewall surface modifying agent 560, for example a silane. In step VIII, a highly selective bottom surface modifying agent 570 (e.g. comprising a phosphate, phosphonate, or catechol) is applied. In some cases, the bottom surface modifying agent will selectively remove unwanted sidewall surface modifying agent from the bottom group 4-5 transition metal oxide layer 520. In step IX, a molecule of interest 590 or group of molecules of interest (not shown) is selectively attached to the bottom of the nanoscale well through a coupling group on the bottom surface modifying agent 570.

[0222] Fig. 6 provides an embodiment of a method of producing a selectively functionalized nanoscale well of the invention. The nanoscale well is formed onto the base substrate 610. In some cases, the layer of the base substrate 610 can be a silicon dioxide layer, and can be, for example, the top cladding layer for an optical waveguide below (not shown). In step I, a group 4-5 transition metal oxide layer 620, e.g. hafnium oxide, is deposited. In step II, a silicon dioxide well-forming layer 630, for example comprising silicon dioxide, is deposited. In step III, an aluminum reflective layer 640 is deposited. In step IV, a nanoscale well 600 is produced by etching through layers 640 and 630 to expose the group 4-5 transition metal oxide layer 620. The group 4-5 transition metal oxide layer 620 can act as an etch stop layer in this process allowing for enhanced control of the etching process. The nanoscale well 600 that is formed comprises silicon dioxide on its sidewalls and a group 4-5 transition metal oxide on the bottom. In step V, a highly selective bottom surface modifying agent 650, (e.g. comprising a phosphate, phosphonate, or catechol) is applied. In step VI, the device is treated with a sidewall surface modifying agent 660, for example a silane. In this embodiment, the sidewall surface modifying agent 660 is somewhat selective. It covers the silicon dioxide sidewall, and some sidewall surface modifying agent is on the bottom group 4-5 transition metal oxide layer 620. In step VII, a selective removal compound is applied. The selective removal compound will remove sidewall surface modifying agent 660 from the group 4-5 transition metal oxide layer 620 and will also selectively remove the bottom surface modifying agent 650 from the top of the aluminum reflective layer 640, while leaving the bottom surface modifying agent 650 on the group 4-5 transition metal oxide layer 620. In some cases, the removal compound will remain on the top of the aluminum reflective layer640. In step VIII, a molecule of interest 690 or group of molecules of interest (not shown) is selectively attached to the bottom of the nanoscale well through coupling groups on the bottom surface modifying agent 650.

[0223] In the above embodiments, in will be understood that the order of steps can be varied. In particular, the order of the addition of the sidewall surface modifying agent and the bottom surface modifying agent may vary. While none of Fig. 4, Fig. 5, or Fig. 6 explicitly show a waveguide core as the bottom of the well, it will be understood by those of skill in the art that the methods described and straightforward variants thereof can be used for such embodiments.

[0224] In some cases, the substrate can be disposed at an angle whereby the surface modifying agent is selectively deposited on the sidewalls without depositing on the bottom of the well.Methods of producing arrays of analytical devices

[0225] As described above, arrays of the invention are useful, for example, in the large-scale sequencing of nucleic acids, including, in particular, genomic sequencing. Such arrays can be produced by a variety of methods. Some aspects of the production of the instant arrays involves the use of microfabrication methods such as semiconductor or MEMS processing methods, which have been highly developed for the production of integrated circuits. Similar processes have been used to create MEMS (micro electromechanical systems) for a variety of applications including inkjet printers, accelerometers, pressure transducers, and displays (such as digital micromirror displays (DMDs)). Microfabrication methods can be applied to a large base substrate such as a wafer, which can later be diced into many devices, allowing for the production of many devices at one time.

[0226] The methods of the invention may, for example, apply resist processes, such as photoresists, to define structural elements on base substrates or other layers. Etching processes can be used to produce three-dimensional structures, including component structures in the integrated analytical device. Deposition processes can be used to add layers onto the devices. Other semiconductor processes such as ashing, polishing, release, liftoff, and wet cleans can also be employed to create the structures of the invention, as described in more detail below.

[0227] For example, lithographic techniques can be used to define a mask layer out of polymeric materials, such as photoresists, using e.g., conventional photolithography, e-beamlithography, or the like. Alternatively, lithographic techniques can be applied in conjunction with layer deposition methods to deposit metal mask layers, e.g., using aluminum, gold, platinum, chrome, or other conventionally used metals, or other inorganic mask layers, e.g., silica based substrates such as silicon, SiCh, or the like. Alternatively, negative tone processes can be employed to define pillars of resists that correspond to, for example, nanowells. See, e g., U.S. Pat. No. 7,170,50, which is incorporated by reference herein in its entirety for all purposes. The mask layer can then be deposited over resist pillars and the pillars are subsequently removed. In some approaches, both the underlying substrate and the mask layer are fabricated from the same material.

[0228] By providing the mask and underlying layers of the same material, one can ensure that the two layers have the same interactivity with the environments to which they are exposed, and thus minimize any hybrid surface interactions.

[0229] In the case of SiCh-based substrates and mask layers, conventional fabrication processes can be employed. For example, a base substrate bearing a surface-exposed feature, such as a waveguide, can have a layer of resist deposited over its surface. A negative of the mask layer is then defined by appropriate exposure and development of the resist layer to provide resist islands where one wishes to retain access to the underlying feature. The mask layer is then deposited over the surface and the remaining resist islands are removed, e.g., through a lift off process, to provide the openings to the underlying feature. In the case of metal layers, deposition can be accomplished through a number of means, including evaporation, sputtering or the like. Such processes are described in, e.g., U.S. Pat. No. 7,170,50. In the case of silica based mask layers, a chemical vapor deposition (CVD) process can be employed to deposit a silicon layer onto the surface. Following lift off of the resist layer, a thermal oxidation process can convert the mask layer to SiCh. Alternatively, etching methods can be used to etch access points to underlying layers using conventional processes. For example, a silicon layer can be deposited over an underlying substrate. A resist layer is then deposited over the surface of the silicon layer and exposed and developed to define the pattern of the mask. The access points are then etched from the silicon layer using an appropriate differential etch to remove silicon but not the underlying SiO2 substrate. Once the mask layer is defined, the silicon layer is again converted to SiO2 using, e.g., a thermal oxidation process.

[0230] In each of the above exemplary microfabrication techniques, the process begins with a clean substrate layer. The substrate layer used in the instant methods can be of any suitable rigid material. The substrate layer material can comprise, for example, an inorganic oxide material such as silica. A preferred substrate layer material comprises a detector layer, such as, for example, a CMOS wafer, i.e., a wafer made up of CMOS sensors or CCD arrays. See, for example, CMOS Imagers From Phototransduction to Image Processing (2004) Yadid-Pecht and Etienne-Cummings, eds.; Springer; CMOS / CCD Sensors and Camera Systems (2007) Holst and Lomheim; SPIE Press.

[0231] As mentioned above, the methods of the invention in some cases use resists for defining and producing structures with lithography. These resists can be, for example, photoresists or e-beam resists. The photoresists can be developed using UV, deep UV, G-line, H-line, I-line or other suitable wavelength or set of wavelengths. The type of resist that is used, and therefore the type of instrumentation that is employed for processing, will depend on the dimensions of the features that are created. In many processes described herein, higher resolution resists and equipment will be used for the production of the nanoscale well which corresponds to the reaction volume, where the size of the nanoscale well can be on the order of 10 nm to 500 nm, and a lower resolution resist and associated instrumentation is used for the creation of the rest of the integrated analytical device, which can have features on the dimensions of 1 micron to 20 microns. Many resists are known in the art, and many are available commercially from companies such as Rohm and Haas and Shipley. The resists used in the processes of the invention can be negative or positive photoresists. Where a process is described herein using a negative photoresist, it is to be understood that a suitable positive photoresist can also be employed where practical, and vice versa. Where appropriate, chemical amplification can also be employed to increase the sensitivity of the resist. The removal of the resist, the cleaning, rinsing, ashing, and drying of the substrate can be performed as appropriate and as taught and known in the art.

[0232] In some cases, the tools used for photolithography of the nanoscale well use a photolithography exposure tool capable of creating structures having feature sizes of about of 10 nm to about 100 nm. Such systems include, for example, an AMSL XT1250 exposure tool.

[0233] Etching processes are used in some aspects of the invention in order to produce the three dimensional features in a substrate or in other layers, to fashion, for example, optical elements or lenses, or reaction volumes such as nanowells. The etching process that is usedwill depend on the type of material used, the dimensions of the features, and the resist system. In some cases, wet etching or wet chemical etching is employed. Electrochemical etching can also be employed. In some embodiments plasma etching or reactive ion etching (RIE) is used as an etching process. Deep reactive ion etching (DRIE) can also be employed, for example, where structures having high aspect ratio are desired. Dry vapor phase etching, for example with xenon difluoride, can also be used. Bulk micromachining or surface micromachining can be used as appropriate to create the device structures of the disclosure. The etching used in the methods of the disclosure can be gray-scale etching. The conditions of the resist formation and etching are controlled to produce side walls having the desired geometries, such as having the desired side-wall angle.

[0234] Some processes of the invention involve the deposition of reflective layers. The deposition of these reflective layers can be accomplished by wet processes including spinning on layers from solution, or by gas-phase processes. Suitable processes include electroplating, sputter deposition, physical vapor deposition, evaporation, molecular beam epitaxy, atomic layer deposition, and chemical vapor deposition. Metals can be used as the reflective layer. Suitable metals include gold, nickel, aluminum, chromium, titanium, platinum, and silver. The reflective layers can comprise aluminum, which can be deposited by sputtering, for example using a commercially available sputter tool available from CVC, Novellus, or MRC.

[0235] Where layers are deposited during the processes of the invention, in some cases, the layers are treated before moving on to the next step in the process. For example, the deposited layer can be annealed, planarized, cleaned, passivated, or lightly etched in order to improve its properties.

[0236] In some methods of the invention, protective layers or sacrificial layers are deposited. The protective layers can be polymeric layers, or can be inorganic layers. Suitable protective or sacrificial layers include germanium (Ge) and amorphous silicon (a-Si). Protective layers can be used to produce features as described herein. The type of material for the protective or sacrificial layer can be chosen for its selective reactivity, for example to wet chemical etchants. For example, in some cases, the ability to selectively etch germanium with heated hydrogen peroxide in the presence of silicon dioxide and aluminum results in its being utilized to produce optical structures combined with nanowells.

[0237] In some processes, a pull-back process can be employed. A pull-back process generally involves etching in from the edges of a feature within a layer in order to reduce thedimensions of the feature. Pull-back can be performed using a wet chemical reagent that selectively reacts with a layer which has exposed edges. In some cases a germanium layer is pulled back using hydrogen peroxide.

[0238] Some methods employ a polishing step. Suitable methods include chemicalmechanical polishing or chemical-mechanical planarization (CMP).

[0239] Some methods of the invention incorporate a planarization layer. The method for depositing the planarization layer depends on the type of material that is used. The planarization layer can be a hard material, such as an inorganic material, for example silicon nitride; it can be a metallic material such as aluminum; or it can be a soft material, such as a polymeric material, e.g. an organic or silicon based polymer. The planarization layer can be a glass, such as a silicon dioxide material. In some cases, the planarization layer comprises a spin-on glass such as a silicate, phosphosilicate or siloxane material. Suitable spin-on glass materials are available, for example, from Honeywell Corporation. The planarization layer can comprise, for example, a glass doped with other agents to control its melting properties, such as boro-phosphoro-silicate glass (BPSG). Suitable polymeric planarization materials include, for example, polyimides.

[0240] Layers of group 4-5 transition metal oxides can be formed using known semiconductor processing methods. In some cases, the layer of group 4-5 transition metal oxide, e.g hafnium oxide, is formed using chemical vapor deposition (CVD). In some cases, the layer of group 4-5 transition metal oxide, e.g hafnium oxide, is formed using atomic layer deposition (ALD). The group 4-5 transition metal oxides can be deposited with sputtering or with ion assisted deposition (IAD).

[0241] The steps that relate to creating the nanoscale wells of the invention are typically some of the later steps in the process, since these layers are on top of the device. In some cases, group 4-5 transition metal oxides layers are used as etch-stop layers for controlling the depth of the nanoscale well that is produced.

[0242] The application of the surface modifying materials is sometimes carried out in a separate facility than in the FAB that produces the rest of the analytical device by semiconductor processing techniques. The surface modifying agents can be applied to the wafer before it is diced into chips or after dicing. Typically the surface modifying agents are applied to the wafer prior to being diced into chips.

[0243] After the arrays of the instant disclosure are complete, the arrays can be further processed, such as, for example, by separating the arrays into individual chips (dicing) and readying them for sequencing. The further processing steps will depend on the situation but can include the following treatments: stacking (a process to protect the top surface of the surface-treated device wafer and, in some cases, creating a well for the sequencing mixture); thinning (a process in which the composite top-plated and surface-treated device wafer can be thinned — including grinding lapping, polishing, or other treatments); dicing (a process in which the composite wafer is divided into individual chips using a standard semiconductor dicing saw); and packaging (a process involving a standard pick and place tool to mount the chips onto a carrier and create electrical / optical outputs for data collection). These further processing steps are either known in the art or are disclosed in references such as U.S. Patent Application Publication Nos. 2008 / 0176769 and 2011 / 0183409, which are incorporated by reference herein in their entireties for all purposes.

[0244] As just noted, the arrays of the invention can be incorporated into analysis systems for analyzing the multiple reactions occurring in the nanoscale wells of the array. The arrays described herein typically have nanoscale wells that are accessible to fluid from the top, and that are accessible for optical analysis from the bottom. The arrays are thus generally incorporated into a vessel into which a reaction mixture of interest is introduced. In some cases, the individual nanowells are all in contact with one volume of fluid, which can have, for example, multiple nucleic acid template molecules which can be analyzed, and which can have the nucleotides, cofactors, and other additives for carrying out the reaction to be analyzed.

[0245] The array can be placed within an instrument which has the appropriate optical components, computer controls, and data analysis systems. The array can be held within the instrument such that the reaction conditions, such as the vessel temperature and vessel atmospheric conditions, can be controlled. The vessel atmospheric conditions can comprise the makeup of the gas above the sample, for example the humidity and the level of other gaseous species such as oxygen.

[0246] The multiplexed optical chips of the instant disclosure typically comprise a plurality of optical waveguides, the optical waveguides configured to receive an optical excitation beam from the at least one input optical coupler. For example, a multiplexed optical chip can comprise at least 2, at least 5, at least 10, at least 50, at least 100, at least 500, at least 1,000, atleast 5,000, at least 10,000, or at least 50,000 optical waveguides. In some embodiments, the chip can comprise no more than 100,000, no more than 50,000, no more than 10,000, no more than 5,000, no more than 1,000, no more than 500, or no more than 100 optical waveguides. In other embodiments, the chip can comprise from 1 to 100,000, from 100 to 10,000, or from 500 to 5,000 optical waveguides. Suitable waveguides are described, for example, in U.S. Patent Applications 2008 / 0128627 and 2010 / 0065726 which are incorporated by reference herein in their entirety for all purposes.

[0247] The optical waveguides of the instant invention typically comprise a core of relatively high refractive index material surrounded by a cladding of a lower reflective index material. Typically channel waveguides are used. In some cases planar waveguides are used. The material for the cladding is typically silicon dioxide. The material for the core can be any suitable material. In some cases, the waveguide core comprises silicon nitride. In some cases, the waveguide core material comprises a group 4-5 transition metal oxide, for example, hafnium oxide. In some cases the nanoscale well extends down such that the bottom of the nanoscale well is close to the top of the core of the optical waveguide. Where the waveguide core comprises a group 4-5 transition metal oxide, the core can extend down such that the top of the waveguide core forms the bottom of the nanoscale well.Arrays of analytic devices and their uses

[0248] The selectively functionalized substrates of the invention can employed to produce arrays of analytic or analytical devices. In various respects, the terms “analytic device”, “analytical device” and “integrated analytical device” refer to components that are functionally connected to carry out an analysis. A multiplexed sequencing chip therefore comprises an array of the above-described integrated analytical devices. In some cases, these arrays of analytic devices are referred to as chips, especially where a significant portion of the devices is produced by semiconductor processing methods. In some cases, the term analytic device as used herein refers to one unit cell comprising, for example a single nanoscale well, a detector and associated optical elements. In some cases, the term analytic device is used herein to refer to a device containing a plurality or an array of nanoscale wells and associated detectors and optical elements, for example, the analytical chip that is placed in the instrument. Those of skill in the art will understand the meaning from the context in which the term used.

[0249] The arrays can be used for any suitable analysis that utilizes the molecules of interest selectively bound to specific regions, for example bottoms of nanoscale wells. In some cases, the devices are used for the analysis of biomolecules. The devices can be microarrays, for example, nucleic acid microarrays, protein microarrays, peptide microarrays, glycan microarrays, antibody microarrays, and aptamer microarrays. See, for example, Aparna et al. Recent Progress in Development and Application of DNA, Protein, Peptide, Glycan, Antibody, and Aptamer Microarrays, Biomolecules 2023, 13(4), 602, which is incorporated herein by reference for all purposes. The analytic devices can be used for protein sequencing, for example single molecule protein / peptide sequencing, especially approaches based on Edman degradation imaging; see, for example:, Floyd et al. Annu Rev Biophys. 2022 May 9; 51 : 181-200, Callahan et al. Trends Biochem Sci. 2020 Jan; 45(1): 76-89, U.S. Patent Application 20200209256, and U.S. Patent Application 2020 / 0219590, each of which is incorporated by reference herein in their entirety for all purposes.

[0250] In preferred embodiments, the instant invention is directed to observing nucleic acid sequencing reactions, e.g., sequencing-by-incorporation reactions or sequencing-by-binding reactions.

[0251] In some embodiments, such an illuminated reaction analyzes a single molecule to generate nucleotide sequence data pertaining to that single molecule. For example, a single nucleic acid template may be subjected to a sequencing-by-incorporation reaction to generate one or more sequence reads corresponding to the nucleotide sequence of the nucleic acid template. For a detailed discussion of such single molecule sequencing, see, e.g., U.S. Patent Nos. 6,056,661, 6,917,726, 7,033,764, 7,052,847, 7,056,676, 7,170,050, 7,361,466, 7,416,844; Published U.S. Patent Application Nos. 2007-0134128 and 2003 / 0044781; and M.J. Levene, J. Korlach, S.W. Turner, M. Foquet, H.G. Craighead, W.W. Webb, SCIENCE 299:682-686, January 2003 Zero-Mode Waveguides for Single-Molecule Analysis at High Concentrations, all of which are incorporated herein by reference in their entireties for all purposes.

[0252] A template / DNA polymerase-primer complex of such a sequencing system is provided, typically immobilized, within a nanoscale well. Preferably, such reaction cells are arrayed in large numbers upon a substrate in order to achieve the scale necessary for genomic or other large-scale DNA sequencing approaches.

[0253] For nucleotide sequencing, typically, in order to enhance the sequencing throughput of the system, multiple polymerase-enzyme complexes are typically independently monitored,where each complex is sequencing a separate template sequence. In the case of genomic sequencing or sequencing of other large DNA components, these templates will typically comprise overlapping fragments of the genomic DNA. By sequencing each fragment, one can then assemble a contiguous sequence from the overlapping sequence data from the fragments.

[0254] In certain aspects, the subject invention provides substrates and methods for performing single-molecule observation. The optical arrays of the invention can provide information on individual molecules whose properties are hidden in the statistical mean that is recorded by ordinary ensemble measurement techniques. In addition, because of multiplexing, the arrays are conducive to high-throughput implementation, requiring small amounts of reagent(s), and taking advantage of the high bandwidth of modern avalanche photodiodes for extremely rapid data collection. Moreover, because single-molecule counting automatically generates a degree of immunity to illumination and light collection fluctuations, single-molecule analysis can provide greater accuracy in measuring quantities of material than bulk fluorescence or light-scattering techniques. As such, the subject substrates and devices may be used in a wide variety of circumstances including sequencing individual human genomes as part of preventive medicine, rapid hypothesis testing for genotype-phenotype associations, in vitro and in situ gene-expression profiling at all stages in the development of a multi-cellular organism, and determining comprehensive mutation sets for individual clones and profiling in various diseases or disease stages. Other applications involve profiling of cell receptor diversity, identifying known and new pathogens, and exploring diversity towards agricultural, environmental and therapeutic goals.

[0255] The analysis typically uses the molecule or molecules of interest that are attached to the bottoms of the nanoscale wells. In some cases, the molecules of interest are detected directly, e.g. by detecting fluorescent moieties on the molecules of interest. In other cases, the analysis method detects molecules that interact with the attached molecules of interest (analytes). For example, a protein or peptide molecule of interest can interact with labeled peptide or amino acid recognizing molecule such as labeled antibodies or labeled aptamers that have a specific affinity for an amino acid in the attached protein or peptide. Recognition of peptides and amino acids is described, for example in US 2020 / 0209256 which is incorporated by reference herein for all purposes. In some cases, an attached enzyme can interact with labeled substrates or other components to provide information about the substrates or enzyme.

[0256] Arrays of analytic devices typically comprise arrays of integrated analytical devices, each having a nanoscale well and the components required for illuminating the nanoscale well and detecting light emitted from it. An embodiment of an analytical device is shown in the unit cell illustrated in Fig. 10. The term unit cell is used here to refer to a section of the chip that is repeated to form the array of analytic devices. The nanoscale well 1000 extends through aluminum layer 1040, sidewall forming layer 1030, down to a group 4-5 transition metal oxide layer 1050 which forms the bottom of the well. The sidewalls and top of the device are coated with silicon dioxide 1020.

[0257] The exemplary device includes from top to bottom, in the following order, a nanoscale well, a waveguide, a third aperture element, a diffractive beam shaping element or lens, a second aperture element, a color filtration layer, a laser rejection filter layer, a first aperture element, and a detector layer, all of which are on a base substrate. The diffractive beam shaping element in this embodiment of the nanoscale integrated analytical device would direct light emitted from the nanoscale well in directions perpendicular to the waveguide.

[0258] Fig. 11 A illustrates an embodiment of a nanoscale well of the invention in a chip having an optical waveguide below the nanoscale well for providing excitation illumination. The elements are shown in cross-section. The nanoscale well 1100 extends through the aluminum layer 1150 and the silicon dioxide well forming layer 1130 to the hafnium oxide layer 1120. The hafnium oxide acts as an etch-stop layer for controlling the etch depth in forming the nanoscale well. A surface layer of 1160 of silicon dioxide covers the sidewalls of the nanoscale well and the top of the chip. The surfaces formed in this way can be selectively functionalized as described herein to localize the molecules of interest to the bottoms of the nanoscale well. Below the nanoscale well 1100 is an optical waveguide 1190 formed from silicon nitride. The optical waveguide 1190 extends in and out of the page for the illumination of multiple nanoscale wells (e g. hundreds or thousands of nanoscale wells) in a row above the optical waveguide as part of an analysis device. In some cases, other optical elements such as apertures, lenses, filters, and photodetectors are integrated into the analysis device below the nanoscale wells.

[0259] Fig. 1 IB illustrates an embodiment of a nanoscale well of the invention in a chip having an optical waveguide below the nanoscale well for providing excitation illumination. The elements are shown in cross-section. The nanoscale well 1101 extends through the aluminum layer 1151 and the silicon dioxide well forming layer 1131 to the hafnium oxidelayer 1121. The hafnium oxide acts as an etch-stop layer for controlling the etch depth in forming the nanoscale well. The sidewalls of the nanoscale well are silicon dioxide, the material of the well-forming layer 1131. On the top of the chip is an aluminum oxide layer, which is a native aluminum oxide layer on the aluminum layer 1151. The surfaces formed in this way can be selectively functionalized as described herein to localize the molecules of interest to the bottoms of the nanoscale well. Below the nanoscale well 1101 is an optical waveguide core 1191 formed from silicon nitride. The optical waveguide 1191 extends in and out of the page for the illumination of multiple nanoscale wells (e.g. hundreds or thousands of nanoscale wells) in a row above the optical waveguide as part of an analysis device. In some cases, other optical elements such as apertures, lenses, filters, and photodetectors are integrated into the analysis device below the nanoscale wells.

[0260] Examples of integrated systems comprising arrays of optical analytical devices are also provided in U.S. Patent Application Publication Nos. 2012 / 0014837; 2012 / 0019828; and 2012 / 0021525.

[0261] Arrays of integrated analytical devices, such as arrays of devices comprising nanoscale wells, can be fabricated at ultra-high density, providing anywhere from 1000 ZMWs per cm2, to 1,000,000 ZMWs per cm2, or more. Thus, at any given time, it can be possible to analyze the reactions occurring in from 100, 1000, 3000, 5000, 10,000, 20,000, 50,000, 100,000, 1 million, 10 million, 25 million, 50 million, 100 million, or even more nanoscale emission volumes or other reaction regions within a single analytical system or even on a single substrate.Instruments and systems

[0262] In various respects, “analytical system”, or “instrument” refers to a larger system including the array of integrated analytical devices (e.g. sequencing chip) and other components for performing an analysis operation. The sequencing chip is preferably removably coupled into the instrument. Liquid samples and / or reagents can be brought into contact with the sequencing chip before or after the sequencing chip is coupled with the system. The system can provide electronic signals and / or illumination light to the sequencing chip and can receive electronic signals from the detectors or other electronic components in the sequencing chip. In some cases, for example where the chip does not include integrated detectors, the instrument can receive optical signals from the chip. The system can alsoprovide mechanical support for and / or thermal exchange with the sequencing chip. The instrument or system can have computers to manipulate, store, and analyze the data from the sequencing chip. For example, the instrument can have the capability of identifying the order of added nucleotide analogs in a nucleic acid sequencing reaction. The identification can be carried out, for example, as described in U.S. Pat. No. 8,182,993, which is incorporated herein by reference for all purposes.

[0263] The systems and instruments of the invention typically have an optical source to provide illumination light to the chip. Optical sources that emit in the visible wavelength range are particularly useful for the analytical systems of the present disclosure, for example, optical sources that emit between 450 nm and 700 nm or from 500 nm to 650 nm. In some embodiments, the instant systems can include more than one optical source.

[0264] In preferred embodiments, the optical source is a laser source. Any suitable type of laser can be used for the instant systems. In some cases, solid state lasers are used, for example, III-V semiconductor lasers. Solid state lasers that emit in the desired wavelength range can be used. Lasers suitable for use in the disclosed systems, including GalnN lasers, are described, for example in Sizov et al., “Gallium Indium Nitride-Based Green Lasers,” J. Lightwave Technol., 30, 679-699 (Mar. 1, 2012), Nakamura, et al. “Current Status and Future Prospects of InGaN-Based Laser Diodes”, JSAP Int. No. 1, January, 2000, Jeong et al. Nature, Scientific Reports, “Indium gallium nitride-based ultraviolet, blue, and green light emitting diodes functionalized with shallow periodic hole patterns”, DOI: 10.1038, and Tagaki et al., “High-Power and High-Efficiency True Green Laser Diodes”, SEI Tech Rev, No. 77, October 2013; which are each incorporated by reference herein for all purposes in their entireties.

[0265] In some embodiments, the optical source is a light emitting diode, for example a superluminescent light emitting diode. In some embodiments, the optical source is a verticalcavity surface-emitting laser (VCSEL), or other comparable optical device. Where the optical source is a small format, modular device, such as a light emitting diode or the like, the source itself can also be considered to be an optical delivery device for purposes of the instant analytical optical systems.

[0266] In some embodiments, the systems of the instant disclosure further comprise a computer that receives at least one electronic signal from an optical detector, or region of an optical detector, for example the detected signals described above, and that analyzes the atleast one electronic signal. More specifically, the analysis performed by the computer can comprise obtaining nucleic acid sequencing information from the electronic signal.

[0267] It is to be understood that the above description is intended to be illustrative and not restrictive. It readily should be apparent to one skilled in the art that various embodiments and-modifi cations may be made to the invention disclosed in this application without departing from the scope and spirit of the invention. The scope of the invention should, therefore, be determined not with reference to the above description, but should instead be determined with reference to the appended claims, along with the full scope of equivalents to which such claims are entitled. All publications mentioned herein are cited for the purpose of describing and disclosing reagents, methodologies and concepts that may be used in connection with the present invention. Nothing herein is to be construed as an admission that these references are prior art in relation to the inventions described herein. Throughout the disclosure various patents, patent applications and publications are referenced. Unless otherwise indicated, each is incorporated by reference in its entirety for all purposes.

Claims

What is claimed is:1 A substrate comprising a plurality of nanoscale wells disposed through a top surface of the substrate, wherein each nanoscale well comprises a sidewall surface that comprises silicon dioxide or aluminum oxide and a bottom surface that comprises a group 4-5 transition metal oxide.

2. The substrate of claim 1, wherein the group 4-5 transition metal oxide is hafnium oxide.

3. The substrate of claim 1, wherein the group 4-5 transition metal oxide is selected from the group consisting of titanium oxide, zirconium oxide, niobium oxide, and tantalum oxide.

4. The substrate of claim 1, further comprising a bottom surface modifying agent that is selectively associated with the bottom surface and that comprises a coupling group.

5. The substrate of claim 4, further comprising a sidewall modifying agent that is associated with the sidewall surface and that passivates the sidewall.

6. The substrate of claim 1, wherein the bottom surface comprises a bottom surface modifying agent comprising one or more phosphonic acid groups, one or more catechol groups, one or more phosphate groups, or a combination thereof.

7. The substrate of claim 1, wherein the bottom surface comprises a bottom surface modifying agent comprising one or more phosphonic acid groups and a coupling group.

8. The substrate of claim 7, wherein the bottom surface modifying agent comprises a biotin- PEG-phosphonic acid.

9. The substrate of claim 1, wherein the bottom surface comprises a bottom surface modifying agent comprising one or more catechol groups and a coupling group.

10. The substrate of claim 9, wherein the bottom surface modifying agent is a biotin-PEG- catechol.

11. The substrate of claim 1, wherein the bottom surface comprises a first bottom surface modifying agent that comprises a coupling group and a second bottom surface modifying agent that lacks the coupling group.

12. The substrate of claim 1, wherein the sidewall surface comprises silicon dioxide, further comprising a silane on the silicon dioxide.

13. The substrate of claim 1, wherein the sidewall surface comprises silicon dioxide, further comprising a PEG-silane on the silicon dioxide.

14. The substrate of claim 1, wherein the bottom surface comprises a coupling group bound thereto, wherein the coupling group is selected from the group consisting of biotin, a biotin-binding protein, an antibody, an antibody fragment, a lectin, and a nucleic acid.

15. The substrate of claim 1, wherein the bottom surface comprises a coupling group bound thereto, wherein the coupling group is selected from the group consisting of an amine group, a carboxyl group, a hydroxyl group, a sulfhydryl group, an azido group, an alkyne group, a cyclooctyne group, a dibenzocyclooctyne (DBCO) group, a trans-cyclooctene (TCO) group, a norbomene group, a tetrazine group, an epoxy group, a glycidyl group, an aldehyde group, an alkenyl group, an acrylate group , a methacrylate group, an anhydride group, an activated carboxylic acid group, and an n-hydroxysuccinimide (NHS) ester group.

16. The substrate of claim 1, wherein the bottom surface comprises a coupling group bound thereto, wherein density of the coupling group on the bottom surface is 100 times or more greater than density of the coupling group on the sidewall surface or the top surface.

17. The substrate of claim 1, wherein the bottom surface comprises a coupling group bound thereto, wherein density of the coupling group on the bottom surface is 1000 times or more greater than density of the coupling group on the sidewall surface or the top surface.

18. The substrate of claim 1, wherein the bottom surface comprises an immobilized molecule or molecules of interest each bound to a coupling group, wherein density of the molecules of interest on the bottom surface is 100 times or more greater than density of the molecules of interest on the sidewall surface and the top surface.

19. The substrate of claim 18, wherein the molecule or molecules of interest comprise a polymerase-template complexes.

20. The substrate of claim 1, wherein the bottom surface comprises an immobilized molecule or molecules of interest, wherein density of the molecules of interest on the bottom surface is 1000times or more greater than density of the molecules of interest on the sidewall surface or the top surface.

21. The substrate of claim 20, wherein molecule or molecules of interest comprise polymerase- template complexes.

22. The substrate of claim 1, wherein the substrate comprises a layer of silicon dioxide overlying a layer of group 4-5 transition metal oxide, wherein the nanoscale wells are nanoscale apertures that penetrate through the layer of silicon dioxide to the layer of group 4-5 transition metal oxide, whereby the base of the nanoscale well comprises the group 4-5 transition metal oxide.

23. The substrate of claim 1, wherein the top surface comprises silicon dioxide.

24. The substrate of claim 1, wherein the top surface comprises aluminum oxide.

25. The substrate of claim 1, wherein the bottom surface of each well has an area less than196,000 nm2.

26. The substrate of claim 1, wherein the bottom surface of each well has an area less than 70,700 nm2.

27. The substrate of claim 1, wherein the bottom surface of the well is the top surface of an optical waveguide comprising a group 4-5 transition metal oxide.

28. An analytic device comprising: the substrate of claim 1; and one or more optical waveguides disposed within the substrate below the plurality of nanoscale wells, each optical waveguide comprising a core; wherein the bottom surface of each nanoscale well is disposed sufficiently proximal to a core of at least one of the one or more optical waveguides to be illuminated by an evanescent field emanating from the core when optical energy is passed through the core.

29. The analytic device of claim 28, wherein the bottom surface of the each nanoscale well comprises a single optically resolvable immobilized molecule of interest.

30. The analytic device of claim 29 wherein the immobilized molecule of interest comprises a polymerase-template complex.

31. The analytic device of claim 29 wherein the immobilized molecule of interest comprises a protein or a peptide.

32. The analytic device of claim 28 further comprising an integrated optical detector disposed below the one or more optical waveguides.

33. The analytic device of claim 32 further comprising one or more integrated optical element below the one or more optical waveguides and above the optical detector.

34. The analytic device of claim 33 wherein the one or more integrated optical element comprises a diffractive optical element, lens, filter, or aperture.

35. An analytic device comprising: a) a substrate comprising i) at least one optical waveguide and ii) at least one nanoscale well that is disposed through a top surface of the substrate and that penetrates into a top of the substrate and extends toward an optical waveguide core of the optical waveguide, wherein the nanoscale well comprises a sidewall surface that comprises silicon dioxide or aluminum oxide and a bottom surface that comprises a group 4-5 transition metal oxide; and b) a detector disposed within the substrate below the at least one nanoscale well to receive photons emitted from within the nanoscale well.

36. The analytic device of claim 35, wherein a single optically resolvable molecule of interest is bound to the bottom surface of the at least one nanoscale well and is sufficiently proximal to the optical waveguide core to be illuminated by an evanescent field emanating from the optical waveguide core when optical energy is passed through the optical waveguide.

37. The analytic device of claim 35, wherein the at least one nanoscale well comprises a plurality of nanoscale wells.

38. The analytic device of claim 37, further comprising one or more integrated optical elements below the one or more optical waveguides and above the detector.

39. The analytic device of claim 38, wherein the one or more integrated optical element comprises a diffractive optical element, lens, fdter, or aperture.

40. The analytic device of claim 35, wherein the group 4-5 transition metal oxide is hafnium oxide.

41. The analytic device of claim 35, wherein the group 4-5 transition metal oxide is selected from the group consisting of titanium oxide, zirconium oxide, niobium oxide, and tantalum oxide.

42. The analytic device of claim 35, comprising a bottom surface modifying agent that is selectively associated with the bottom surface and that comprises a coupling group.

43. The analytic device of claim 42, comprising a sidewall surface modifying agent that is associated with the sidewall surface.

44. The analytic device of claim 35, wherein the bottom surface comprises a bottom surface modifying agent comprising one or more phosphonic acid groups, one or more catechol groups, one or more phosphate groups, or a combination thereof.

45. The analytic device of claim 35, wherein the bottom surface comprises a bottom surface modifying agent comprising one or more phosphonic acid groups and a coupling group.

46. The analytic device of claim 45, wherein the bottom surface modifying agent comprises a biotin-PEG-phosphonic acid.

47. The analytic device of claim 35, wherein the bottom surface comprises a bottom surface modifying agent comprising one or more catechol groups and a coupling group.

48. The substrate of claim 47, wherein the bottom surface modifying agent comprises a biotin- PEG-catechoL49. The analytic device of claim 35, wherein the bottom surface comprises a first bottom surface modifying agent that comprises a coupling group and a second bottom surface modifying agent that lacks the coupling group.

50. The analytic device of claim 35, wherein the sidewall surface comprises silicon dioxide, further comprising a silane coating on the silicon dioxide.

51. The analytic device of claim 35, wherein the sidewall surface comprises silicon dioxide, further comprising a PEG-silane coating on the silicon dioxide.

52. The analytic device of claim 35, wherein the bottom surface comprises a coupling group bound thereto, wherein the coupling group is selected from the group consisting of biotin, a biotinbinding protein, an antibody, an antibody fragment, a lectin, and a nucleic acid.

53. The analytic device of claim 35, wherein the bottom surface comprises a coupling group bound thereto, wherein the coupling group is selected from the group consisting of an amine group, a carboxyl group, a hydroxyl group, a sulfhydryl group, an azido group, an alkyne group, a cyclooctyne group, a dibenzocyclooctyne (DBCO) group, a trans-cyclooctene (TCO) group, a norbomene group, a tetrazine group, an epoxy group, a glycidyl group, an aldehyde group, an alkenyl group, an acrylate group , a methacrylate group, an anhydride group, an activated carboxylic acid group, and an n-hydroxysuccinimide (NHS) ester group.

54. The analytic device of claim 35, wherein the bottom surface comprises a coupling group bound thereto, wherein density of the coupling group on the bottom surface is 100 times or more greater than density of the coupling group on the sidewall surface and the top surface.

55. The analytic device of claim 35, wherein the bottom surface comprises a coupling group bound thereto, wherein density of the coupling group on the bottom surface is 1000 times or more greater than density of the coupling group on the sidewall surface and the top surface.

56. The analytic device of claim 35, wherein the bottom surface comprises an immobilized molecule or molecules of interest, wherein density of the molecule or molecules of interest on the bottom surface is 100 times or more greater than density of the molecules of interest on the sidewall surface and the top surface.

57. The analytical device of claim 56 wherein the molecule or molecules of interest comprise immobilized polymerase-template complexes.

58. The analytical device of claim 56 wherein the molecule or molecules of interest comprise immobilized proteins or peptides.

59. The analytic device of claim 35, wherein the bottom surface comprises an immobilized molecule or molecules of interest, wherein density of the molecule or molecules of interest on thebottom surface is 1000 times or more greater than density of the molecules of interest on the sidewall surface and the top surface.

60. The analytical device of claim 59 wherein the molecule or molecules of interest comprise immobilized polymerase-template complexes.

61. The analytic device of claim 35, wherein the substrate comprises a layer of silicon dioxide overlying a layer of group 4-5 transition metal oxide, wherein the at least one nanoscale well is a nanoscale aperture that penetrates through the layer of silicon dioxide to the layer of group 4-5 transition metal oxide.

62. The analytic device of claim 35, wherein the top surface comprises silicon dioxide.

63. The analytic device of claim 35, wherein the bottom surface of the at least one nanoscale well has an area less than 196,000 nm2.

64. The analytic device of claim 35, wherein the bottom surface of the at least one nanoscale well has an area less than 70,700 nm2.

65. The analytic device of claim 35, wherein the optical core comprises a group 4-5 transition metal oxide, and the at least one nanoscale well penetrates to the top of the of the optical core such that the top of the optical core forms the bottom of the nanoscale well.

66. A method of detecting a molecule of interest, comprising: a) providing an optical waveguide substrate comprising: i) an optical waveguide core surrounded by a lower refractive index cladding including a optical waveguide upper cladding above the core, and ii) a nanoscale well that penetrates into the optical waveguide upper cladding and extends toward the optical waveguide core, the nanoscale well comprising a molecule of interest on its bottom surface, the bottom surface disposed sufficiently proximal to the optical waveguide core to be illuminated by an evanescent field emanating from the optical waveguide core when optical energy is passed through the optical waveguide core, wherein the bottom surface of the nanoscale well comprises a single optically resolvable immobilized molecule of interest,wherein the nanoscale well comprises a sidewall surface that comprises silicon dioxide or aluminum oxide, and a bottom surface that comprises a group 4-5 transition metal oxide; b) exposing the substrate to fluid comprising an analyte comprising a fluorescent or fluorogenic moiety that emits a signal in response to the evanescent field; c) directing optical energy through the optical waveguide core, thereby generating the evanescent field and illuminating the single optically resolvable immobilized molecule of interest; and d) detecting a signal from the analyte upon interaction of the analyte with the single optically resolvable immobilized molecule of interest.

67. The method of claim 66, wherein the optical waveguide substrate comprises two or more optical waveguides and a plurality of nanoscale wells, the bottom surface of each nanoscale well disposed sufficiently proximal to an optical waveguide core of one of the two or more optical waveguides to be illuminated by an evanescent field emanating from the optical waveguide core when optical energy is passed through the optical waveguide core.

68. The method of claim 66, wherein the detecting a signal from the analyte comprises disposing a detector within the substrate below the optical waveguide cores; and, during the directing optical energy of c), detecting fluorescent or fluorogenic emissions from the analyte.

69. The method of claim 66, wherein the single optically resolvable immobilized molecule of interest comprises an enzyme.

70. The method of claim 69, wherein the enzyme is a polymerase.

71. The method of claim 66, wherein the analyte comprises a nucleotide analog.

72. The method of claim 66, wherein the single optically resolvable immobilized molecule of interest comprises a polypeptide and the analyte comprises an amino acid recognition molecule.

73. The method of claim 66, wherein the group 4-5 transition metal oxide is hafnium oxide.

74. The method of claim 66, wherein the group 4-5 transition metal oxide is selected from the group consisting of titanium oxide, zirconium oxide, niobium oxide, and tantalum oxide.

75. The method of claim 66, further comprising a bottom surface modifying agent that is selectively associated with the bottom surface and that comprises a coupling group.

76. The method of claim 75, further comprising a sidewall surface modifying agent that is associated with the sidewall surface and that passivates the sidewall.

77. The method of claim 66, wherein the bottom surface comprises a bottom surface modifying agent comprising one or more phosphonic acid groups, one or more catechol groups, one or more phosphate groups, or a combination thereof.

78. The method of claim 66, wherein the bottom surface comprises a bottom surface modifying agent comprising one or more phosphonic acid groups and a coupling group.

79. The method of claim 78, wherein the bottom surface modifying agent comprises a biotin- PEG-phosphonic acid.

80. The method of claim 66, wherein the bottom surface comprises a bottom surface modifying agent comprising one or more catechol groups and a coupling group.

81. The method of claim 80, wherein the bottom surface modifying agent comprises a biotin- PEG-catechol.

82. The method of claim 66, wherein the bottom surface comprises a first bottom surface modifying agent that comprises a coupling group and a second bottom surface modifying agent that lacks the coupling group.

83. The method of claim 66, wherein the sidewall surface comprises silicon dioxide, further comprising a silane coating on the silicon dioxide.

84. The method of claim 66, wherein the sidewall surface comprises silicon dioxide, further comprising a PEG-silane coating on the silicon dioxide.

85. The method of claim 66, wherein the bottom surface comprises a coupling group bound thereto, wherein the coupling group is selected from the group consisting of biotin, a biotin-binding protein, an antibody, an antibody fragment, a lectin, and a nucleic acid.

86. The method of claim 66, wherein the bottom surface comprises a coupling group bound thereto, wherein the coupling group is selected from the group consisting of an amine group, a carboxyl group, a hydroxyl group, a sulfhydryl group, an azido group, an alkyne group, a cyclooctyne group, a dibenzocyclooctyne (DBCO) group, a trans-cyclooctene (TCO) group, a norbomene group, a tetrazine group, an epoxy group, a glycidyl group, an aldehyde group, an alkenyl group, an acrylate group , a methacrylate group, an anhydride group, an activated carboxylic acid group, and an n-hydroxysuccinimide (NHS) ester group.

87. The method of claim 66, wherein the substrate comprises a layer of silicon dioxide overlying a layer of group 4-5 transition metal oxide, wherein the nanoscale well is a nanoscale aperture that penetrates through the layer of silicon dioxide to the layer of group 4-5 transition metal oxide.

88. The method of claim 66, wherein the top surface comprises silicon dioxide.

89. The method of claim 66, wherein the top surface comprises aluminum oxide.

90. The method of claim 66, wherein the bottom surface of the well has an area less than196,000 nm2.

91. The method of claim 66, wherein the bottom surface of the well has an area less than 70,700 2 nm .

92. A method of performing sequencing-by-synthesis comprising: providing an analytic device comprising: a substrate comprising an optical waveguide and a nanoscale well disposed through a top surface of the substrate and extending toward an optical waveguide core of the optical waveguide, wherein the nanoscale well comprises a sidewall surface that comprises silicon dioxide or aluminum oxide and a bottom surface that comprises a group 4-5 transition metal oxide, and wherein the nanoscale well comprises a polymerase-template complex immobilized to the bottom surface of the well; providing illumination light into the optical waveguide core whereby the polymerase- template complex is illuminated by an evanescent field emanating from the optical waveguide core; providing fluorescently labeled nucleotide analogs; and monitoring stepwise addition of the fluorescently labeled nucleotide analogs as they are added to a nascent strand of the template.

93. The method of claim 92, wherein the group 4-5 transition metal oxide is hafnium oxide.

94. The method of claim 92, wherein the group 4-5 transition metal oxide is selected from the group consisting of titanium oxide, zirconium oxide, niobium oxide, and tantalum oxide.

95. The method of claim 92, comprising a bottom surface modifying agent that is selectively associated with the bottom surface and that comprises a coupling group.

96. The method of claim 95, comprising a sidewall surface modifying agent that is associated with the sidewall surface and that passivates the sidewall.

97. The method of claim 92, wherein the bottom surface comprises a bottom surface modifying agent comprising one or more phosphonic acid groups, one or more catechol groups, one or more phosphate groups, or a combination thereof.

98. The method of claim 92, wherein the bottom surface comprises a bottom surface modifying agent comprising one or more phosphonic acid groups and a coupling group.

99. The method of claim 98, wherein the bottom surface modifying agent comprises a biotin- PEG-phosphonic acid.

100. The method of claim 92, wherein the bottom surface comprises a bottom surface modifying agent comprising one or more catechol groups and a coupling group.

101. The method of claim 100, wherein the bottom surface modifying agent comprises a biotin- PEG-catechol.

102. The method of claim 92, wherein the bottom surface comprises a first bottom surface modifying agent that comprises a coupling group and a second bottom surface modifying agent that lacks the coupling group.

103. The method of claim 92, wherein the sidewall surface comprises silicon dioxide, further comprising a silane coating on the silicon dioxide.

104. The method of claim 92, wherein the sidewall surface comprises silicon dioxide, further comprising a PEG-silane coating on the silicon dioxide.

105. The method of claim 92, wherein the bottom surface comprises a coupling group bound thereto, wherein the coupling group is selected from the group consisting of biotin, a biotin-binding protein, an antibody, an antibody fragment, a lectin, and a nucleic acid.

106. The method of claim 92, wherein the bottom surface comprises a coupling group bound thereto, wherein the coupling group is selected from the group consisting of an amine group, a carboxyl group, a hydroxyl group, a sulfhydryl group, an azido group, an alkyne group, a cyclooctyne group, a dibenzocyclooctyne (DBCO) group, a trans-cyclooctene (TCO) group, a norbomene group, a tetrazine group, an epoxy group, a glycidyl group, an aldehyde group, an alkenyl group, an acrylate group , a methacrylate group, an anhydride group, an activated carboxylic acid group, and an n-hydroxysuccinimide (NHS) ester group.

107. The method of claim 92, wherein the substrate comprises a layer of silicon dioxide overlying a layer of group 4-5 transition metal oxide, wherein the nanoscale well is a nanoscale aperture that penetrates through the layer of silicon dioxide to the layer of group 4-5 transition metal oxide.

108. The method of claim 92, wherein the top surface comprises silicon dioxide.

109. The method of claim 92, wherein the top surface comprises aluminum oxide.

110. The method of claim 92, wherein the bottom surface of the well has an area less than196,000 nm2.

111. The method of claim 92, wherein the bottom surface of the well has an area less than 70,700 2 nm112. A method of preparing a substrate for selective immobilization of a molecule or molecules of interest thereon, comprising: providing a substrate comprising a plurality of nanoscale wells disposed through a top surface of the substrate, wherein each nanoscale well comprises a sidewall surface that comprises silicon dioxide or aluminum oxide and a bottom surface that comprises a group 4-5 transition metal oxide; and contacting the substrate with a bottom surface modifying agent that comprises a coupling group and that selectively associates with the group 4-5 transition metal oxide, thereby selectively locating the coupling group on the bottom surface of the nanoscale wells.

113. The method of claim 112, wherein the bottom surface modifying agent comprises one or more phosphonic acid groups, one or more catechol groups, one or more phosphate groups, or a combination thereof.

114. The method of claim 112, wherein the bottom surface modifying agent comprises a biotin- PEG-phosphonic acid.

115. The method of claim 112, wherein the bottom surface modifying agent comprises a biotin- PEG-catechol.

116. The method of claim 112, wherein the sidewall comprises silicon dioxide, comprising contacting the substrate with a sidewall surface modifying agent that associates with and passivates the silicon dioxide.

117. The method of claim 112, wherein the sidewall comprises silicon dioxide, comprising contacting the substrate with a sidewall surface modifying agent that comprises one or more silane groups and that associates with and passivates the silicon dioxide.

118. The method of claim 117, wherein the sidewall surface modifying agent comprises a PEG- silane.

119. The method of claim 117, wherein the substrate is contacted with the bottom surface modifying agent and then contacted with the sidewall surface modifying agent.

120. The method of claim 119, comprising, after contacting the substrate with the sidewall surface modifying agent, contacting the substrate with a removal compound that preferentially removes the sidewall surface modifying agent from the group 4-5 transition metal oxide.

121. The method of claim 117, wherein the substrate is contacted with the sidewall surface modifying agent and then contacted with the bottom surface modifying agent.

122. The method of claim 112, comprising coupling the molecule or molecules of interest to the coupling group, thereby selectively immobilizing the molecule or molecules of interest on the bottom surface of the nanoscale wells.

123. The method of claim 112, wherein the group 4-5 transition metal oxide is hafnium oxide.

124. The method of claim 112, wherein the group 4-5 transition metal oxide is selected from the group consisting of titanium oxide, zirconium oxide, niobium oxide, and tantalum oxide.

125. The method of claim 112, wherein the coupling group is selected from the group consisting of biotin, a biotin-binding protein, an antibody, an antibody fragment, a lectin, and a nucleic acid.

126. The method of claim 112, wherein the coupling group is selected from the group consisting of an amine group, a carboxyl group, a hydroxyl group, a sulfhydryl group, an azido group, an alkyne group, a cyclooctyne group, a dibenzocyclooctyne (DBCO) group, a trans-cyclooctene (TCO) group, a norbornene group, a tetrazine group, an epoxy group, a glycidyl group, an aldehyde group, an alkenyl group, an acrylate group , a methacrylate group, an anhydride group, an activated carboxylic acid group, and an n-hydroxysuccinimide (NHS) ester group.

127. The method of claim 112, wherein the density of the coupling group on the bottom surface is 100 times or more greater than density of the coupling group on the sidewall surface and the top surface.

128. The method of claim 112, wherein the density of the coupling group on the bottom surface is 1000 times or more greater than density of the coupling group on the sidewall surface and the top surface.

129. The method of claim 112, wherein the substrate comprises a layer of silicon dioxide overlying a layer of metal oxide, wherein the nanoscale wells are nanoscale apertures that penetrate through the layer of silicon dioxide to the layer of group 4-5 transition metal oxide.

130. The method of claim 112, wherein the top surface comprises silicon dioxide.

131. The method of claim 112, wherein the top surface comprises aluminum oxide.

132. The method of claim 112, wherein the bottom surface of each well has an area less than196,000 nm2.

133. The method of claim 112, wherein the bottom surface of each well has an area less than 70,700 nm2.

134. A method for producing an array of analytic devices on a substrate, comprising:providing a substrate comprising an optical waveguide core layer that comprises one or more optical waveguide cores; depositing an optical waveguide upper cladding layer comprising silicon dioxide on the optical waveguide core layer; depositing a group 4-5 transition metal oxide on the optical waveguide upper cladding layer to form a group 4-5 transition metal oxide layer; depositing a silicon dioxide layer on the group 4-5 transition metal oxide layer to form a well forming layer; and patterning and etching to form an array of nanoscale apertures penetrating through the well forming layer to the group 4-5 transition metal oxide layer, thereby forming an array of nanoscale wells that each comprise a silicon dioxide sidewall surface and a group 4-5 transition metal oxide bottom surface.

135. The method of claim 134, wherein the group 4-5 transition metal oxide layer is an etch-stop layer to control the depth of the apertures.

136. The method of claim 134, wherein the optical waveguide core layer overlies a detector layer.

137. The method of claim 134, wherein the group 4-5 transition metal oxide is hafnium oxide.

138. The method of claim 134, wherein the group 4-5 transition metal oxide is selected from the group consisting of titanium oxide, zirconium oxide, niobium oxide, and tantalum oxide.

139. The method of claim 134, wherein the bottom surface of each well has an area less than 196,000 nm2.

140. The method of claim 134, wherein the bottom surface of each well has an area less than 70,700 nm2.

141. The method of claim 134, further comprising depositing a reflective layer on the well forming layer prior to patterning and etching the array of nanoscale apertures.

142. A substrate with selectively functionalized regions, the substrate comprising: a plurality of active regions that each comprise a group 4-5 transition metal oxide, each active region having a molecule or molecules of interest attached thereto; andsurrounding regions around the plurality of active regions, the surrounding regions comprising silicon dioxide or aluminum oxide.

143. The substrate of claim 142 wherein the molecule or molecule or molecules of interest are attached to the plurality of active regions through a coupling group on an active surface region surface modifying agent.

144. The substrate of claim 142 wherein the surrounding regions comprise a surrounding region surface modifying agents that passivates the surrounding regions.

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