Discriminatory Bio-Adhesion Over Nano-Patterned Polymer Brushes

226 Pages·2016·3.42 MB·English

UUnniivveerrssiittyy ooff MMaassssaacchhuusseettttss AAmmhheerrsstt
SScchhoollaarrWWoorrkkss@@UUMMaassss AAmmhheerrsstt
Open Access Dissertations
9-2013
DDiissccrriimmiinnaattoorryy BBiioo--AAddhheessiioonn OOvveerr NNaannoo--PPaatttteerrnneedd PPoollyymmeerr
BBrruusshheess
Saugata Gon
University of Massachusetts Amherst
Follow this and additional works at: https://scholarworks.umass.edu/open_access_dissertations
Part of the Biomedical Engineering and Bioengineering Commons, Chemical Engineering Commons,
and the Nanoscience and Nanotechnology Commons
RReeccoommmmeennddeedd CCiittaattiioonn
Gon, Saugata, "Discriminatory Bio-Adhesion Over Nano-Patterned Polymer Brushes" (2013). Open Access
Dissertations. 796.
https://doi.org/10.7275/fxcc-ec94 https://scholarworks.umass.edu/open_access_dissertations/796
This Open Access Dissertation is brought to you for free and open access by ScholarWorks@UMass Amherst. It
has been accepted for inclusion in Open Access Dissertations by an authorized administrator of
ScholarWorks@UMass Amherst. For more information, please contact [email protected].
DISCRIMINATORY BIO-ADHESION OVER
NANO-PATTERNED POLYMER BRUSHES

A Dissertation Presented
by
SAUGATA GON

Submitted to the Graduate School of the
University of Massachusetts Amherst in partial fulfillment of the
requirement for the degree of

DOCTOR OF PHILOSOPHY

September 2013
Department of Chemical Engineering

i
©
Copyright by Saugata Gon 2013
All Rights Reserved

ii
DISCRIMINATORY BIO-ADHESION OVER
NANO-PATTERNED POLYMER BRUSHES

A Dissertation Presented
by
SAUGATA GON

Approved as to style and content by:

Maria M. Santore, Chair

Jeffrey M. Davis, Member

David M. Ford, Member

Harry Bermudez, Member

T. J. Mountziaris, Department Head
Department of Chemical Engineering.

iii
DEDICATION

To my wife and my family members.

iv
ACKNOWLEDGEMENTS

It was a great experience working in the Santore lab at the PSE department at
UMass Amherst. The work done in this thesis would not have been possible without
active support and guidance by my advisor Prof. Maria M. Santore. Whenever I had any
questions or needed anything in the lab I could just walk into her office and she was
always there to help me out. I do not have enough words to thank her and I would not try
it here.
Another person who helped me getting here today is Prof. Lakis Mountziaris, the
departmental head of the Chemical Engineering dept. I would like to take this opportunity
to thank Prof. Mountziaris for his encouraging advice and suggestions which were really
helpful.
I want to thank the group members in the Santore group in Polymer Science and
the Davis group in Chemical Engineering department. Their help, suggestions and
friendship were instrumental in achieving the goal for this PhD thesis.
Next in the thank list are my friends in Amherst and in India. Without their
presence and support this journey would have been really a boring one.
The support and company of my wife Somdatta throughout this journey made it a
really enjoyable one. Her presence always motivated me to work harder and that made
this goal reachable. At last I would like to mention about my parents, my brother,
nephew, sister in law and all my family members in India whose caring words and faith
in me helped me to reach the finish-line finally.

v
ABSTRACT
DISCRIMINATORY BIO-ADHESION OVER NANO-
PATTERNED POLYMER BRUSHES
SEPTEMBER 2013
SAUGATA GON, B. TECH. HIT INDIA
M.TECH., IIT BOMBAY, INDIA
M.S., UNIVERSITY OF MASSACHUSETTS AMHERST
PhD., UNIVERSITY OF MASSACHUSETTS AMHERST
Directed by Professor Maria M. Santore

Surfaces functionalized with bio-molecular targeting agents are conventionally
used for highly-specific protein and cell adhesion. This thesis explores an alternative
approach: Small non-biological adhesive elements are placed on a surface randomly,
with the rest of the surface rendered repulsive towards biomolecules and cells. While the
adhesive elements themselves, for instance in solution, typically exhibit no selectivity for
various compounds within an analyte suspension, selective adhesion of targeted objects
or molecules results from their placement on the repulsive surface. The mechanism of
selectivity relies on recognition of length scales of the surface distribution of adhesive
elements relative to species in the analyte solution, along with the competition between
attractions and repulsions between various species in the suspension and different parts of
vi
the collecting surface. The resulting binding selectivity can be exquisitely sharp;
however, complex mixtures generally require the use of multiple surfaces to isolate the
various species: Different components will be adhered, sharply, with changes in collector
composition. The key feature of these surface designs is their lack of reliance on
biomolecular fragments for specificity, focusing entirely on physicochemical principles at
the lengthscales from 1 – 100 nm. This, along with a lack of formal patterning, provides
the advantages of simplicity and cost effectiveness.
This PhD thesis demonstrates these principles using a system in which cationic
poly-L-lysine (PLL) patches (10 nm) are deposited randomly on a silica substrate and the
remaining surface is passivated with a bio-compatible PEG brush. TIRF microscopy
revealed that the patches were randomly arranged, not clustered. By precisely controlling
the number of patches per unit area, the interfaces provide sharp selectivity for adhesion
of proteins and bacterial cells. For instance, it was found that a critical density of patches
(on the order of 1000/m2) was required for fibrinogen adsorption while a greater density
comprised the adhesion threshold for albumin. Surface compositions between these two
thresholds discriminated binding of the two proteins. The binding behavior of the two
proteins from a mixture was well anticipated by the single- protein binding behaviors of
the individual proteins.
The mechanism for protein capture was shown to be multivalent: protein
adhesion always occurred for averages spacings of the adhesive patches smaller than the
dimensions of the protein of interest. For some backfill brush architectures, the spacing
between the patches at the threshold for protein capture clearly corresponded to the major
dimension of the target protein. For more dense PEG brush backfills however, larger
vii
adhesion thresholds were observed, corresponding to greater numbers of patches
involved with the adhesion of each protein molecule. . The thesis demonstrates the
tuning of the position of the adhesion thresholds, using fibrinogen as a model protein,
using variations in brush properties and ionic strength. The directions of the trends
indicate that the brushes do indeed exert steric repulsions toward the proteins while the
attractions are electrostatic in nature.
The surfaces also demonstrated sharp adhesion thresholds for S. Aureus bacteria,
at smaller concentrations of adhesive surfaces elements than those needed for the protein
capture. The results suggest that bacteria may be captured while proteins are rejected
from these surfaces, and there may be potential to discriminate different bacterial types.
Such discrimination from protein-containing bacterial suspensions was investigated
briefly in this thesis using S. Aureus and fibrinogen as a model mixture. However, due to
binding of fibrinogen to the bacterial surface, the separation did not succeed. It is still
expected, however, that these surfaces could be used to selectively capture bacteria in the
presence of non-interacting proteins.
The interaction of these brushes with two different cationic species PLL and
lysozyme were studied. The thesis documents rapid and complete brush displacement by
PLL, highlighting a major limitation of using such brushes in some applications. Also
unanticipated, lysozyme, a small cationic protein, was found to adhere to the brushes in
increasing amounts with the PEG content of the brush. This finding contradicts current
understanding of protein-brush interactions that suggests increases in interfacial PEG
content increase biocompatibility.

viii
TABLE OF CONTENTS
Page
ABSTRACT ....................................................................................................................... vi
LIST OF TABLES ........................................................................................................... xiii
LIST OF FIGURES ......................................................................................................... xiv

CHAPTER
1. INTRODUCTION ...........................................................................................................1
1.1 Surface heterogeneity and its role in adhesion ..................................................1
1.1.1 Specific and nonspecific interaction ........................................................3
1.2 Adhesion on tunable surface patterns ................................................................4
1.3 Effect of flow on adhesion .................................................................................8
1.4 Surface patterning methods..............................................................................10
1.5 Motivation and objective .................................................................................12
1.5.1 Thesis objective ......................................................................................13
1.6 Research strategy .............................................................................................13
1.7 Thesis overview ...............................................................................................15
1.8 References ........................................................................................................18

2. ARCHITECTURE AND STABILITIES OF PLL-PEG BRUSHES: CASE STUDIES
WITH CATIONIC AND ANIONIC PROTEINS AND A STRONG CATIONIC
POLYELECTROLYTE PLL ............................................................................................28
2.1 Introduction ......................................................................................................28
2.2 Synthesis and characterization of brushes .......................................................30
2.2.1 Brush synthesis ......................................................................................30
2.2.2 Brush characterization using NMR ........................................................31
2.3 Experimental methodology ..............................................................................32
2.4 Results ..............................................................................................................34
2.4.1 Calculation of brush height .....................................................................34
2.4.2 Interaction with globular proteins ...........................................................37
2.4.3 Interaction with PLL ...............................................................................42
2.5 Discussion .......................................................................................................48
2.6 Conclusions .....................................................................................................52
2.7 References .......................................................................................................54

ix