Reaction-based indicator displacement assay (RIA) for the development of a triggered release system capable of biofilm inhibition

Article abstract of DOI:10.1039/c9cc07759f

Here, a reaction-based indicator displacement hydrogel assay (RIA) was developed for the detection of hydrogen peroxide (H₂O₂) via the oxidative release of the optical reporter Alizarin Red S (ARS). In the presence of H₂O₂, the RIA system displayed potent biofilm inhibition for Methicillin-resistant Staphylococcus aureus (MRSA), as shown through an in vitro assay quantifying antimicrobial efficacy. This work demonstrated the potential of H₂O₂-responsive hydrogels containing a covalently bound diol-based drug for controlled drug release.

Full text of DOI:10.1039/c9cc07759f

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Reaction-based indicator displacement assay (RIA)
for the development of a triggered release system
capable of biofilm inhibition†
Cite this: Chem. Commun., 2019,
55, 15129
a
a
Bethany L. Patenall, ‡a George T. Williams‡,
Lauren Gwynne,
Received 3rd October 2019,
Accepted 5th November 2019
a
a
b
Liam J. Stephens,
Emma V. Lampard,
Hollie J. Hathaway,
c
d
b
Naing T. Thet,a Amber E. Young,
Mark J. Sutton,
Robert D. Short,
DOI: 10.1039/c9cc07759f
a
Steven D. Bull,
Tony D. James, *a Adam C. Sedgwick *e and
a
rsc.li/chemcomm
A. Toby A. Jenkins
*
Here, a reaction-based indicator displacement hydrogel assay (RIA) competitive displacement of ARS was observed with concomitant
was developed for the detection of hydrogen peroxide (H2O2) via observation of an increase in absorption at 513 nm in solution
the oxidative release of the optical reporter Alizarin Red S (ARS). In (ARS wavelength).
the presence of H2O2, the RIA system displayed potent biofilm
Aryl boronic acids/boronate esters are well known to
inhibition for Methicillin-resistant Staphylococcus aureus (MRSA), undergo hydrogen peroxide (H2O2)-mediated oxidation to form
as shown through an in vitro assay quantifying antimicrobial the corresponding phenol.13 This unique synthetic transformation
efficacy. This work demonstrated the potential of H2O2-responsive has been exploited in organic synthesis and fluorescence
hydrogels containing a covalently bound diol-based drug for con- sensing.13 We thus envisaged that modification of the previously
trolled drug release.
developed ARS hydrogel bound indicator displacement assay
(IDA) would yield a multimodal detection platform for the
Dye displacement assays exploit the chemoselective reactivity of
certain chemical moieties and the reversible binding of dye
molecules to a specific receptor.1 Such chemistry has begun to
find widespread use with marked enhancement over traditional
sensing assays.1–8 More complex systems containing multiple
dyes also offer new paradigms for microarray development.9
Not surprisingly, dye displacement assays have been elegantly
employed by a number of research groups. These constructs
often rely on boronic acid systems as the receptor (host)
subunit with a 1,2- and 1,3-diol guest.10
Previously our group has developed boronate-based hydro-
gel systems as dye displacement assays (borogel) for mono-
saccharide detection.11,12 As shown in Scheme 1, the commercially
available 1,2-diol dye Alizarin Red S (ARS) was shown to successfully
bind to the boronate hydrogel and result in a colour change from
red to orange. Upon the addition of a monosaccharide, the
a Department of Chemistry, University of Bath, Bath, BA2 7AY, UK.
E-mail: t.d.james@bath.ac.uk, A.T.A.Jenkins@bath.ac.uk
b Department of Chemistry, Lancaster University, UK
c The Scar Free Foundation Centre for Children’s Burns Research,
The Bristol Royal Hospital for Children, Bristol, UK
d Public Health England, Porton Down, Salisbury, Wiltshire SP4 0JG, UK
e Department of Chemistry, University of Texas at Austin, 105 East 24th Street
A5300, Austin, Texas 78712-1224, USA. E-mail: a.c.sedgwick@utexas.edu
† Electronic supplementary information (ESI) available. See DOI: 10.1039/
c9cc07759f
Scheme 1 (a) Previously developed hydrogel bound dye displacement
assay (IDA) for the detection of monosaccharides.11,12 (b) Present work –
the development of a hydrogel bound reaction-based indicator displacement
(RIA) assay for the detection of H2O2 and for the inhibition of MRSA biofilm
formation.
‡ Equal contribution.
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detection of H2O2 with attendant antimicrobial activity
(Scheme 1).14–16 Here, we report the construction of a covalently
incorporated ARS polyacrylamide hydrogel that undergoes
oxidative activation in the presence of H2O2 to release ARS and
afford a reaction-based indicator displacement assay (RIA).15
In vitro antibacterial assays with Methicillin-resistant Staphylococcus
aureus (MRSA) indicated significant activity against biofilm
formation for the combination of ARS and H2O2.17,18
In brief, phenylboronic acid (PBA) and benzoxaborole (BOB)
acrylamide monomers were synthesised as previously reported.11,12
Polyacrylamide hydrogels were synthesised using water (60% w/w),
acrylamide (38% w/w), methylene bisacrylamide (1% w/w), and
BOB (1% w/w) or PBA (1% w/w). For qualitative purposes, hydrogel
slabs containing BOB and PBA were immersed in 2.5 Â 10À4 M ARS
(PBS solution). Covalent incorporation was qualitatively measured
via the observed colour change from red to orange, as measured
against a blank hydrogel (Fig. S1 and S2, ESI†). For quantitative
purposes, hydrogel cylinders (0.1 g) were immersed in a
2.5 Â 10À4 M ARS solution (1 mL) and the UV-Vis absorption
at 513 nm was measured over time. As shown in Fig. S3 and S4
(ESI†), a decrease in absorbance at 513 nm was observed, which
corresponded to ARS uptake into the gel. After approximately
Fig. 2 UV-Vis absorption per gram of BOB upon addition of various
concentrations of H2O2 (0–4 mM) in PBS (pH 7.4, PBS = 0.01 M) over
time (minutes). Absorbance was measured at 513 nm at 25 1C. Error bars
indicate standard deviation (n = 3).
5 h, both PBA and BOB gels were saturated with ARS, which was the concentration of H2O2 led to an increased release of ARS
indicated by no further decrease in absorption at 513 nm. Each from the borogels, as seen in the higher absorption at 513 nm.
gel was then placed into a solution of PBS (1 mL) to wash out Interestingly, the greatest sensitivity and ARS release was
any unbound ARS, which was shown by an increase in absorp- observed for the PBA-based gels, indicative of a greater reactivity
tion at 513 nm (Fig. S5 and S6, ESI†). No further increase in towards H2O2 over the BOB-based gels (see Fig. S7 and S8, ESI†).
absorption was observed after 3 h, which indicated the full This change in sensitivity is rationalised as the BOB moiety
release of any unbound ARS from each gel.
displays an enhanced binding affinity towards 1,2-diols due to
The prepared gels were then used to evaluate the response an adjacent alkyl alcohol coordinating to the boron atom.12,19
towards H2O2. Each gel (PBA and BOB) was placed into a Therefore, we believe the adjacent methyl alcohol retards oxida-
solution of PBS (1 mL) and then exposed to various concentra- tion of ARS bound-boronic acid by H2O2.
tions of H2O2 (0–4 mM). As shown in Fig. 1 and 2, increasing
Recent efforts by Lee and co-workers have demonstrated that
alizarin (10 mg mLÀ1) is an effective inhibitor of biofilm formation
for three Staphylococcus aureus (S. aureus) strains and one
Staphylococcus epidermidis strain.20,21 Biofilms are complex bacterial
communities that can facilitate antibiotic resistance and impair
wound healing.22 Hence, the development of new systems that can
effectively treat or inhibit biofilm formation are highly desirable.
H2O2 is a commonly used disinfectant and antiseptic in
wound care. Therefore, we explored the potential of this system
in the development of a H2O2-responsive hydrogel for the
triggered release of ARS for biofilm inhibition against the three
key stages of bacterial growth: lag, exponential and stationary.
Due to the PBA-based gel displaying the greatest sensitivity
towards H2O2 over BOB-based gels (see Fig. S7 and S8, ESI†),
only PBA gels were evaluated for biofilm inhibition. Control
studies showed that the minimum inhibitory concentration (MIC)
of H2O2 was 3.5–7 mM for Staphylococcus aureus (S. aureus) H560
and MRSA252, 0.8–1.6 mM for Pseudomonas aeruginosa PAO1
(P. aeruginosa PAO1) and 3–6 mM for Escherichia coli NCTC 10418
(E. coli NCTC 10418). Unfortunately, due to poor solubility, no MIC
was determined for ARS against all the bacterial strains used in this
study (see Fig. S9–S11, ESI†).23–25
Fig. 1 UV-Vis absorption per gram of PBA upon addition of various
concentrations of H2O2 (0–4 mM) in PBS (pH 7.4, PBS = 0.01 M) over
time (minutes). Absorbance was measured at 513 nm at 25 1C. Error bars
indicate standard deviation (n = 3).
ARS was able to inhibit biofilm formation for S. aureus
MRSA252 and S. aureus H560 at 100 mM when added during
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detection of H2O2 with concomitant release of ARS for anti-
microbial application. The greatest reactivity towards H2O2 was
observed for the PBA-based gel compared to the BOB-based gel,
attributed to attenuated reactivity of the cyclic BOB-boronate
ester. In addition, the antimicrobial efficacy of each assay
component was evaluated with the aim of developing a ‘‘triggered
release’’ antimicrobial hydrogel. ARS was discovered to be a
potent biofilm inhibitor in combination with hydrogen peroxide
against S. aureus MRSA252, with the ARS loaded PBA-based gel
successfully inhibiting biofilm formation. These results lead us
to suggest that PBA-based gels, in combination with an early
bacterial detection system for a MRSA biomarker, might find
use as a ‘‘smart’’ wound dressing capable of preventing MRSA
biofilm formation.26
Fig. 3 Biofilm inhibition of S. aureus MRSA252 when treated with 2 mM
H2O2 and solution containing released ARS from ARS-PBA-based gel using
2 mM H2O2 (3 h incubation). Experiments were repeated using three
biological replicates, and error bars indicate standard deviation. Statistical
significance of biofilm inhibition was assessed by performing a one-way
ANOVA using GraphPad 7.0. ****p r 0.001 relative to untreated control.
The authors would like to thank the EPSRC for grant EP/
R003556/1. BLP would also like thank James Tudor and Mr and
Mrs Watson for additional funding. GTW would like to thank
the EPSRC and Public Health England. ACS, ATAJ and NTT wish
to thank the EPSRC for funding on Smartwound plasma –
the lag phase of growth (0 h), similar to other reports in the EP/R003939/1. TDJ wishes to thank the Royal Society for a
literature for Alizarin.21 However, ARS was unsuccessful in the Wolfson Research Merit Award. We would like to thank James
inhibition of P. aeruginosa PAO1 and E. coli NCTC 10418 T. Brewster II at Harvard college for helpful discussions and
biofilms at concentrations below 100 mM (see Fig. S14–S16, suggestions to improve the manuscript.
ESI†). Additionally, H2O2 inhibited biofilm formation, albeit
at much higher concentrations, prevents growth at 5 mM for
S. aureus MRSA252 and S. aureus H560, 10 mM for E. coli NCTC
Conflicts of interest
10418, and 100 mM for P. aeruginosa PAO1 (see Fig. S17–S19,
ESI†). H2O2 (2 mM) with ARS (50 mM) acted synergistically,
effecting biofilm inhibition of S. aureus MRSA252 when added
during the lag phase (Fig. S20, ESI†). Unfortunately, this combi-
nation was unable to inhibit biofilm formation at all other growth
phases for each bacterial strain (Fig. S21 and S22, ESI†).
No conflicts of interest.
Notes and references
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