Multiplexed in-gel microfluidic immunoassays: characterizing protein target loss during reprobing of benzophenone-modified hydrogels
Abstract
From whole tissues to single-cell lysate, heterogeneous immunoassays are widely utilized for analysis of protein targets in complex biospecimens. Recently, benzophenone-functionalized hydrogel scaffolds have been used to immobilize target protein for immunoassay detection with fluorescent antibody probes. In benzophenone-functionalized hydrogels, multiplex target detection occurs via serial rounds of chemical stripping (incubation with sodium-dodecyl-sulfate (SDS) and β-mercaptoethanol at 50-60 °C for ≥1 h), followed by reprobing (interrogation with additional antibody probes). Although benzophenone facilitates covalent immobilization of proteins to the hydrogel, we observe 50% immunoassay signal loss of immobilized protein targets during stripping rounds. Here, we identify and characterize signal loss mechanisms during stripping and reprobing. We posit that loss of immobilized target is responsible for ≥50% of immunoassay signal loss, and that target loss is attributable to disruption of protein immobilization by denaturing detergents (SDS) and incubation at elevated temperatures. Furthermore, our study suggests that protein losses under non-denaturing conditions are more sensitive to protein structure (i.e., hydrodynamic radius), than to molecular mass (size). We formulate design guidance for multiplexed in-gel immunoassays, including that low-abundance proteins be immunoprobed first, even when targets are covalently immobilized to the gel. We also recommend careful scrutiny of the order of proteins targets detectedmore »
- Authors:
-
- Univ. of California, Berkeley, CA (United States)
- Univ. of California, Berkeley, CA (United States); Chan Zuckerberg BioHub, San Francisco, CA (United States)
- Publication Date:
- Research Org.:
- Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)
- Sponsoring Org.:
- USDOE Office of Science (SC); National Institutes of Health (NIH); National Sciences and Engineering Research Council of Canada (NSERC)
- OSTI Identifier:
- 1591820
- Grant/Contract Number:
- AC02-05CH11231; R33 CA2259296; PGS-D 487496
- Resource Type:
- Accepted Manuscript
- Journal Name:
- Scientific Reports
- Additional Journal Information:
- Journal Volume: 9; Journal Issue: 1; Journal ID: ISSN 2045-2322
- Publisher:
- Nature Publishing Group
- Country of Publication:
- United States
- Language:
- English
- Subject:
- 59 BASIC BIOLOGICAL SCIENCES
Citation Formats
Gopal, Anjali, and Herr, Amy E. Multiplexed in-gel microfluidic immunoassays: characterizing protein target loss during reprobing of benzophenone-modified hydrogels. United States: N. p., 2019.
Web. doi:10.1038/s41598-019-51849-8.
Gopal, Anjali, & Herr, Amy E. Multiplexed in-gel microfluidic immunoassays: characterizing protein target loss during reprobing of benzophenone-modified hydrogels. United States. https://doi.org/10.1038/s41598-019-51849-8
Gopal, Anjali, and Herr, Amy E. Mon .
"Multiplexed in-gel microfluidic immunoassays: characterizing protein target loss during reprobing of benzophenone-modified hydrogels". United States. https://doi.org/10.1038/s41598-019-51849-8. https://www.osti.gov/servlets/purl/1591820.
@article{osti_1591820,
title = {Multiplexed in-gel microfluidic immunoassays: characterizing protein target loss during reprobing of benzophenone-modified hydrogels},
author = {Gopal, Anjali and Herr, Amy E.},
abstractNote = {From whole tissues to single-cell lysate, heterogeneous immunoassays are widely utilized for analysis of protein targets in complex biospecimens. Recently, benzophenone-functionalized hydrogel scaffolds have been used to immobilize target protein for immunoassay detection with fluorescent antibody probes. In benzophenone-functionalized hydrogels, multiplex target detection occurs via serial rounds of chemical stripping (incubation with sodium-dodecyl-sulfate (SDS) and β-mercaptoethanol at 50-60 °C for ≥1 h), followed by reprobing (interrogation with additional antibody probes). Although benzophenone facilitates covalent immobilization of proteins to the hydrogel, we observe 50% immunoassay signal loss of immobilized protein targets during stripping rounds. Here, we identify and characterize signal loss mechanisms during stripping and reprobing. We posit that loss of immobilized target is responsible for ≥50% of immunoassay signal loss, and that target loss is attributable to disruption of protein immobilization by denaturing detergents (SDS) and incubation at elevated temperatures. Furthermore, our study suggests that protein losses under non-denaturing conditions are more sensitive to protein structure (i.e., hydrodynamic radius), than to molecular mass (size). We formulate design guidance for multiplexed in-gel immunoassays, including that low-abundance proteins be immunoprobed first, even when targets are covalently immobilized to the gel. We also recommend careful scrutiny of the order of proteins targets detected via multiple immunoprobing cycles, based on the protein immobilization buffer composition.},
doi = {10.1038/s41598-019-51849-8},
journal = {Scientific Reports},
number = 1,
volume = 9,
place = {United States},
year = {Mon Oct 28 00:00:00 EDT 2019},
month = {Mon Oct 28 00:00:00 EDT 2019}
}
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