Three-dimensional surface for protein and small molecule microarrays
Inventors
Liu, Jun O. • Cheng, Zhiqiang • Zhu, Heng • Guo, Zufeng • Peng, Hanjing
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Assignees
MemberJohns Hopkins UniversityJohns Hopkins UniversityFounded in 1876, Johns Hopkins University is recognized as the first research university in the United States. It advances interdisciplinary education, high-impact research, and global outreach, supporting knowledge translation, technological innovation, and community partnerships. The university fosters academic excellence, innovation incubation, outreach, and inclusion across multiple campuses in Baltimore, integrating into the city's social, economic, and cultural life.
Founded in 1876, Johns Hopkins University is recognized as the first research university in the United States. It advances interdisciplinary education, high-impact research, and global outreach, supporting knowledge translation, technological innovation, and community partnerships. The university fosters academic excellence, innovation incubation, outreach, and inclusion across multiple campuses in Baltimore, integrating into the city's social, economic, and cultural life.
Publication Number
US-12649980-B2
Publication Date
2026-06-09
Expiration Date
Abstract
Surface structures that enable the preparation of three-dimensional microarrays of proteins or small molecules or other types of macromolecules are disclosed. The three-dimensional microarrays possess higher sensitivity for detecting protein-macromolecule and small molecule-protein interactions in a high-throughput fashion.
Core Innovation
The invention relates to a three-dimensional microarray comprising a surface-modified substrate with a scaffold having a specified molecular structure in which m and n are each independently an integer from 1 to 1000. The surface is modified to provide a 3D microarray surface chemistry, including polymer chains with diazirine functional groups or ethacrynic acid covalently coupled to form a 3D-EA surface.
The scaffold includes polymer chains with diazirine functional groups, and the diazirine functional groups are photoactivated to form reactive carbene species for covalent capture and immobilization of small molecules on the three-dimensional microarray surface. A printed small-molecule library is immobilized onto the 3D scaffold through photocrosslinking to diazirine functional groups, and the workflow includes contacting the microarray with cells and detecting bound glucose transporter protein interactions with immobilized rapafucins.
The document further describes an EA covalent interaction and the impact of using a 3D-EA surface rather than a 2D-EA surface. The discussion includes protein immobilization via GST-EA interaction, protein microarrays, and covalent protein-ligand pair strategies, including haloTag, SNAP, and CLIP, with example results using a rapafucins and rapaglutin A binder to identify GLUT1 and characterize downstream cellular effects.
Claims Coverage
The independent claim set centers on a three-dimensional microarray with a surface-modified scaffold having a specified molecular structure defined by m and n from 1 to 1000. Three inventive features are carried through the claim set: photocrosslinking of a printed small-molecule library to diazirine functional groups, and a cell-based binding and detection assay for identifying a glucose transporter inhibitor, narrowed to GLUT isoforms and specifically GLUT1.
Three-dimensional microarray with scaffold-defined molecular structure
A three-dimensional microarray comprising a surface-modified substrate comprising a scaffold having the specified molecular structure, where m and n are each independently an integer from 1 to 1000.
Photocrosslinked printed small-molecule library on diazirine functional groups
A library of small molecules printed at one or more locations and immobilized to the surface through photocrosslinking to diazirine functional groups.
Cell-based identification of a glucose transporter inhibitor by binding and detection
A method for identifying a glucose transporter inhibitor by contacting the three-dimensional microarray with cells expressing a glucose transporter protein, allowing the glucose transporter protein to bind to rapafucins on the microarray, and detecting the bound glucose transporter protein.
Glucose transporter protein restricted to GLUT isoforms
The glucose transporter protein is one of GLUT1, GLUT3, and GLUT4.
Glucose transporter protein restricted to GLUT1
The glucose transporter protein used is GLUT1.
Overall, the claim coverage is centered on a three-dimensional microarray having a scaffold with a defined molecular structure (m and n from 1 to 1000), combined with photocrosslinking-based immobilization of printed small-molecule libraries and a cell-based binding and detection method for identifying glucose transporter inhibitors, narrowed to GLUT isoforms and specifically GLUT1.
Stated Advantages
Increased sensitivity for detecting protein-macromolecule and small molecule-protein interactions on the three-dimensional microarray versus 2D.
Higher signal-to-background ratio compared with 2D surfaces in the disclosed rapafucin diazirine surface optimization.
Improved response and signal-to-noise for GST-based protein detection and protein-protein interaction screening on the disclosed 3D ethacrynic acid surfaces versus alternative surfaces.
Improved protein detection signals on the 3D-EA surface versus a 2D-EA surface.
Documented Applications
Screening workflows using cell lysates and detection by antibodies or tags on the three-dimensional microarray platform for protein-ligand interactions.
Identification of a glucose transporter inhibitor by contacting a three-dimensional microarray with cells expressing a glucose transporter protein, detecting bound glucose transporter protein, and using rapafucins as binders.
Glucose transporter inhibitor identification and validation for GLUT1, including detection and interaction with rapaglutin A from a 3,918-rapafucin 3D microarray.
Protein immobilization and detection using GST-EA interactions on an ethacrynic acid functionalized three-dimensional surface.
Protein-protein interaction screening formats using covalent protein-ligand pairs, including haloTag, SNAP-tag, and CLIP-tag, in connection with the disclosed three-dimensional microarray formats.
A three-dimensional microarray platform used in assays to identify glucose transporter inhibitors by contacting the microarray with cells expressing a glucose transporter protein, binding to rapafucins, and detecting the bound glucose transporter protein.
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