title: Direct Write Protein Patterns for Multiplexed Cytokine Detection from Live Cells Using Electron Beam Lithography.
abstract: Simultaneous detection of multiple biomarkers, such as extracellular signaling molecules, is a critical aspect in disease profiling and diagnostics. Precise positioning of antibodies on surfaces, especially at the micro- and nanoscale, is important for the improvement of assays, biosensors, and diagnostics on the molecular level, and therefore, the pursuit of device miniaturization for parallel, fast, low-volume assays is a continuing challenge. Here, we describe a multiplexed cytokine immunoassay utilizing electron beam lithography and a trehalose glycopolymer as a resist for the direct writing of antibodies on silicon substrates, allowing for micro- and nanoscale precision of protein immobilization. Specifically, anti-interleukin 6 (IL-6) and antitumor necrosis factor alpha (TNFα) antibodies were directly patterned. Retention of the specific binding properties of the patterned antibodies was shown by the capture of secreted cytokines from stimulated RAW 264.7 macrophages. A sandwich immunoassay was employed using gold nanoparticles and enhancement with silver for the detection and visualization of bound cytokines to the patterns by localized surface plasmon resonance detected with dark-field microscopy. Multiplexing with both IL-6 and TNFα on a single chip was also successfully demonstrated with high specificity and in relevant cell culture conditions and at different times after cell stimulation. The direct fabrication of capture antibody patterns for cytokine detection described here could be useful for biosensing applications.
authors.affiliation: Department of Bioengineering, University of California, Los Angeles , 410 Westwood Plaza, Los Angeles, California 90095, United States.
authors.forename: Uland Y
authors.initials: UY
authors.lastname: Lau
authors.affiliation: Department of Chemistry and Biochemistry and the California NanoSystems Institute, University of California, Los Angeles , 607 Charles E. Young Drive East, Los Angeles, California 90095, United States.
authors.forename: Sina S
authors.initials: SS
authors.lastname: Saxer
authors.affiliation: Department of Chemistry and Biochemistry and the California NanoSystems Institute, University of California, Los Angeles , 607 Charles E. Young Drive East, Los Angeles, California 90095, United States.
authors.forename: Juneyoung
authors.initials: J
authors.lastname: Lee
authors.affiliation: Department of Chemistry and Biochemistry and the California NanoSystems Institute, University of California, Los Angeles , 607 Charles E. Young Drive East, Los Angeles, California 90095, United States.
authors.forename: Erhan
authors.initials: E
authors.lastname: Bat
authors.affiliation: Department of Bioengineering, University of California, Los Angeles , 410 Westwood Plaza, Los Angeles, California 90095, United States.|Department of Chemistry and Biochemistry and the California NanoSystems Institute, University of California, Los Angeles , 607 Charles E. Young Drive East, Los Angeles, California 90095, United States.
authors.forename: Heather D
authors.initials: HD
authors.lastname: Maynard
mesh_terms: D000818:Animals; D000906:Antibodies / Q000737:chemistry*; D055910:Antibodies, Immobilized / Q000737:chemistry; D015374:Biosensing Techniques / Q000295:instrumentation / Q000379:methods*; D002460:Cell Line; D055664:Electrochemical Techniques; D015850:Interleukin-6 / Q000302:isolation & purification*; D008262:Macrophage Activation / Q000276:immunology; D008264:Macrophages / Q000166:cytology / Q000276:immunology / Q000378:metabolism; D051379:Mice; D011108:Polymers / Q000737:chemistry; D011327:Printing / Q000379:methods*; D020349:Surface Plasmon Resonance; D014199:Trehalose / Q000737:chemistry; D014409:Tumor Necrosis Factor-alpha / Q000302:isolation & purification*
chemical_list: D000906:Antibodies; D055910:Antibodies, Immobilized; D015850:Interleukin-6; D011108:Polymers; D014409:Tumor Necrosis Factor-alpha; C577686:interleukin-6, mouse; D014199:Trehalose
keywords: antibody patterning; biosensor; dark-field microscopy; electron beam lithography; localized surface plasmon resonance; trehalose glycopolymer