# US 8,772,202 B2 · Articles having localized molecules disposed thereon and methods of producing same

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- Number: US 8,772,202 B2
- Document ID: US8772202B2
- Status: Granted
- Kind: B2
- Application number: 13/487,978
- Filed: 2012-06-04
- Published: 2012-11-29
- Granted: 2014-07-08
- Earliest claimed: 2006-03-30
- Assignees: Pacific Biosciences of California, Inc.
- Applicants: David R. Rank; Jeffrey Wegener; Jonas Korlach; Daniel Roitman; Yue Xu; John Lyle; Stephen Turner; Paul Peluso; Geoff Otto; Ronald L. Cicero
- Inventors: David R. Rank; Jeffrey Wegener; Jonas Korlach; Daniel Roitman; Yue Xu; John Lyle; Stephen Turner; Paul Peluso; Geoff Otto; Ronald L. Cicero
- Web page: https://patentgrep.com/patent/US8772202B2

## CPC

- C12Q 1/6874: involving nucleic acid arrays, e.g. sequencing by hybridisation
- B01J 19/0046: {Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays (synthesis methods per se C40B50/00)}
- B82Y 20/00: Nanooptics, e.g. quantum optics or photonic crystals
- B82Y 30/00: Nanotechnology for materials or surface science, e.g. nanocomposites
- C12Q 1/6837: using probe arrays or probe chips (C12Q1/6874 takes precedence)
- B01J 2219/00427: using masks
- B01J 2219/0045: using optical fibres
- B01J 2219/00605: the compounds being directly bound or immobilised to solid supports
- B01J 2219/0061: The surface being organic
- B01J 2219/00612: the surface being inorganic
- B01J 2219/00617: by chemical means
- B01J 2219/00626: Covalent
- B01J 2219/00635: by reactive plasma treatment
- B01J 2219/00637: by coating it with another layer
- B01J 2219/00639: the compounds being trapped in or bound to a porous medium
- B01J 2219/00659: Two-dimensional arrays
- B01J 2219/00711: Light-directed synthesis
- B01J 2219/00722: Nucleotides
- Y10T 29/49002: Electrical device making
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- B01J: CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B82Y: SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- Y10T: TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION

## Abstract

Methods of producing substrates having selected active chemical regions by employing elements of the substrates in assisting the localization of active chemical groups in desired regions of the substrate. The methods may include optical, chemical and/or mechanical processes for the deposition, removal, activation and/or deactivation of chemical groups in selected regions of the substrate to provide selective active regions of the substrate.

## Claims

### Claim 1

A method of preparing a substrate for selective immobilization of an enzyme thereon, comprising:
providing a substrate having a first surface component and a second surface component where the first and second surface components have different surface characteristics, wherein the substrate comprises a layer of the second surface component on a layer of the first surface component, and wherein the substrate comprises a zero mode waveguide disposed through the second surface component layer to the first surface component layer and exposing a portion of the first surface component layer, the portion of the first surface component layer thereby exposed forming a bottom reading surface of the zero mode waveguide; and
contacting the substrate with a composition that selectively associates with the second surface component, which composition comprises a compound comprising one or more phosphonic acid groups, thereby passivating the second surface component and preparing the substrate for selective immobilization of the enzyme atop the bottom reading surface in the zero mode waveguide.

### Claim 2 (depends on 1)

The method of claim 1, wherein the first surface component comprises SiO2 and the second surface component comprises a metal or metal oxide.

### Claim 3 (depends on 1)

The method of claim 1, wherein the differing surface characteristics comprise surface charge.

### Claim 4 (depends on 3)

The method of claim 3, wherein the first surface component has a negative surface charge and the second surface component has a positive surface charge.

### Claim 5 (depends on 1)

The method of claim 1, comprising contacting the substrate with a silane that associates with the first surface component.

### Claim 6 (depends on 5)

The method of claim 5, wherein the substrate is contacted with the composition that selectively associates with the second surface component and then contacted with the silane.

### Claim 7 (depends on 1)

The method of claim 1, comprising selectively coupling the enzyme atop the bottom reading surface in the zero mode waveguide.

### Claim 8 (depends on 6)

The method of claim 6, wherein selectively coupling the enzyme atop the bottom reading surface comprises contacting the substrate with poly(L-lysine)-poly(ethylene glycol) or poly(L-lysine)-poly(ethylene glycol)-biotin that associates with the first surface component and coupling the enzyme to the poly(L-lysine)-poly(ethylene glycol) or poly(L-lysine)-poly(ethylene glycol)-biotin.

### Claim 9 (depends on 1)

The method of claim 1, wherein the composition that selectively associates with the second surface component is polyvinylphosphonic acid.

### Claim 10 (depends on 1)

The method of claim 1, wherein the composition that selectively associates with the second surface component is selected from the group consisting of:

### Claim 11 (depends on 1)

The method of claim 1, wherein the composition that selectively associates with the second surface component is selected from the group consisting of: 2-carboxyethyl phosphonic acid; amino tri(methylene phosphonic acid); 1-hydroxyethylidene-1,1,-diphosphonic acid; hexamethylenediaminetetra(methylenephosphonic acid); diethylenetriamine penta(methylene phosphonic acid); ethylenediamine tetra(methylene phosphonic acid); bis(hexamethylene triamine penta(methylenephosphonic acid)); 2-phosphonobutane-1,2,4-tricarboxylic acid; and monoethanloamine diphosphonate.

### Claim 12 (depends on 1)

The method of claim 1, wherein the composition that selectively associates with the second surface component comprises an alkyl phosphonate.

### Claim 13 (depends on 1)

The method of claim 1, wherein the composition that selectively associates with the second surface component is selected from the group consisting of: octyl phosphonic acid, decyl phosphonic acid, dodecyl phosphonic acid, hexadecyl phosphonic acid, octadecyl phosphonic acid, docosyl phosphonic acid, hydroxy-dodecyl phosphonic acid, hydroxy-undecenyl-phosphonic acid, and decanediylbis(phosphonic acid).

### Claim 14 (depends on 1)

The method of claim 1, wherein the composition that selectively associates with the second surface component comprises a second coupling group.

### Claim 15 (depends on 7)

The method of claim 7, wherein the enzyme comprises a polymerase.

### Claim 16

A method of analyzing an enzyme that is selectively immobilized on a substrate, comprising:
a) selectively immobilizing the enzyme on the substrate by:
providing a substrate having a first surface component and a second surface component where the first and second surface components have different surface characteristics, wherein the substrate comprises a layer of the second surface component on a layer of the first surface component, and wherein the substrate comprises a zero mode waveguide disposed through the second surface component layer to the first surface component layer,
contacting the substrate with a composition that selectively associates with the second surface component, which composition comprises a compound comprising one or more phosphonic acid groups, and
selectively coupling the enzyme to the first surface component in the zero mode waveguide; and
b) after selectively immobilizing the enzyme on the substrate, providing illumination to and receiving electromagnetic radiation from an observation volume of the zero mode waveguide through the first surface component layer.

### Claim 17 (depends on 16)

The method of claim 16, wherein the first surface component comprises SiO2 and the second surface component comprises a metal or metal oxide.

### Claim 18 (depends on 16)

The method of claim 16, wherein selectively coupling the enzyme to the first surface component comprises contacting the substrate with a silane that associates with the first surface component and coupling the enzyme to the silane.

### Claim 19 (depends on 18)

The method of claim 18, wherein the silane comprises a first coupling group, and wherein selectively coupling the enzyme to the first surface component comprises coupling the enzyme to the first coupling group.

### Claim 20 (depends on 18)

The method of claim 18, wherein the substrate is contacted with the composition that selectively associates with the second surface component and then contacted with the silane.

### Claim 21 (depends on 16)

The method of claim 16, wherein the composition that selectively associates with the second surface component is polyvinylphosphonic acid.

### Claim 22 (depends on 16)

The method of claim 16, wherein the enzyme comprises a polymerase.