Ratiometric electrochemical detection of hydrogen peroxide and glucose

Article abstract of DOI:10.1039/c7ob00211d

Hydrogen peroxide (H₂O₂) detection is of high importance as it is a versatile (bio)marker whose detection can indicate the presence of explosives, enzyme activity and cell signalling pathways. Herein, we demonstrate the rapid and accurate ratiometric electrochemical detection of H₂O₂ using disposable screen-printed electrodes through a reaction-based indicator assay. Ferrocene derivatives equipped with self-immolative linkers and boronic acid ester moieties were synthesised and tested, and, through a thorough assay optimisation, the optimum probe showed good stability, sensitivity and selectivity towards H₂O₂. The optimised conditions were then applied to the indirect detection of glucose via an enzymatic assay, capable of distinguishing 10 μM from the background within minutes.

Full text of DOI:10.1039/c7ob00211d

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DOI: 10.1039/C7OB00211D
Organic & Biomolecular Chemistry
ARTICLE
Ratiometric Electrochemical Detection of Hydrogen Peroxide and
Glucose
Sean Goggins,a* Ellen A. Apsey,a Mary F. Mahon,a and Christopher G. Frosta
Received 00th January 20xx,
Accepted 00th January 20xx
Hydrogen peroxide (H2O2) detection is of high importance as it is a versatile (bio)marker whose detection can indicate the
presence of explosives, enzyme activity and cell signalling pathways. Herein, we demonstrate the rapid and accurate
ratiometric electrochemical detection of H2O2 using disposable screen-printed electrodes through a reaction-based indicator
assay. Ferrocene derivatives equipped with self-immolative linkers and boronic acid ester moieties were synthesised and
tested, and, through a thorough assay optimisation, the optimum probe showed good stability, sensitivity and selectivity
towards H2O2. The optimised conditions were then applied to the indirect detection of glucose via an enzymatic assay,
capable of distinguishing 10 μM from the background within minutes.
DOI: 10.1039/x0xx00000x
www.rsc.org/
Introduction
Hydrogen peroxide (H2O2) is an important small molecule used
in many industrial applications, such as paper-bleaching and in
the manufacture of disinfectants and explosives.1 In nature, it
has been recently established that H2O2 is involved within a
number of crucial roles related to cell signalling.2 Abnormally
high concentrations of H2O2 can be cytotoxic to cells,3 but more
significantly however, it is a precursor to the indiscriminately
reactive hydroxyl radical (•OH).4 This, along with other reactive
oxygen species (ROS),5 have been shown to contribute to
oxidative stress, a major factor in the onset of various diseases.6
Additionally, H2O2 is produced as a by-product in numerous
enzyme-catalysed processes such as the aerobic oxidation of
alcohols,7 urates,8 amino acids,9 and certain carbohydrates,10
including glucose.11 Moreover, H2O2 has become a popular
signal propagator within signal amplification methodologies
used to enhance the sensitivity of diagnostic assays.12,13 Since
H2O2 can be used as a reactive biomarker for effective disease
diagnosis, and disease monitoring, as well as to infer the
presence of trace amounts of explosives,14 there is therefore a
significant interest in the development of accurate and reliable
hydrogen peroxide detection methods.
at the point-of-need setting. Electrochemistry is therefore
gaining
Fig 1. Design approach towards the ratiometric electrochemical detection of
hydrogen peroxide.
Conventional H2O2 detection is typically achieved through
optical techniques, namely using luminescent,15 or fluorescent
methods.16 However, the need for transparent samples as well
as the use of expensive optical equipment, prohibits their use
increasing popularity due to its low-cost, inherent
miniaturisation capability and simple incorporation into point-
of-care (POC) devices.17 Despite this, the adoption of
electrochemical (bio)sensors into POC devices is hampered by
inaccurate and unreliable results caused by a number of issues;
primarily significant variations in screen-printed electrode
surface areas. One method to improve the accuracy and
reliability of electrochemical (bio)sensors is through the
employment of dual-reporter or ratiometric detection systems.
Over recent years, the electrochemical sensing community have
developed a variety of such innovative protocols for the
a.Department of Chemistry, University of Bath, Claverton Down, Bath, BA2 7AY, UK.
* Corresponding author: s.goggins@bath.ac.uk
†
Electronic Supplementary Information (ESI) available: General information,
materials, electrochemical methods, synthetic routes and procedures for ferrocene
probes, additional differential pulse voltammograms, additional assay figures, full
details of the oxidant screen and copies of NMR spectra for novel compounds. CCDC
1528331. For ESI and crystallographic data in CIF or other electronic format, see
DOI: 10.1039/x0xx00000x
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detection of DNA,18 enzyme activity,19 proteins,20 heavy crystallography (Fig 3.)),29 compound
Journal Name
2
containeVdiewaArrteiccleeOnntllinye-
DOI: 10.10393/Cc7oOnBt0a0i2n1e1Dd
metals,21 and small molecules,22 among others.23 Continuing described allyl carbamate linker,30 and compound
our endeavour into developing ratiometric electrochemical no linker at all. Compounds , structural analogs of
methods towards more accurate and more reliable compounds without the boronic acid trigger, were designed
4–5
1–2
electrochemical biosensors, we describe herein, the to determine and confirm that the specificity of the reactivity
development of a ferrocene-derived probe specifically designed towards H2O2 arises from the boronic acid ester trigger unit.
for the facile ratiometric electrochemical detection of H2O2 and
demonstrate its application towards a reliable electrochemical
glucose chemodosimeter.
Results and Discussion
Optimisation of Probe Structure
H2O2 is itself electrochemically active but only at a high
oxidation potential.24 In order to decrease this overpotential
Fig 3. X-ray crystal structure of probe
and C–H hydrogen atoms have been omitted for clarity. Atom colours: C, green;
O, red; B, purple; N, blue; Fe, orange.§§
1. Ellipsoids are depicted at 30% probability
and increase specificity, previous electrochemical methods for
the detection of H2O2 have focussed upon modifying electrodes
using advanced materials.25 However, the use of modified
electrodes is unfeasible at the point-of-need setting as they can
be expensive and difficult to manufacture, and are currently
unable to be easily mass produced. To obtain a ratiometric
detection method at facile oxidation potentials,§ without the
need for modified electrodes, we looked to begin our
investigation by utilising ferrocene as a redox-active label. Also,
by coupling a boronic acid trigger moiety into the design of the
ferrocene-based probe, we hypothesised that oxidation and
subsequent hydrolysis of the boronic acid trigger would only
occur selectively in the presence of H2O2,26 allowing for an
irreversible reaction-based detection method to be achieved
(Fig. 1).27
With probes 1–5 in hand, 100 μM concentrations of the
probes in pH 8.1 tris(hydroxymethyl)methylamine (Tris) buffer
were exposed to a solution containing 1 mM (10 equivalents) of
H2O2 and the assay analysed after 20 minutes using differential
pulse voltammetry (DPV). When the reaction assays regarding
probes
1 and 2 were analysed, complete disappearance of the
oxidation peak corresponding to the substrate and a new peak,
at a significantly lower oxidation potential, was observed (Fig.
4). This peak was found to be at an identical oxidation potential
as that of aminoferrocene 6, which was synthesised separately
according to a literature procedure.31 As such, this observation
of a ratiometric electrochemical detection method can be
attributed to the as-designed H2O2-mediated oxidation of the
boronic acid moiety to its corresponding alcohol, which is
followed by subsequent linker elimination and carbamate
decarboxylation to release aminoferrocene 6.
2.5
2.0
1.5
1.0
0.5
0.0
Fig. 2 Structures of ferrocene-derived ratiometric electrochemical H2O2 probes 1–
5.
Towards this end, compounds
1–5 were designed and
synthesised (see ESI) with the aim of identifying the optimum
structural criteria needed to attain a selective ratiometric
electrochemical detection method for H2O2 (Fig. 2). Since
different self-immolative linkers exhibit different elimination
-400
-200
0
200
400
kinetics in response to H2O2,28 compounds
to determine the linker that delivered the quickest release of an
electron-rich ferrocene reporter unit. Specifically, compound
1–3 were designed
Potential (mV)
1
Fig. 4 Differential pulse voltammogram (DPV) overlays of probe 1 after exposure
to 10 eq. of H2O2
utilised the commonly employed p-benzyl carbamate linker (the
definitive structure of which was confirmed by X-ray
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The analysis of the peroxide assay with probe
revealed the disappearance of the probe peak but no expected greater ΔEox observed between substrate and product, which is
peak at a lower oxidation potential was seen. This could be due likely caused by its increased hydrophobicity.34
to H2O2-mediated oxidation of the ferroceneboronic acid probe
ARTICLE
3
also further optimisation due to its increased reactiVvieitwyAraticnledOntlhinee
DOI: 10.1039/C7OB00211D
3
to hydroxyferrocene
aqueous conditions,32 rapid decomposition of the product
occurred. When probes and were exposed to H2O2, no peak
corresponding to aminoferrocene was observed showing that
7 but due to the known instability of 7 in
4
5
6
the carbamate functionality is stable towards alkaline peroxide
and confirms that the boronic acid ester trigger moieties are
essential for achieving peroxide selectivity within reaction-
based assays.
Two separate equimolar solutions (100 μM total ferrocene
concentration) containing probe
1 and aminoferrocene 6 (Fig.
5), and probe and (see ESI) were analysed by DPV.
2
6
Differences in oxidation potentials (ΔEox) between probe and
reporter were found to be 268 mV (±14 mV)§§§ and 232 mV (±4
mV)§§§ respectively. These values can be considered more than
sufficient to deliver a ratiometric detection method as an ideal
difference in oxidation potential between substrate and
product has previously been suggested to be between 100–200
mV to ensure both compounds are not oxidised at the same
oxidation potential.33 This allows for, through integration of the
peaks on the voltammogram, reaction conversions to be
calculated via:
Fig. 6 Conversion of probe
1 (100 µM) to aminoferrocene 6 in 50 mM pH 8.1 Tris
buffer in the presence of various concentrations of H2O2.
Assay Optimisation
In order to improve the sensitivity of the assay, a number of
reaction parameters were investigated. First, a range of
different alkaline buffers were tested to determine if the buffer
type had any effect on the reactivity of the probe.
Unfortunately, any diversion away from the originally chosen
Tris buffer either led to the appearance of significant artefacts
on the voltammogram or caused substantial decomposition of
the ferrocene probes. As such, ideal like-for-like comparisons
could not be made since accurate peak integrations, and
therefore precise reaction conversions, were unable to be
obtained.
ퟔ
∫
(
퐶표푛푣. %
)
=
× 100
(
)
ퟔ + ퟏ 표푟 ퟐ
∫
∫
8.0
6.0
4.0
2.0
0.0
-500 -400 -300 -200 -100
0
100 200 300 400 500
Potential vs Ag/AgCl (mV)
Fig. 5 DPV of probe
buffer.
1
(50 µM) and aminoferrocene (50 µM) in 50 mM pH 8.1 Tris
6
In order to determine which probe showed the greatest
reactivity towards H2O2, as well as the quickest elimination
kinetics, 100 μM concentrations of the probes were exposed to
differing concentrations of H2O2 and monitored over time
through ratiometric electrochemical analysis (Fig. 6 and ESI).
Pleasingly, all concentrations of 1 eq. H2O2 and higher afforded
positive production of compound
6. Importantly, a <2%
background rate was also observed exemplifying the excellent
stability of probes with carbamate linkages in aqueous buffers.
This allowed a 100 μM concentration of H2O2 to be determined
from the background. Despite improved solubility in the
Fig. 7 Conversion of probe
1 (100 µM) to aminoferrocene 6, varying the pH of 50
mM Tris buffer in the presence of 500 µM of H2O2.
aqueous medium, probe
1 was taken forward over probe 2 for
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The pH of the assay was next to be studied since peroxide μM (2.5 eq.) H2O2. However, at these elevated temperatures,
View Article Online
DOI: 10.1039/C7OB00211D
reactivity and linker elimination can both be affected through fluctuating conversions can be seen thought to be due to the
changes in pH.35 The useful working pH range of Tris buffer is 7– slow disappearance of the aminoferrocene
6 product peak and
9 and as such, the assay was conducted at various pH values thus indicating decomposition of the product. This can be
within this range (Fig. 7).36 As expected, decreasing the pH of rationalised by the increased oxidation rate, and subsequent
the assay medium led to a significant reduction in conversion fragmentation, of aminoferrocene 6 at these high temperatures
due to the shift in equilibrium from the hydroxide anion (HOO−) via known oxidation pathways.37 To minimise product oxidation,
to its neutral species (H2O2) and also due to the increase in all subsequent assays were performed at room temperature.
stability of the resultant phenol intermediate post-oxidation of Importantly however, at all temperatures tested, minimal
probe
enhancing the reactivity of H2O2 towards probe
quantitative conversion to aminoferrocene
within 20 minutes in the presence of 500 μM (5 eq.) of H2O2.
1
. Increasing the pH above 8.1 had the desired effect of background conversions were observed (<2%), which reinforces
, enabling near the high stability of the carbamate functionality to undesired
to be obtained background hydrolysis.
The optimised assay parameters were then applied to the
detection of different H2O2 concentrations using probe (Fig. 8)
1
6
1
and a calibration curve obtained (Fig. 9). Overall, the
optimisation allowed for near quantitative conversions to
aminoferrocene
6 to be achieved in just 20 minutes in the
presence of 10 equivalents of H2O2 and importantly, in the
absence of any peroxide, no conversion was seen. A linear
dynamic range between 0 and 800 µM of H2O2 could also be
observed.
80%
70%
60%
50%
40%
30%
20%
10%
0%
Fig. 8 Conversion of probe
1 (100 µM) to aminoferrocene 6 in 50 mM pH 9 Tris
buffer in the presence of various concentrations of H2O2.
Oxidant
Fig. 9 Calibration curve for the conversion of probe
after 20 minutes at varying H2O2 concentrations.
1 (100 µM) to aminoferrocene
6
Fig. 10 Conversion of probe
buffer in the presence of 500 µM of varying oxidants at room temperature.
Abbreviations: mCPBA meta-Chloroperbenzoic Acid, Oxone® Potassium
1 (100 µM) to aminoferrocene 6 in 50 mM pH 9.0 Tris
=
=
Peroxymonosulfate, TEMPO
= 2,2,6,6-Tetramethyl-1-piperidinyloxy, CHP =
Finally, in a bid to improve the sensitivity of the assay
Cumene Hydroperoxide, NMO = 4-Methylmorpholine N-Oxide, Luperox® = Di-tert-
butyl Peroxide, TBHP = tert-Butylhydrogen Peroxide.
further, the temperature of the reaction was next to be looked
at (see ESI). Increasing the temperature of the assay from room
temperature to 37°C was found to only slightly increase
reaction conversion. Increasing the temperature further led to
a dramatic rate increase with near quantitative conversions
being obtained within 20 minutes in the presence of only 250
Peroxide Selectivity Studies
To determine the selectivity of the probe for H2O2, a range of
different peroxides, oxidants and salts were screened (Fig. 10).
Specifically, a 100 μM solution of probe
1 was exposed to 5
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equivalents of the oxidant and after 20 minutes of vigorous greatly reduced. Importantly, in the absence of GOx, neither
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stirring, a sample of the assay was taken and subjected to DPV conversion of
1
to aminoferrocene 6, orDaOnI:y10s.h10if3t9i/nC7oOxBid00a2ti1o1Dn
analysis. Of all oxidants screened, sodium percarbonate was the potential,45 was observed showing that conversion of the probe
only oxidant other than H2O2 to give >15% conversion of probe was not occurring through a supramolecular interaction
1
to aminoferrocene
6
. Sodium carbonate itself delivered 0% between the boronic acid ester and glucose.46
As both high sensitivity for the analyte and a quick time-to-
confirming that the conversion observed for sodium
percarbonate is caused by the ≈66% contained H2O2. meta- response are both critical factors in the implementation of
Chloroperbenzoic acid (mCPBA) furnished 14% conversion over diagnostic assays within point-of-need biosensors, we chose to
the same time period but significant decomposition of both
1
take forward the highest concentration of GOx previously
and was observed in this case, presumably due to oxidation of tested. Thus, to attain the sensitivity of the ratiometric
6
the iron (II) centre. Other peroxides, such as Oxone®, cumene electrochemical assay towards glucose, a range of glucose
hydroperoxide (CHP), Luperox® and tert-butylhydrogen concentrations were screened in the presence of 100 UmL−1
peroxide (TBHP), all gave little to no conversion showing the GOx (Fig. 11).
excellent selectivity of probe
1 to H2O2. Other ROS, including
free radicals (TEMPO), N-oxides (NMO), hypochlorite and
nitrite, among others (see ESI for full oxidant screen), also
afforded minimal conversion if any.
Ratiometric Electrochemical Glucose Detection
According to the World Health Organisation, an estimated 422
million people suffer with diabetes globally and approximately
90% suffer with Type II; the type most commonly caused by
behavioural and environmental risk factors.38 Left unregulated,
elevated blood glucose levels can inflict significant capillary
vessel damage and, depending on its location in the body, can
go on to cause retinopathy, kidney failure and the onset of
cardiovascular disease.39 Evidently, glucose concentrations in
blood needs to be regularly measured reliably and to a high
level of accuracy (±20% for concentrations above 5.6 mM or
within ±0.83 mM for below).40 Surprisingly however, many
currently commercially available glucose biosensors do not
meet this standard.41 There is therefore still significant room for
improvement regarding improving the accuracy and reliability
of glucose detection.
Fig. 11 Conversion of probe 1 (100 µM) to aminoferrocene 6 in 50 mM pH 9.0 Tris
buffer in the presence of 100 UmL−1 GOx with varying concentrations of α-
D-
glucose.
Selective enzymatic reactions are often employed within
electrochemical analyte detection methods to minimise noise
from the possible presence of electrooxidisable interferents in
the sample matrix.42 For electrochemical glucose sensing,
glucose oxidase (GOx) is most commonly chosen,43 and in the
process of oxidising glucose to
D-glucono-δ-lactone, the
mechanism of action for GOx also reduces molecular oxygen to
hydrogen peroxide (H2O2).44 As such, we hypothesised that we
could also apply probe
1 under our previously optimised
conditions towards the ratiometric electrochemical detection
of glucose.
Initially, we sought to investigate the level of GOx activity
needed to achieve full conversion of probe
1 in the shortest
time (see ESI), to help facilitate implementation of the
methodology into a point-of-need device in the future. The Fig. 12 Calibration curve for the conversion of probe
1 (100 µM) to
aminoferrocene
6 after 10 minutes.
concentration of glucose within the assay was chosen to be 5
mM (50 eq.) to ensure that enough H2O2 would be released to
Similar to previous, high concentrations of glucose (>5 mM)
were able to successfully achieve full conversion of probe to
within the 20-minute timeframe. Again, in the absence of the
sugar, no conversion was observed which highlights the
selectivity of the probe towards the enzyme-catalysed
production of H2O2 and not through any undesired interaction
deliver full conversion of
1
. As expected, high levels of GOx
to within
1
6
activity (>50 UmL−1) delivered full conversion of
1
6
the 20-minute assay time. Lowering the concentration of the
enzyme led to a much slower conversion since the rate of
glucose oxidation, and therefore H2O2 production, would be
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§§ Crystal Data for compound
1. C24H28NO4FeB, M =461.13,
with the enzyme. The assay also demonstrated a good dynamic
range over two orders of magnitude allowing for glucose
concentrations between 10 µM and 1 mM to be easily
distinguishable after just 10 minutes (Fig. 12). Glucose
concentrations in blood, and other bodily fluids, are typically
found within this range,47 which lends this ratiometric detection
method towards such application if desired. As all ratiometric
electrochemical conversions have been determined through
the use of screen-printed carbon electrodes, subsequent work
will involve the implementation of the methodology into a
point-of-need biosensor with the aim of developing a device
with an improved accuracy over those which are currently
commercially available.
triclinic, space group P-1 (no. 2), a = 9.8839(4), b = V9i.e9w2A3rt3ic(le4O),nclin=e
DOI: 10.1039/C7OB00211D
13.1134(5) Å, α = 109.219(4), β = 93.721(3), γ = 115.017(4)°, U =
1068.78(7) Å3, Z = 2, T = 150 K, μ(Cu Kα) = 5.914 mm-1, Dc =
1.433 gcm-3, 10333 reflections measured (10.32° ≤ 2ϴ ≤ 143.94°),
4176 unique (Rint = 0.0448) which were used in all calculations.
The final R1 was 0.0421 (I>2 σ (I)) and wR2 was 0.1112 (all data).
CCDC 1528331 contains the supplementary crystallographic data
for 1.
§§§ Differences in oxidation potentials (ΔEox) were calculated as
the mean average from a set of 8 separate electrochemical
experiments (DPV) performed on the same sample solution. ±
errors are the standard deviation from this mean (n = 8).
1
2
3
C. W. Jones, Applications of Hydrogen Peroxide and
Derivatives, The Royal Society of Chemistry, Cambridge, 1999,
ch. 6, pp. 231–251.
Conclusions
(a) Z. A. Wood, L. B. Poole and P. A. Karplus, Science, 2003,
300, 650; (b) S. G. Rhee, Science, 2006, 312, 1882; (c) E. A.
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Richter-Landsberg, J. Neurochem., 1999, 73, 2501; (e) P.
Daroui, S. D. Desai, T.-K. Li, A. A. Liu and L. F. Liu, J. Biol. Chem.,
2004, 279, 14587.
In conclusion, two ferrocene-derived probes were designed for
the ratiometric electrochemical detection of hydrogen peroxide
(H2O2) and synthesised to contain two different self-immolative
linkers for comparison. The probe containing
a benzyl
carbamate linker was found to give a larger difference in
oxidation potential between probe and product and exhibited
faster elimination kinetics than the probe containing an allyl
carbamate linker. An optimisation of the diagnostic assay
parameters found that pH 9 tris(hydroxymethyl)methylamine
(Tris) buffer at room temperature afforded efficient conversion
of probe to product while minimising electrochemical artefacts
and any undesired product oxidation. The probe was also found
to be very selective for H2O2 compared with a range of other
common oxidants.
The ratiometric electrochemical detection methodology
was then applied to the detection of glucose through the
concomitant glucose oxidase (GOx)-catalysed reduction of O2 to
H2O2. The optimum enzyme concentration was initially found
prior to the determination of the sensitivity of the glucose
assay. Glucose concentrations as low as 50 µM could be
determined from the background rate within 20 minutes and
the assay also exhibited a good dynamic range over 2 orders of
magnitude.
4
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Acknowledgements
165.
12 For reviews: (a) P. Scrimin and L. J. Prins, Chem. Soc. Rev.,
2011, 40, 4488; (b) O. V. Makhlynets and I. V. Korendovych,
The authors would like to thank Atlas Genetics Ltd. for the kind
donation of a potentiostat and for providing the screen-printed
carbon electrodes. They would also like to acknowledge the use
of nuclear magnetic resonance (NMR) and mass spectrometry
(MS) facilities within the Chemical Characterisation and Analysis
Facility (CCAF) at the University of Bath for compound
characterisation.
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Notes and references
§ Facile oxidation potentials are considered to be 0 mV (±500 mV)
vs. Ag/AgCl as this minimises the risk of interfering redox
reactions from taking place and where the background current is
lowest.
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Organic & Biomolecular Chemistry
Page 8 of 8
View Article Online
DOI: 10.1039/C7OB00211D
Ferrocene-derived probes equipped with self-immolative linkers and boronic ester trigger moieties
enable selective electrochemical detection of hydrogen peroxide, and glucose.