US-20260125746-A1 - METHODS AND COMPOSITIONS FOR SUBSTRATE SURFACE CHEMISTRY

Abstract

The present disclosure relates in some aspects to methods and compositions for generating modified surfaces for microfluidic device (e.g., flow cells) and for characterizing the surface chemistry of the modified surfaces, such as the relative density of biomolecules immobilized on the surfaces.

Inventors

• Aijie Han
• Wei Li
• Liangliang Qiang

Assignees

• ESBIOLAB, LLC

Dates

Publication Date

20260507

Application Date

20231004

Claims (20)

1. 1 . A method for density and/or quality characterization of surface-immobilized oligonucleotide molecules, comprising: a) contacting a flow cell substrate with a plurality of oligonucleotide molecules, wherein: the plurality of oligonucleotide molecules comprise fluorescently labeled oligonucleotide molecules that account for no more than 10% of the plurality of oligonucleotide molecules; b) allowing oligonucleotide molecule immobilization on a surface of the flow cell substrate; c) separating oligonucleotide molecules that are not immobilized on the surface from the flow cell substrate; and d) detecting signals associated with fluorescent labels of the fluorescently labeled oligonucleotide molecules among oligonucleotide molecules that are immobilized on the surface, thereby characterizing the density and/or quality of the oligonucleotide molecules immobilized on the surface of the flow cell substrate.
2. 2 - 5 . (canceled)
3. 6 . The method of claim 1 , wherein the flow cell substrate does not comprise biomolecules immobilized on the surface prior to the oligonucleotide molecule immobilization.
4. 7 . (canceled)
5. 8 . The method of claim 1 , wherein the flow cell substrate is pretreated with a silane prior to the oligonucleotide molecule immobilization.
6. 9 . The method of claim 1 , wherein the flow cell substrate comprises a hydrogel on the surface.
7. 10 . (canceled)
8. 11 . The method of claim 1 , wherein fluorescently labeled oligonucleotide molecules account for no more than 10% of the plurality of oligonucleotide molecules.
9. 12 . The method of claim 1 , wherein fluorescently labeled oligonucleotide molecules account for no more than 5% of the plurality of oligonucleotide molecules.
10. 13 . The method of claim 1 , wherein fluorescently labeled oligonucleotide molecules account for no more than 3% of the plurality of oligonucleotide molecules.
11. 14 . The method of claim 1 , wherein fluorescently labeled oligonucleotide molecules account for no more than 2% of the plurality of oligonucleotide molecules.
12. 15 . The method of claim 1 , wherein fluorescently labeled oligonucleotide molecules account for no more than 1% of the plurality of oligonucleotide molecules.
13. 16 . The method of claim 1 , wherein fluorescently labeled oligonucleotide molecules account for no more than 0.5% of the plurality of oligonucleotide molecules.
14. 17 - 19 . (canceled)
15. 20 . The method of claim 1 , wherein the plurality of oligonucleotide molecules comprise partial or full-length primer sequences.
16. 21 - 22 . (canceled)
17. 23 . The method of claim 1 , wherein for each fluorescently labeled oligonucleotide molecule, the fluorescent label and the oligonucleotide molecule are covalently or non-covalently conjugated.
18. 24 . (canceled)
19. 25 . The method of claim 23 , wherein the fluorescent label is covalently conjugated to the oligonucleotide molecule, and the fluorescent label is chemically or enzymatically cleavable.
20. 26 - 34 . (canceled)

Description

CROSS-REFERENCE TO RELATED APPLICATION This application claims the priority benefit of U.S. Provisional Patent Application Ser. No. 63/413,462, filed Oct. 5, 2022, the contents of which are incorporated herein by reference in its entirety. FIELD The present disclosure relates in some aspects to methods and compositions for manufacturing substrates (e.g., in microfluidic devices such as flow cells) having surface modifications, and/or for characterizing surface chemistry of manufactured substrates. Methods of using the manufactured and/or characterized substrates are also disclosed, e.g., for nucleic acid analysis including DNA sequencing. BACKGROUND Microfluidic devices with surface treatment, e.g., surface immobilized DNA primers, antibodies and peptides, etc. are useful in biomolecular assay, DNA sequencing, protein sequencing and other biomolecular target detections. For instance, the density and/or quality of immobilized molecules on the surface of microfluidic device need to be characterized or measured to guarantee consistent results for downstream applications such as DNA clustering and sequencing, protein/antibody quantification, and DNA PCR reaction. However, existing methods for manufacturing modified surfaces and characterizing surface chemistry can be time consuming and expensive and can suffer from low accuracy. There remains a need for improved microfluidic surface treatment and characterization methods. The present disclosure addresses such and other needs. SUMMARY The most common way to measure the density of surface molecules (e.g., DNA oligos) is by hybridization with complementary DNA oligo probes with detectable tags such as fluorescent dyes. However, this process is very time consuming, and the accuracy of measurement can be affected by the sequence of probe, salinity, pH, and/or temperature and concentration of probes. In some embodiments, provided herein are compositions and methods for measuring the surface density of molecules on a substrate (e.g., a flow cell) in a fast, accurate, and cost-effective manner. In some embodiments, the methods provided herein can achieve fast and more accurate measurements of surface immobilized biomolecule density with more than 40% cost reduction. BRIEF DESCRIPTION OF THE DRAWINGS The drawings illustrate certain features and advantages of this disclosure. These embodiments are not intended to limit the scope of the appended claims in any manner. FIG. 1 shows a schematic for characterization of surface molecular density by hybridization with fluorescent DNA oligo probes. FIG. 2 shows a schematic for characterization of surface molecular density using internal reference oligos with fluorescent tags. FIGS. 3A-3B show fluorescent counts of surface chemistry with different amounts oligo-dye added. FIG. 3A shows fluorescent signal intensities measured from internal immobilized oligo-dye. FIG. 3B shows fluorescent signal intensities measured by hybridization with an oligo-dye. FIG. 4 shows the correlation of the fluorescent counts using the hybridized probe method and the internal dye for surfaces with different immobilized molecular densities. FIG. 5 compares workflows for measuring oligo primer density on a flow chip surface. FIG. 5A shows a flowchart for the hybridization-based method. FIG. 5B shows a flowchart for the internal oligo-dye method. FIG. 6 shows representative images of fluorescent probe staining of flow chip surfaces. FIG. 6A shows non-uniform staining of fluorescent probes on flow chip surface under low flow rate. FIG. 6B shows uniform staining of fluorescent probes on flow chip surface. DETAILED DESCRIPTION All publications, comprising patent documents, scientific articles and databases, referred to in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication were individually incorporated by reference. If a definition set forth herein is contrary to or otherwise inconsistent with a definition set forth in the patents, applications, published applications and other publications that are herein incorporated by reference, the definition set forth herein prevails over the definition that is incorporated herein by reference. The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. Biological arrays, ordered or random, can be used to detect and analyze molecules, including DNA and RNA. In these applications, the arrays can be engineered to include probes for nucleotide sequences present in genes of humans and other organisms. In certain applications, for example, individual DNA and RNA probes may be attached at locations on a substrate, randomly or in a geometric grid (e.g., on a substrate with pre-patterned micro-structures, such as depressions/wells). Biological arrays may be used for nucleic acid sequencing. In general, genetic sequencing involves determining the order of nucleotides