Peptide-Based Stimuli-Responsive Materials for Bioanalytical Applications

López, Gabriel P

Chilkoti, Ashutosh

Li, Linying

2017-05-16T17:27:28Z

2019-04-26T08:17:12Z

2017

Biomedical Engineering

Surfaces with switchable properties in response to external stimuli (e.g., temperature and pH) have attracted substantial research interest because of their ability to modulate biomolecule activity, protein immobilization, and cell adhesion. These stimuli-responsive substrates offer versatile platforms for developing biosensors, cell culture substrates, diagnostic systems, and drug delivery systems. In this work, we controllably functionalized substrates with genetically engineered polypeptides to fabricate thermally responsive surfaces for various bioanalytical applications. Genetically engineered elastin-like polypeptides (ELPs) are one class of thermally responsive biopolymers that are characterized by their lower critical solution temperature (LCST) phase behavior in water; ELPs at a given concentration in aqueous solvent phase separate to form protein-rich coacervates above the cloud point transition temperature (Tt). ELPs present an attractive alternative to synthetic, stimuli-responsive polymers due to their biocompatibility, monodispersity, and controlled physicochemical properties.

To fabricate ELP-modified surfaces with desired structure and functionality, we first investigated the adsorption behavior of ELP homopolymers and ELP block copolymers onto silica surfaces. We provided an in-depth understanding of adsorption kinetics, mechanism and surface conformation for the “canonical” ELP sequence (Val-Pro-Gly-Val-Gly), which enabled precise conformational control of the adsorbed ELPs. We also showed that genetically incorporating the silaffin R5 peptides into ELP chains significantly enhanced the binding affinity of ELPs to silica surfaces, leading to thicker ELP layers with a higher surface coverage. To extend this work, we also explored the adsorption behavior of ELP block copolymers onto silica surfaces using theoretical and experimental approaches. Our results showed that the silaffin tag not only enhanced the binding of ELP block copolymers to silica surfaces, but also directed micelle adsorption, leading to close-packed micellar arrangements dissimilar to the sparse and patchy arrangements observed for ELP micelles lacking a silaffin tag. In addition, the surface-grafted ELP unimers exhibited interfacial phase transition behavior, while the adsorbed ELP micelles were no longer thermally-responsive. These studies provided insight into the design of ELP based smart surfaces with controlled structure-architecture-function relationship.

After achieving programmable adsorption of ELPs onto surfaces, we exploited these thermally responsive surfaces for several bioanalytical applications, including cell culture and diagnostic assays. We first developed a simple approach to pattern cells on gold patterned silicon substrates using ELPs with cell- and gold-binding domains. Cell patterning was achieved by exploiting orientation of the adsorbed ELP to either enhance (gold regions) or impede (silicon oxide regions) cell adhesion at particular locations on the patterned surface. Along a similar vein, we fabricated a thermoresponsive cell culture substrate using rationally designed ELP coatings with precisely spaced cell-adhesive motifs. The reversible swelling and collapse of ELPs thermally modulated the accessibility of cell-binding domains to enable cell adhesion at T > Tt and efficient cell recovery at T < Tt.

In addition, we have utilized ELP-modified particles to develop smart diagnostics. We demonstrated proof-of-concept for an acoustofluidic, chip-based method that enables the rapid capture and isolation of biomarkers from blood for off-chip quantification. We showed that biomarkers were rapidly immobilized onto the surfaces of ELP-modified particles via co-aggregation, and continuously separated from the blood cells using an acoustofluidic device. The captured biomarkers can then be quantified using flow cytometry, or released from the surfaces of particles for further analysis. By designing ELP fusion proteins that can capture target bioactive materials, this platform system can be readily extended to separate a range of biological materials (e.g., cells, viruses and cell-free DNA) from complex biofluids.

In summary, we achieved controlled adsorption of ELP homopolymers and block copolymers onto surfaces with tailored architecture and functionality. These ELP-modified smart surfaces have been utilized to create cellular patterns, a thermoresponsive cell culture substrate, and a biomarker separation and detection platform.

https://hdl.handle.net/10161/14386

Biomedical engineering

Materials Science

Molecular biology

Cell culture

Diagnostic assay

Elastin-like polypeptides

Microfluidics

Protein adsorption

Stimuli-responsive biomaterials

Peptide-Based Stimuli-Responsive Materials for Bioanalytical Applications

Dissertation

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