(188cf) Investigation of Lectin-Functionalized Surfaces As Biosensors Towards Pathogen Capture Using Azlactone-Based Block Copolymers As a Reactive Platform

Investigation
of lectin-functionalized surfaces as biosensors towards pathogen capture using
azlactone-based block copolymers as a reactive platform

Mohammadali
Masigol¹, Niloy Barua¹, B.S. Lokitz², R.R.
Hansen¹

¹Chemical
Engineering Department,

 Kansas State University, Manhattan, KS
66506 2

²Center for
Nanophase Materials Sciences,

Oak Ridge National Laboratory Oak Ridge,
TN 37831

rrhansen@ksu.edu

Simple, rapid and
inexpensive biosensors are required for pathogen isolation and detection in food
safety, water quality, and disease diagnostics. Affinity molecules (antibodies,
nucleic acid aptamers, peptides, and lectins) that recognize epitopes on the extracellular
surface of bacteria can be functionalized on sensor surfaces for detection.
Lectins, a group of carbohydrate-binding proteins, are promising recognition
molecules for this application because of their ability to recognize complex
carbohydrates and discriminate them based on slight difference in structure. However,
they are limited in use by highly reversible binding (K_D ~ 1-10 mM),
leading to poor capture efficiency. Immobilizing higher surface densities of
lectins may promote multivalent binding, ultimately improving detection
sensitivity.

In this work, we are investigating
a new sensor surface treatment that may enhance the capture of bacteria with
affinity molecules such as lectins. The surfaces contain azlactone-functionalized
polymer films (poly(glycidyl methacrylate)-block-poly(vinyldimethyl
azlactone) designed to immobilize high, tunable protein densities. We first investigated
the critical coupling parameters that affected lectin loading into the polymer
films, quantified by ATR-FTIR characterization.  Parameters found to influence
the azlactone-lectin coupling reaction were (1) polymer density and film structure,
(2) coupling conditions (pH, lectin concentration, and lectin incubation time),
and (3) structure and molecular weight of the lectin. As the first step, different
concentrations of PGMA-b-PVDMA in chloroform (0.25-1% wt.) were spin coated
over the plasma-cleaned silicon substrates and the resulting polymer films were
incubated with wheat germ agglutinin (WGA)-FITC and Helix pomatia (HPA)-FITC
lectins. It was found that high densities of brushlike polymers (corresponding
to 0.75% wt) were optimal for maximum lectin coupling, while crosslinked
polymer films (corresponding to 1.0% wt) resulted in diminished levels, as
shown in Figure 1A.  pH was also a critical factor, higher pH (8.5-10) was
desired for amine based coupling, however, this can also increase the rate of
hydrolysis of azlactone groups. A pH of 9.3 was found to be the optimum for
achieving maximum coupling of WGA with azlactones (Fig 2B). Fig 2B was obtained
by the comparison of peak height (C=O of azalctone group) by using ATR-FTIR
before and after lectin functionalization. Our current investigation aims to
quantify the resulting gains in sensitivity, capture efficiency, and detection
limits that can be obtained using optimized surfaces. Figure C and D describe surface-bound 
Ecoli K12 using WGA, HPA, and Concanvalin A (ConA), which show
lectin-specific capture levels over the polymer films. Each response is related
to the lectin sugar specificity to the EPS content of Ecoli K12 cell
surface.

Figure
1. (A) Fluorescent intensity of WGA and
HPA functionalized surfaces coated with different concentrations of
PGMA-b-PVDMA (control= PGMA-b-PVDMA coated on Si) (B) Effect of pH on azlactone
reaction conversion, contribution of hydroxyl (OH) group (Hydrolysis) and WGA amine groups (Aminolysis) for opening the azlactone ring (C) E. coli K12 capture on the
lectin-functionalized surfaces (D) Number of E.coli K12 captured on the lectin-functionalized
surfaces (shown in part C).