Mediated glucose biosensors for application in continuous glucose monitoring
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2023-04-19Author
Jayakumar, Kavita
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Abstract
Diabetes affects 1 in 11 people globally and requires monitoring of blood glucose
levels as a part of its treatment. Initial blood glucose monitoring devices involved
finger prick testing at regular intervals daily, a self-monitoring strategy reliant on
patient compliance. Advances in technology have facilitated continuous glucose
monitors (CGMs), (semi-) implantable devices that can monitor glucose levels in vivo
and transmit the data to mobile applications. Majority of commercial CGMs are
electrochemical in nature and meet the requirements for a commercial sensing implant.
There are three main factors that limit CGMs for in vivo use – namely oxygen
dependence, low molecular weight (LMW) materials and foreign body response (FBR)
– which negatively affect the lifetime and accuracy of CGMs. This thesis aims to detail
strategies to combat these issues, building on previous work performed in the field of
enzymatic electrochemical glucose sensors.
Chapter 2 details the use of design of experiments (DoE) to optimise enzyme electrode
components – Osmium complex-based redox polymer, commercial glucose oxidising
enzyme (glucose oxidase, GOx) and crosslinker (polyethylene glycol diglycidyl ether,
PEGDGE). Previous work established high current and stability of a similar system
which also incorporated acid treated multiwalled carbon nanotubes (MWCNTs) as a
nanosupport. The MWCNTs enable high currents and surface coverages, but the
quantities required were quite high, which can be detrimental for in vivo applications.
In this chapter, the grafting of enzyme to nanosupport was carried out to allow
minimisation of MWCNT amounts while retaining high currents and operational
stability. DoE facilitated the determination of electrode component amounts for
optimal current density and stability. The optimised enzyme electrodes show a current
density of 3.18 ± 0.30 mA cm−2
, representing a 146% increase in current density in 50
mM phosphate-buffered saline at 37 °C containing 5 mM glucose when compared to
similar systems where enzyme and nanosupport are not grafted to each other. Using
the predictive DoE model, component amounts were then modified to minimise the
quantity of the enzyme-MWCNT nanoconjugate, resulting in a biosensor which
showed similar electrochemical behaviour and current density to the optimised system
while using 93% less of the nanoconjugate Commercial GOx shows excellent behaviour for glucose oxidation but uses oxygen in
its half reaction to regenerate. This is problematic as in vivo oxygen levels can
fluctuate resulting in errors in measurement. Additionally, enzyme regeneration by
oxygen reduction gives hydrogen peroxide as a product. Peroxide can cause enzyme
instability as it oxidises the methionine residues of the enzyme, decreasing its activity.
To combat this use, GOx was replaced with engineered cellobiose dehydrogenase
(CDH) in Chapter 3. CDH is a dehydrogenase and thus does not use oxygen in its half
reactions and has been modified to selectively choose glucose as its substrate. The
enzyme electrodes comprising osmium complex-based redox polymer, CDH and
PEGDGE were optimised with DoE, while a direct electron transfer (DET) based
system was also optimised through conventional methods. The resulting sensors had
sensitivities in the same order of magnitude as those in literature. Most importantly,
sensor signals showed no difference in the presence and absence of oxygen. The
sensors derived from CDH were shown to be specific to glucose over other clinically
relevant in vivo sugars and selective, i.e., capable of glucose sensing in the presence
of interfering species present in complex media.
While no individual species is classified as an interferent in complex media they
seemed to exhibit a cooperative effect resulting in a minimisation of current (43%).
This is usually overcome with the use of polymer coatings. However, polymer coatings
themselves lead to reduced sensor signals on their application, due to the formation of
a diffusion barrier. Chapter 4 focuses on the design of polymer coatings to enable
protection against biofouling while retaining current density. This was done by
designing polymer coatings with a compatible epoxy crosslinking moiety on the
polymer backbone that could crosslink with the redox polymer used in the sensing
layer. The polymers selected in this study were zwitterionic in nature because of their
inherent ability to minimise biofouling. Protein adsorption and cell adhesion studies,
using fibrinogen and fibroblasts respectively, allowed a screening to select the most
effective of the synthesised polymers for biofouling resistance. This poly(2-
methacryloyloxyethyl phosphorylcholine-co-glycidyl methacrylate (MPC)-type
polymer showed similar biofouling resistance compared to commercial polymer
Lipidure with ~50% reduction in fibrinogen adsorption and ~80% reduction in
fibroblast adhesion. When used as coatings for glucose biosensors fabricated in Chapter 3, MPC showed ability to resist protein adsorption while retaining current
density similar to a non-coated system with 1.5-fold increase in sensitivity.
MPC polymers showed ability to impart biofouling resistance while maintaining
current signals. Nevertheless, their ability to resist LMW materials was not proved. In
Chapter 5, a series of different protective strategies were explored to determine which
approach would be the best to protect from biological and LMW interferences.
Enzymatic scavenging using enzymes that target LMW species was investigated but
showed inefficient scavenging, likely due to low enzymatic activity. Polymer
multilayer approach with successive anionic and MPC polymer layers was utilised and
showed the best potential. MPC as the outerlayer showed biofouling resistance
whereas an anionic interlayer ([Poly(1-vinylimidazole-co-4-styrene sulfonic acid
sodium salt hydrate], P(VI1
-SSNa1
)) inhibited anionic LMW species such as uric acid
and ascorbic acid which cause interference. Moreover, due to the compatible
crosslinking sites, the layers intermix at the boundary between them, minimising
diffusional barrier and allowing current signals similar to a non-coated system. This
multilayer protection system extends linear range and enables higher current and
stability than a non-coated system in 50 mM phosphate-buffered saline and artificial
plasma. Chapter 6 summarises the results from Chapter 2-5 and highlights the
significant conclusions that can be drawn from these results. Future directions that can
improve on the strategies in this thesis are also discussed.