Extracellular hydrogen peroxide measurements using a flow injection system in combination with microdialysis probes – Potential and challenges

Article abstract of DOI:10.1016/j.freeradbiomed.2018.05.089

There is a strong need for techniques that can quantify the important reactive oxygen species hydrogen peroxide (H₂O₂) in complex media and in vivo. We combined chemiluminescence-based H₂O₂ measurements on a commercially available flow injection analysis (FIA) system with sampling of the analyte using microdialysis probes (MDPs), typically used for measurements in tissue. This allows minimally invasive, quantitative measurements of extracellular H₂O₂ concentration and dynamics utilizing the chemiluminescent reaction of H₂O₂ with acridinium ester. By coupling MDPs to the FIA system, measurements are no longer limited to filtered, liquid samples with low viscosity, as sampling via a MDP is based on a dynamic exchange through a permeable membrane with a specific cut-off. This allows continuous monitoring of dynamic changes in H₂O₂ concentrations, alleviates potential pH effects on the measurements, and allows for flexible application in different media and systems. We give a detailed description of the novel experimental setup and its measuring characteristics along with examples of application in different media and organisms to highlight its broad applicability, but also to discuss current limitations and challenges. The combined FIA-MDP approach for H₂O₂ quantification was used in different biological systems ranging from marine biology, using the model organism Exaiptasia pallida (light stress induced H₂O₂ release up to ~ 2.7 μM), over biomedical applications quantifying enzyme dynamics (glucose oxidase in a glucose solution producing up to ~ 60 μM H₂O₂ and the subsequent addition of catalase to monitor the H₂O₂ degradation process) and the ability of bacteria to modify their direct environment by regulating H₂O₂ concentrations in their surrounding media. This was shown by the bacteria Pseudomonas aeruginosa degrading ~ 18 μM background H₂O₂ in LB-broth. We also discuss advantages and current limitations of the FIA-MDP system, including a discussion of potential cross-sensitivity and interfering chemical species.

Full text of DOI:10.1016/j.freeradbiomed.2018.05.089

Author’s Accepted Manuscript
Extracellular Hydrogen Peroxide Measurements
Using a Flow Injection System in Combination with
Microdialysis Probes – Potential and Challenges
Maria Moßhammer, Verena Schrameyer, Peter Ø.
Jensen, Klaus Koren, Michael Kühl
www.elsevier.com
PII:
S0891-5849(18)30952-3
https://doi.org/10.1016/j.freeradbiomed.2018.05.089
FRB13791
DOI:
Reference:
To appear in:
Free Radical Biology and Medicine
Received date: 10 January 2018
Revised date: 14 May 2018
Accepted date: 29 May 2018
Cite this article as: Maria Moßhammer, Verena Schrameyer, Peter Ø. Jensen,
Klaus Koren and Michael Kühl, Extracellular Hydrogen Peroxide Measurements
Using a Flow Injection System in Combination with Microdialysis Probes –
Potential
and
Challenges, Free Radical Biology and Medicine,
https://doi.org/10.1016/j.freeradbiomed.2018.05.089
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Extracellular Hydrogen Peroxide Measurements Using a Flow Injection
System in Combination with Microdialysis Probes – Potential and
Challenges
Maria Moßhammer1, Verena Schrameyer1, Peter Ø. Jensen2,3, Klaus Koren1,4* and Michael Kühl1,5*
1
Marine Biological Section, Department of Biology, University of Copenhagen, Denmark
Department of Clinical Microbiology, Rigshospitalet, Denmark
Department of Immunology and Microbiology, Costerton Biofilm Center, Faculty of Health and Medical Sciences,
2
3
University of Copenhagen, Denmark
Department of Bioscience – Microbiology, University of Aarhus, Denmark
Climate Change Cluster, University of Technology Sydney, Australia
4
5
*
Correspondence: mkuhl@bio.ku.dk, klaus.koren@bios.au.dk
Abstract
There is a strong need for techniques that can quantify the important reactive oxygen species hydrogen peroxide (H2O2)
in complex media and in vivo. We combined chemiluminescence-based H2O2 measurements on a commercially
available flow injection analysis (FIA) system with sampling of the analyte using microdialysis probes (MDPs),
typically used for measurements in tissue. This allows minimally invasive, quantitative measurements of extracellular
H2O2 concentration and dynamics utilizing the chemiluminescent reaction of H2O2 with acridinium ester. By coupling
MDPs to the FIA system, measurements are no longer limited to filtered, liquid samples with low viscosity, as sampling
via a MDP is based on a dynamic exchange through a permeable membrane with a specific cut-off. This allows
continuous monitoring of dynamic changes in H2O2 concentrations, alleviates potential pH effects on the measurements,
and allows for flexible application in different media and systems. We give a detailed description of the novel
experimental setup and its measuring characteristics along with examples of application in different media and
organisms to highlight its broad applicability, but also to discuss current limitations and challenges. The combined
FIA-MDP approach for H2O2 quantification was used in different biological systems ranging from marine biology,
using the model organism Exaiptasia pallida (light stress induced H2O2 release up to ~2.7 µM), over biomedical
applications quantifying enzyme dynamics (glucose oxidase in a glucose solution producing up to ~60 µM H2O2 and the
subsequent addition of catalase to monitor the H2O2 degradation process) and the ability of bacteria to modify their
direct environment by regulating H2O2 concentrations in their surrounding media. This was shown by the bacteria
Pseudomonas aeruginosa degrading ~18 µM background H2O2 in LB-broth). We also discuss advantages and current
limitations of the FIA-MDP system, including a discussion of potential cross-sensitivity and interfering chemical
species.
Keywords:
Hydrogen peroxide, H2O2, ROS, Flow injection analysis, FIA, Microdialysis probe, Extracellular measurement, Optical
sensor, Chemiluminescence
1. Introduction
Hydrogen peroxide (H2O2) is a reactive oxygen species (ROS) [1] and a strong oxidant, but relatively unreactive
in comparison to other ROS [2]. The peroxide bond is nonetheless prone to cleaving due to heating, photolysis or
*
Abbreviations: H2O2, hydrogen peroxide; ROS, reactive oxygen species; FIA, flow injection analysis; MDP, microdialysis probe; MDP-FIA, flow
injection system coupled to a microdialysis probe; GOX, glucose oxidase; CL, chemiluminescent reagent; AE, acridinium ester; PMT,
photomultiplier tube; ID, inner diameter; OD, outer diameter; LOD, limit of detection; LOQ, limit of quantification; DI, deionized water; LB medium,
Luria-Bertani broth; PBS, saline phosphate buffer, F2 medium, enriched seawater medium; ASW, artificial seawater; FSW, filtered seawater
* Corresponding authors.
E-mail address: klaus.koren@bios.au.dk, mkuhl@bio.ku.dk
1

contact with redox metals [2,3]. These cleaving reactions can form the highly reactive hydroxyl radical (HO●). Its
reactions have very low activation energy barrier and is one of the strongest known oxidants [2,4]; thus it is very
important to understand the production and degradation processes of H2O2 as one of the main sources of HO●.
Furthermore, H2O2 is of great importance in cell biology, where it plays a key role in important biochemical processes
such as oxidative stress and as a transmitter of redox signals [2,5]. However, the exact role of H2O2 is often not fully
understood due to uncertainties in the determination of real concentrations in vivo [6].
Some organisms such as algae [7] or human granulocytes (PMN) [8] release H2O2, while most cells and
organism can sense and respond to H2O2 in their environment [5,9]. In many systems it is, however, still not possible to
determine exact concentrations or monitor actual H2O2 dynamics, due to a lack of suitable techniques [6,9]. In an
environmental context, H2O2 is released as an oxidative stress response by e.g. algae [10–15] or corals harboring
microalgal symbionts [6,16]. H2O2 can also be formed via different (photo)chemical pathways in lakes [17,18], rivers
[19,20], rainwater [17,19,21–25], geothermal springs [26] and open ocean seawater [19,27–30], and H2O2 has even
been measured in thousands of years old ice cores [31,32]. In humans, H2O2 has been measured in exhaled air [33–35],
urine [36,37], blood [38–40] or ocular fluids [41], and it functions as an important signaling agent between cells [42].
Hydrogen peroxide has also been linked to various diseases such as neurodegenerative diseases, Alzheimer’s or lung
infections [33,43,44] underlining the concentration-dependent dual character of H2O2 as a both harmful and important
compound in biological systems. There is thus a strong need for methods that can quantify concentrations of H2O2 in
vivo or under in vivo-like conditions with minimal sample manipulation. Such measurements are paramount for our
understanding of the processes regulating the release and degradation of H2O2 in different environmental and
biomedical systems.
Quantification of H2O2 is frequently attempted with optical techniques based on irreversible luminescent probes.
Such approaches can be used to detect H2O2 via the luminescence intensity upon probe exposure giving valuable
insights into hotspots and the presence of a species. However, correlating such changes in luminescence intensity to real
H2O2 concentrations is difficult and often impossible [6]. A frequently used and well established technique for the
determination of H2O2 in liquid samples is based on flow injection analysis (FIA) in combination with a H2O2-specific
chemiluminescent reaction (Figure 1) [6,19]. This approach enables dynamic H2O2 concentration measurements in
retrieved samples in near real time, where FIA overcomes the issue of the irreversibility of the underlying reaction
scheme. Typically, such FIA-based systems consist of a valve system, which can switch between two modes (load and
injection), a peristaltic pump ensuring a continuous flow of sample and reagents, and a photomultiplier tube coupled to
a mixing cell for sensitive detection of the chemiluminescence intensity caused by the reaction of a chemiluminescent
Figure 1: Schematic representation of a flow injection analysis (FIA) system used to quantify H2O2 in the load
mode. The aggregate free sample is acquired with a sample tube and filled in the sample loop. When switching to
injection mode, the sample is mixed with the chemiluminescent reagent (CL) and flushed by the carrier solution into a
mixing chamber, where it is mixed with a buffer solution to adjust the pH and facilitate the reaction with the
chemiluminescent reagent. The mixing chamber is optically coupled to a photomultiplier tube (PMT) detector that
monitors the analyte-dependent chemiluminescence signal. Even flow of sample and reagents is facilitated with a
peristaltic pump.
reagent (CL reagent) with the analyte of interest. Different chemistries such as luminol with a Co(II) catalyst [17,29,45–
47] or the acridinium ester (10-methyl-9-(p-formylphenyl)-acridinium carboxylate trifluoromethanesulfonate) [19,48–
51] have been used for H2O2 detection. Both chemiluminescence reaction schemes are pH dependent and show cross-
sensitivities to Fe(II), which can however be overcome by working with buffered solutions and addition of FerroZineTM
to complex freely available Fe(II) [45,48]. Temperature effects have to be taken into consideration as well, as
chemiluminescent reactions are temperature dependent [52]. Luminol-based measurements are also influenced by other
2

analytes [45], while acridinium ester (AE) based measurements (employed in this study) are described as highly
selective towards H2O2 in the literature [19,49]. However, we observed various media effects indicative of potential
cross-sensitivity and interfering species, and we conducted a detailed study of cross-sensitivity and potential interfering
species (see Supplementary information, results and discussion sections).
A major shortcoming of the overall FIA technique is its restriction to aggregate-free, liquid samples of low
viscosity, as connectors and tubes are very narrow and block easily. Thus, the sample often needs a pretreatment step,
such as filtering. Additionally, a relatively large sample volume (up to a few mL) is typically required, which can
impose dramatic changes and artefacts in systems with a restricted volume and limits the spatio-temporal resolution. To
overcome these limitations, we combined the FIA system with sampling via so-called microdialysis probes (MDPs)
(Figure 2). Such probes are minimally invasive as analytes are retrieved from the sample via diffusion across a
microdialysis membrane with a defined molecular cut-off into a perfusion solution. MDPs consist of a semipermeable
membrane, which can be used to dialyze liquid, semisolid or solid media [53]; they were first developed for minimally
invasive in vivo measurements of drug concentrations in tissue and organs of animals or humans [54]. MDPs can be
applied in vivo as well as in vitro; they have been frequently coupled to different analytical systems, such as liquid
chromatography, flow-through biosensors and capillary and microchip electrophoresis [55] and also allow
measurements beyond liquid samples [53].
The analyte recovery by the MDPs is dependent on various factors, such as MDP membrane length, perfusion
speed of the internal carrier solution or temperature, making it a tunable system for specific applications with a variety
of sampling schemes [54]. In combination with a dynamic measurement technique such as the FIA system, MPDs can
thus facilitate flexible measurements in a range of media and systems. In this study, we give a detailed presentation of a
MDP-based FIA system for minimally invasive quantification of H2O2 concentration and dynamics. We give a detailed
description of the novel experimental setup and its measuring characteristics along with different medical and biological
applications to highlight its broad applicability, but also to discuss current limitations and challenges.
2. Materials and Methods
2.1. Chemicals and Stock Solution Preparation
10-Methyl-9-(phenoxycarbonyl)acridinium fluorosulfonate (catalog number: 68617) (AE), hydrogen peroxide
(H2O2) solution, 30 % (w/w) (catalog number: 216763), catalase from bovine liver (catalog number: 1001937057),
glucose oxidase from Aspergillus niger (catalog number: G7141-10KU), and FerroZineTM (catalog number: 160601)
were purchased from Sigma Aldrich (sigmaaldrich.com). Na2CO3 was purchased from Riedel-deHaën
(riedeldehaen.com; catalog number: 31432), HCl (37%) was purchased from Merck (merck-chemicals.com, catalog
number: 1.01834.2500). The Pseudomonas aeruginosa strain used in this work was a catalase A negative PAO1 strain
(∆katA) [56]. Luria-Bertani (LB) broth was prepared from 5 g L-1 yeast extract (oxoid.com; catalog number: LP0021),
10 g L-1 tryptone-broth purchased from Oxoid (oxoid.com; catalog number: LP0042), and 10 g L-1 NaCl
(merckmillipore.com; catalog number: 106404) at a pH of 7.5. F2 medium, i.e., an enriched seawater medium
commonly used to culture marine microalgae, was prepared from filtered and autoclaved natural seawater supplemented
.
with previously prepared stock solutions (final concentrations indicated in brackets): NaNO3 (88.25 µM), NaH2PO4
H2O (31.26 µM), a trace metal stock solution containing (FeCl3 (11.30 µM), Na2EDTA (12.97 µM), CuSO4 (0.040 µM),
Na2MoO4 (0.025 µM), ZnSO4 (0.077 µM), CoCl2 (0.077 µM), MnCl2 (1.430 µM)), as well as vitamin stock solution
containing thiamine.HCl (vitamin B1; 296.5 nM), biotin (vitamin H; 2.047 nM), cyanocobalamin (vitamin B12; 0.3689
nM). All ingredients for these solutions were purchased from Sigma Aldrich). Artificial Seawater (ASW) was prepared
from de-ionized water (DI water) and probiotic reef salt, purchased from Aquaforest (aquaforest.eu) and mixed
according to the manufacturer’s instructions, where the final ASW had a salinity of 35. The ASW was autoclaved
before usage. MilliQ water, DI water and other carrier solutions were treated with 3 mg catalase L-1 for at least 30
minutes before further use, and were used to make all solutions. HCl as well as Na2CO3 and AE stock solution were
added after the treatment of the DI water with catalase (3 mg L-1, left for at least 30 minutes) to make a 0.01 M HCl
solution, the 0.1 M Na2CO3 buffer solution (pH ~11.3) and the AE solutions. In experiments where free Fe(II) was
expected, FerroZineTM was added to the AE solution to a final concentration of 250 nM. H2O2 stock solutions were
freshly prepared every day, using untreated DI water. SI: NaCl, CuCl2 . 2 H2O, FeCl3 . 6 H2O, tert-butyl peroxide, urea,
L-cysteine, glutathione, NaOCl and PBS were purchased from Sigma Aldrich (sigmaaldrich.com); ONOONa was
.
purchased from BioNordika (bionordika.dk), NaOH as well as MnSO4
H2O were purchased from Merck
.
(merckmillipore.com), Agar was bought from PanReac AppliChem (applichemus.com) and FeCl2 4 H2O from Fluka
(fishersci.dk). The concentration of peroxynitrite (ONOONa) was determined via UV/VIS spectrophotometer (max
=
302 nM;  = 1670 M-1 cm-1) according to the product information sheet.
2.2. Instrumentation
3

We used a flow injection analysis system (FeLume; Waterville Analytical) with the dedicated analysis software.
The FIA system was coupled to a peristaltic pump (Dynamax, Rainin Instrument Company; shoprainin.com) and a
syringe pump (Aladdin, World Precision Instruments; wpiinc.com) using PVC pump tubes (Tygon R3607, red/red)
obtained from Glass Expansion (geicp.com). The tubing in-between valves in the FIA system consisted of
fluoropolymer tubing (Upchurch Scientific® FEP; idex-hs.com). Microdialysis probes (CMA 7 Metal Free, 2 mm
membrane length, with a cut-off of 6 kDa cut-off, cuprophane membrane (used for all measurements unless specified
otherwise) and CMA 12 Metal Free, 4 mm membrane length, 20 kDa cut-off, polyarylethersulfone membrane
polyarylethersulfone membrane (used for sub µM calibration, Figure S8 B)) were purchased from CMA
(microdialysis.se, reference number: 8010772) and handled according to the manufacturer’s guidelines, flushing it with
ethanol prior to the first usage. Measurements of O2 concentration were done with a FireStingGO2 meter in combination
with an OXR430 fiber-optic O2 sensor or a respiration vial equipped with an O2 sensor spot (OXVIAL20) that was
monitored via the transparent glass wall of the vial with a bare fiber connected to the meter; all items were obtained
from Pyroscience (pyro-science.com). Electrochemical measurements of H2O2 were done with a H2O2 microsensor
(ISO-HPO-100) coupled to a free radical analysis system (both obtained from World Precision Instruments;
wpiinc.com). Solutions were filtered through syringe filters (Whatman, 0.02 µm, 10 mm; AnatopTM 10,
gelifesciences.com; catalog number: 6809-1102). Samples were illuminated with a fiber-optic tungsten-halogen lamp
equipped with a branched flexible light guide (KL1500, Schott.com), and incident light levels (400-700 nm) for
different lamp settings were quantified in the samples with a submersible, spherical mini quantum sensor (US-SQS/L)
coupled to a calibrated quantum irradiance meter (ULM-500) both purchased from Walz (walz.com).
2.3. Flow Injection Analysis (FIA) System Combined MDPs
The commercially available FIA system has a 10-port valve, which can be switched between load mode and
injection mode (Figure 2) via the PC-controlled software. The system can be set up to also include a washing loop (e.g.
diluted HCl) in order to clean the system tubings; this is e.g. relevant when using seawater in order to dissolve
precipitates formed during the measurement [57]. The chemistry and chemiluminescent reaction for H2O2
measurements was described by King et al. [58]. Briefly, the chemiluminescent acridinium ester reagent (AE) reacts
irreversibly with H2O2 forming an instable dioxetane complex, which releases energy in the form of light upon
degradation to N-methylacridone under alkaline conditions. The chemiluminescence is detected by the PMT as a signal
peak for a particular sample. In order to not over-saturate the PMT, signal intensities above ~1 million counts were
avoided by adjusting the concentration of the chemiluminescent reagent. The peak integral can then be correlated – after
prior calibration – to a specific H2O2 concentration. The MDPs allow the diffusive exchange of the analyte between the
external sample medium and the inner, analyte-free probe perfusion medium, which are separated by a microdialysis
membrane, with a specific molecular cut-off. A perfusion fluid (untreated DI water) is pumped through the dialysis
probe at a certain speed, and is then transported into the 20 cm long sample loop of the flow injection system. The inner
diameter (ID) of the FEP tubing used for the sample loop is 0.75 mm, which means the minimal sample volume is 88.4
µL (a small excess is however recommended to make sure the loop as well as the connectors are filled with bubble free
sample solution). The sample loop is then flushed out by a catalase treated carrier solution, mixed with the AE reagent
and transported into the reaction chamber in front of the PMT, where the pH is adjusted by a buffer solution (0.1 M
Na2CO3, pH ~11.3). Switching back to ‘sample mode’ flushes the system with HCl (0.01 M) to keep the system from
clogging due to precipitates (Figure 2.2).
4

Figure 2: Load and injection mode of the FIA system coupled with the microdialysis probe (MDP). 1) Load mode.
H2O2 is sampled via the microdialysis probe and guided into the sample loop. Excess solution will be flushed into the
waste. Once the sample loop is filled, the 10-port valve can be switched to 2) injection mode. The sample solution
containing H2O2 is flushed out of the sample loop, is mixed with the acridinium ester (AE) reagent and then transported
into the detector mixing chamber by the carrier solution (such as catalase treated MilliQ water or seawater), where it is
mixed with a buffer solution adjusting the pH. The microdialysis probe is operated by a syringe pump, where different
speeds can be set. All other solutions (carrier solution, HCl solution / acid wash, chemiluminescent reagent (acridinium
ester) and the Na2CO3 buffer are pumped through the system using a peristaltic pump.
2.4. Calibration With and Without MDPs
Calibration of the FIA system without the MDP connected (Figure 3A and Figure 4A) were done by adding
defined volumes of the H2O2 stock solution (made in DI water) to the measurement medium. The same medium was
used as carrier solution but treated with 3 mg L-1 catalase and filtered if necessary. For calibrations with the MDP
connected to the FIA system (Figure 3A and Figure 4B), the perfusion-fluid pumped from the syringe through the MDP
was untreated DI water, while DI water treated with 3 mg L-1 catalase was used as a reagent carrier solution and
calibration solutions were stirred. As H2O2 has to enter the MDP via diffusion, a diffusion boundary layer establishes
around the probe making it sensitive to stirring. The H2O2 stock solution was made in DI water and added to the stirred
sample medium. For calibrations at elevated temperatures, vials containing the medium were heated to the required
temperature, and H2O2 was only added right before measurement to avoid loss due to degradation. Real zero values
were determined by adding catalase to a medium blank. The limit of detection (LOD) and limit of quantification (LOQ)
were determined according to literature [59,60] by taking 3.3 times the standard deviation of the catalase treated blank
divided by the slope of the calibration curve (LOD; equation 1) or 10 times the standard deviation of the catalase treated
blank divided by the slope of the calibration curve (LOQ; equation 2).
5

Where σ is the standard deviation of the catalase treated blank (n = 3) and k is the slope of the calibration curve.
2.5. Calibration in Agar
1 wt% agar was dissolved in DI water using the microwave and 1 mL of the solution was pipetted in the plates to
result in agar plates of 0.5 cm height. After cooling to room temperature, H2O2 stock solutions were pipetted on top of
the agar plates and left to equilibrate for 3 h. H2O2 concentrations were measured within the agar plate and in the
medium column above it.
2.6. Cross-Sensitivity Studies
Cross-sensitivity studies were mainly conducted with the direct inlet mode of the FIA system without MDPs.
First, pH effects of the sample were tested (see Figure S2). A potential salinity effect was investigated by measuring
H2O2 calibration curves with different NaCl backgrounds in DI water (0 g / 100 g, 5.1 g / 100 g, 15 g / 100 g and 21 g /
100 g DI water)(see Figure S3). Other potentially interfering species (CuCl2, FeCl2, FeCl3, MnSO4, tert-butyl peroxide,
1
urea, L-cysteine, glutathione, NaOCl; ONOONa and O2) were investigated by first measuring in a 50 µM H2O2 stock
solution, and then in DI water treated with the potentially interfering species. Subsequently, the potentially interfering
species was added to catalase treated MQ water to rule out the production of H2O2, which would interfere with the
signal as well, and lastly the interfering species was measured with a 50 µM H2O2 background to rule out the reaction of
the investigated species with H2O2 or quenching of the chemiluminescence (Figure S4-7).
2.7. Enzymatic Reaction Measurements
The experiment was conducted at room temperature in 200 mL of a stirred PBS buffered, glucose solution (1
mM), wherein H2O2 and O2 were measured simultaneously (Figure 5) using a similar protocol of enzyme addition as in
Koren et al. [61]. The sample solution was kept in a container sealed with a rubber stopper, to avoid O2 leakage. The
rubber stopper had three holes, one for the O2 sensor, one for the MDP mounted on a glass rod, and one for the addition
of the chemicals, which was sealed in-between reagent additions. Glucose oxidase (GOX) and catalase stock solutions
were prepared in DI water (5 mg mL-1). After baseline measurements, 0.125 mg GOX was added. The reaction was
followed for 30 minutes. Then 0.125 mg catalase was added and the reaction was followed for 20 minutes. 0.250 mg
GOX was added and the reaction monitored for 15 minutes. 0.500 mg catalase was added and the reaction followed for
15 minutes. Finally, 0.250 mg GOX was added together with additional 1mM glucose, and the reaction was followed
until O2 was consumed completely and no more H2O2 was produced. O2 input via the MDP membrane was tested for in
Figure S3.
2.8. H2O2 Dynamics in Light Stressed Sea Anemones
The sea anemone Exaiptasia pallida (strain H2) was transferred to a beaker containing aerated artificial seawater
heated to 32°C. The organism was first kept in the dark for 60 minutes, while monitoring H2O2 (Figure 6) and O2 levels.
Subsequently, the sea anemone was exposed to high light (~1500 µmol photons m-2 s-1; 400-700 nm) by irradiation
from the side and top with a 2-branch flexible light guide connected to a fiber-optic tungsten-halogen lamp (KL1500,
Schott). After ~80 minutes, the MDP was positioned in closer proximity to the animal than in the first position. The
H2O2 build up due to light stress was followed for approximately 1 hour.
2.9. H2O2 Levels in LB Medium
The bacterium Pseudomonas aeruginosa was grown overnight at 37°C in an Erlenmeyer flask shaken at 170
rpm. Three falcon tubes were filled with 20 mL autoclaved and diluted LB medium (4mL LB + 16 mL DI) heated to
37°C and stirred within a heating block to ensure constant temperature. One tube was used to monitor H2O2 dynamics
using the MDP - FIA system. The second tube was used to measure O2 dynamics using a FireStingGO2. Every 10
minutes during the experiment, 1 mL samples were taken from this second tube, filtered via a 0.02 µm syringe filters
(Whatman) into Eppendorf tubes and stored at 4°C over-night before measuring their H2O2 content via direct injection
(FIA without a MDP) the following day. The third falcon tube was used to measure O2 and H2O2 dynamics
simultaneously using an electrochemical microsensor (ISO-HPO-100) coupled to a free radical analysis system (World
Precision Instruments; wpiinc.com). Results are shown in the SI (Figure S2)). 2 mL of bacterial overnight culture were
added to each of the stirred diluted LB media (37°C) resulting in a final density of approx. 2 x 108 CFU mL-1. H2O2 and
O2 concentrations were monitored for 2 hours.
3. Results
We present the measuring characteristics of the combined MDP-based FIA measurement of H2O2, and illustrate
various measurements optimization steps regarding reagent concentrations, syringe pump speeds and choice of sample
6

media. The design of the set-up allows minimally invasive sampling without removing sample volume, which enables a
wide variety of application in environmental and biomedical science. We demonstrate the application of the combined
MDP-FIA system for minimally invasive measurements of H2O2 dynamics in almost real time.
3.1. Calibration
In the commercially available FIA system, the aggregate-free sample is sampled via a tube and then guided
through a FEP tube alongside the carrier, the buffer, the HCl wash and the AE solution. They are transported via the
peristaltic pump with the same pump speed through the valve system – to avoid differences in pressure (e.g. buildup of
B
A
Figure 3: Comparison of H2O2 calibration curves measured with direct injection (DI) into the FIA system and
via a microdialysis probe (MDP) coupled to the FIA system (MDP-FIA) using the same chemiluminescent
reagent concentration. A) Shows the difference between calibrating with MDPs (blue and green) and without MDPs
(black, n=3). The inset shows the effect of the syringe pump speed on the calibration curve using MDPs. The two
shown pump speeds are 75 µL min-1 (blue; n=3) and 50 µL min-1 (green; n=1). B) Shows the decrease in in signal
intensities between DI and MDP-FIA measurements, by plotting the ratio of the Peak Integral of the DI signal divided
by the Peak Integral of the MDP-FIA signal at the respective pump speed for different H2O2 concentrations.
backpressure) within the system and to secure even flow speeds. A calibration curve of the ‘direct injection’ method
(DI) is depicted in Figure 3A (black), using a 1 nM AE solution, a 0.1 M Na2CO3 buffer at pH 11.3, a 0.01 M HCl wash
and MilliQ as carrier. All reagents were pre-treated with catalase according to literature [58] as described in the
methods section. H2O2 standard solutions were prepared in untreated DI water and diluted in the measurement medium.
In comparison, 2 calibration curves are depicted for the measurement with a MDP coupled to the FIA system (Figure
3A, blue and green). Sampling via a MDP connected to the FIA system (MDP-FIA) decreased the signal intensities, as
the analyte recovery is diffusion limited and strongly dependent on external factors such as the syringe pump speed and
MDP membrane characteristics (Figure 3A and B).
The measurement time was mainly influenced by the syringe pump speed, which is responsible for filling the 20
cm long sample loop, with a volume of ~88.4 µL (Table S1). The time needed for the sample solution to reach the
sample loop also needs to be taken into consideration and this depends on the used tubes, their IDs and lengths. In our
setup, the volume before the sample loop was ~50 µL. Thus the system response time - the time needed between
sampling and signal out-put - comprises the time required to fill the sample loop plus the tubes connecting the MDP
with the sample loop. However, once a continuous reaction is monitored only the time required for flushing the sample
into the detector and refilling the sample loop needs to be taken into account in between measurements. The time
necessary for flushing the sample out of the sample loop and through the detector can be monitored by observing the
signal peak in the analysis software. The time depends on the pump speed of the peristaltic pump (here 1.8 mL min-1),
the length of tubing in between the sample loop and the detector as well as the geometry and volume of the flow cell. In
our set-up the operational flushing time of the system was ~30 – 35 seconds.
7

Figure 4: Effect of the chemiluminescent reagent (AE) concentration and the measurement medium composition
on the H2O2 calibration curve. It is shown for DI water (black-grey), MilliQ water (light blue), PBS (blue), filtered
seawater (FSW; green), F2 medium (purple), LB broth (red) and agar (1wt % in DI water; dark orange) as well as the
liquid medium above the agar (orange) for the MDP-FIA system. A) Calibrations via direct injection (n=3). B)
Calibrations using a MDP for sampling (n=3).
It is well known [54] that the analyte recovery with MDPs depends among other factors on the syringe pump
speed. Figure 3B shows the difference in analyte recovery over different pump speeds for a 100 µM and a 50 µM H2O2
solution for the MDP-FIA system. However, the slope of the linear calibration curves cannot only be modified by
changing the syringe pump speeds, but also by changing the concentration of the AE reagent or the measurement
medium (Figure 4A and B). We performed calibrations in DI water, MilliQ water, filtered seawater (FSW), artificial
seawater (ASW), F2 medium, PBS and LB broth as well as in agar (1wt% in DI water) and the overlaying medium
using the MDP-FIA system. The AE reagent concentration was gradually increased until signal intensities in
comparable ranges could be achieved, in order to compensate for medium effects and the reduced analyte recovery
caused by the MDP. The DI water used for preparing the H2O2 stock solution was untreated to avoid degradation of the
calibration solutions prior to measurement and each measurement was repeated 3 times (n=3). H2O2 background values
were determined by measuring in untreated blank medium and catalase treated medium samples.
Measurements of H2O2 using direct injection into the FIA or using the combined MDP-FIA set-up exhibited
highly reproducible and linear calibrations curves with small error bars for each particular setting of the various system
parameters. If the system is switched off, it is however recommendable to recalibrate before measurement.
Characteristics of the calibration curves in the different investigated media (shown in Figure 4) are given in Table 1 for
the direct injection (DI) method as well as the MDP-FIA system, together with the used AE reagent concentrations
aiming for an operational measuring range of 1 – 100 µM. The slope, R2 value as well as the determined LOD and LOQ
are given. LOD (Equation 1) and LOQ (Equation 2) were determined according to literature [59,60]. The slopes allow a
comparison between the different calibrations and chosen chemiluminescent reagent (AE) concentrations. It can be used
to estimate the AE reagent concentration necessary to get a calibration curve of a certain steepness, which however does
not oversaturate the PMT at higher H2O2 concentrations. Here we aimed for a slope of ~ 1.2*104 – 3*104 counts · s per
µM H2O2 for a calibration range between 1 – 100 µM.
8

Table 1: Overview of calibration curve characteristics in different media at room temperature using direct injection
(DI) or MDP-FIA (the same membrane was used for all measurements) based measurements at a range of acridinium
ester (AE) reagent concentrations enabling a measurement range of 1-100 µM H2O2. k denotes the slope of the
calibration curve, while σ is the standard deviation of the baseline, i.e. the signal measured in the catalase treated blank.
Slope of the calibration
LOD
(3.3*σ/k)
[µM H2O2]
LOQ
(10*σ/k)
[µM H2O2]
Injection
Mode
AE reagent
[nM]
curve
(k)
Medium
R2
[counts · s · (µM H2O2)-1]
DI
1
10
40
100*
1
3.34*104
2.85*103
1.01*104
1.24*105
2.95*104
2.54*104
1.69*104
2.89*104
1.87*104
5.11*103
3.69*104
1.57*104
7.84*102
1.87*103
1.18*104
1.47*104
3.03*104
1.57*104
1.23*104
9.20 * 103
0.994
0.999
0.980
0.999
0.997
0.996
0.999
0.999
0.990
0.999
0.999
0.991
0.994
0.998
0.997
0.979
0.999
0.998
0.997
0.995
0.05
0.56
0.15
0.18
0.03
0.16
0.03
0.07
0.60
0.09
0.01
0.60
0.13
0.40
0.05
1.5
0.14
1.7
DI water
MDP-FIA
0.47
0.55
0.08
0.49
0.09
0.20
1.8
DI
MilliQ water
PBS buffer
MDP-FIA
40
1
DI
2
MDP-FIA
DI
40
1
0.26
0.02
1.8
Filtered Seawater
(FSW)
6
MDP-FIA
40
1
0.40
1.2
DI
2
F2 medium
10
40
10**
10
40
40
0.14
4.5
MDP-FIA
DI
0.02
0.05
0.71
0.58
0.06
0.14
2.2
LB broth (1 + 4)
Agar
MDP-FIA
MDP-FIA
7.75
Medium Column
above Agar
MDP-FIA
-***
-***
40
9.15 * 103
0.999
* Calibration between 0 – 50 µM H2O2; signal intensities too high for 100 µM due to PMT saturation.
** Calibration temperature was 37 °C and it was calibrated between 0 – 25 µM.
*** n=1; σ is not determined.
3.2. Cross-Sensitivity Studies
Different potentially interfering species on the AE-based H2O2 measurement were tested and the results are
summarized in Table 2. Surprisingly, such tests are apparently lacking in the literature, albeit the AE method is
promoted as being specific for H2O2 [48,57].
Table 2: Cross-sensitivities of potentially interfering chemical species on the AE-based H2O2 detection with the FIA
system. Measurements were done in DI water, catalase treated MQ water as well as in combination with a 50 µM H2O2
9

background. Concentrations were chosen in regard to biological / physiological relevance as judged from the scientific
literature. “-“ indicates conditions that were not tested.
Signal Change in
Catalase Treated MQ
water
Signal Change in DI
water
Signal Change in Combination
with 50 µM H2O2
Investigated Species
Data
Yes. Concentration
dependent
NaCl
-
Yes. Concentration dependent
Figure S3
Cu(II), 100 µM
Fe(III), 200 µM
Fe(II), 200 µM
No change
No change
Increase
No change
No change
Increase
No change
Increase
Decrease
Figure S4
Fe(II), 200 µM + FerroZine 250
nM
Increase
Increase
Decrease
Mn(II), 10 µM
Ferrozine, 250 nM
No change
-
No change
-
No change
Increase
Tert-butyl peroxide, 50 µM
Urea, 10 mM
No change
No change
Decrease
Decrease
No change
Increase
No change
No change
Decrease
Decrease
No change
-
No change
No change
No signal
Figure S5
L-cysteine, 100 µM
Glutathione, 5 mM
NaOCl, 50 µM
No signal
Increase (1O2 production)
-
Figure S6
Figure S7
ONOONa, 50 µM
ONOONa, 50 µM in PBS buffer
In PBS: Increase
In PBS: -
In PBS: -
3.3. Applications
We tested the combined MDP-FIA system in a range of different samples in order to demonstrate its broad
applicability and to discuss some of the current limitations of this new H2O2 measurement approach. The microdialysis
probe was successfully applied to monitor the enzymatic reactions of glucose oxidase and catalase in a PBS buffered
glucose solution (Figure 5). The MDP-FIA system was also applied for measurements of H2O2 production due to light-
induced, photo-oxidative stress within an important model organism in symbiosis research; the sea anemone Exaiptasia
pallida that harbors photosynthetic microalgae as symbionts in its tissue [62] (Figure 6). The MDP-FIA approach has
the advantage here, that it allows the positioning in close proximity to the animal / in the bulk water close to the animal,
without the danger of accidentally attaching the tube to the moving animal due to the suction of the tube or blocking the
tube by aggregates in the water. Figure 6 shows the difference the positioning of the MDP in relation to the animal
makes concerning a stable signal output; especially in a bubbled system. Finally, we looked at H2O2 dynamics in a
bacterial culture (Figure 7). Calibration measurements in several media show high background levels of H2O2 in the
blank; especially the commonly used bacterial culture medium LB showed elevated levels of H2O2 after autoclaving.
Therefore, we investigated how bacteria (in this case Pseudomonas aeruginosa) deal with the external H2O2.
3.3.1. Enzyme reactions.
Glucose oxidase (GOX) was added to 200 mL stirred, PBS buffered glucose solution (1mM) at room
temperature, resulting in a decrease in O2 concentration approximately correlating with the stoichiometrically expected
increase in H2O2 concentration (Figure 5). The observed decrease in O2 concentration (taking a ~ 6 minute delay due to
loading times in account) was ~63 µM O2 while the H2O2 concentration increased by ~57 µM H2O2. When adding
catalase, the H2O2 concentration decreased again, while the decrease in O2 concentration was slowed down due to the
O2 produced by the reaction of catalase and H2O2. When reaching anoxia, the H2O2 production stopped and a decrease
towards 0 µM could be observed due to continuous catalase activity.
10

Figure 5: Monitoring of O2 and H2O2 concentration dynamics in a PBS buffered 1 mM glucose solution upon
alternating additions of glucose oxidase (GOX) and catalase. The enzymatic reaction schemes are shown above the data
plot.
3.3.2. H2O2 dynamics in the sea anemone Exaiptasia pallida.
The sea anemone was kept for one hour in darkness (Figure 6; grey) where there was a visible decrease in the
background H2O2 concentration from ~ 1.3 µM to 0.6 µM. When switching to high light, an immediate increase in H2O2
concentration was observed. In one approach the MDP was placed in proximity to the moving anemone (>1 cm
distance). In a second approach, the MDP was in close proximity to the tissue of one of the sea anemone’s tentacles
(<0.5 cm). H2O2 increased up to similar apparent steady-state concentration in light of ~2.7 µM in both positions.
Figure 5: Monitoring of H2O2 dynamics at 32°C in the symbiont-bearing sea anemone Exaiptasia pallida in the dark
(grey) and under light stress (~60 – 140 min) in two different measuring positions, with the MDP positioned >1 cm
(orange) and <0.5 cm (yellow) from the tissue surface. The MDP was positioned in the bulk water surrounding the
animal, but not in direct contact.
Measurements closer to the sea anemone tissue surface were less fluctuating.
3.3.3. H2O2 levels in LB medium.
A solution of the bacterium Pseudomonas aeruginosa was pipetted into stirred, heated (37°C) LB medium
(indicated at ~10 minutes in Figure 7), which was monitored by an optical O2 sensor, while H2O2 was measured either
11

via direct injection into the FIA or via a MDP inserted in the solution. Both H2O2 measurements via direct injection and
via the MDP-FIA set-up showed a similar behavior, however, the direct injection measurement did not include blank
values measured prior to bacteria addition. Therefore, the blank value of the calibration curve, which is LB medium
without the addition of bacteria measured right before the experiment, is displayed to visualize the determined
background concentration. The O2 was depleted within 60 min, while the H2O2 concentration was depleted within 2-3
hours due to an apparent catalase activity in the bacterial solution. We note that the apparent blank value in the medium
contained a significant amount of H2O2, which was then consumed by the bacteria.
Figure 7: Monitoring of bacterial H2O2 and O2 depletion in diluted, stirred LB growth medium at 37°C. The O2
concentration (blue) was monitored by an optode, while H2O2 was measured with the combined MDP-FIA system (light
blue) and with FIA system using direct sample injection (dark green). For the direct injection, the solution needed to be
filtered (0.02 µm syringe filter) and was stored at 4°C in the fridge overnight before measurement. For the direct
injection (dark green), no sample was taken and filtered for the blank before the addition of the bacteria, the blank from
the calibration is depicted instead.
4. Discussion
We developed a flexible measuring setup for quantifying the important reactive oxygen species H2O2 by
combining sampling via microdialysis probes (MDPs) with sensitive chemiluminescence-based detection of H2O2 on a
flow injection analysis (FIA) system. In its current configuration, the combined MDP-FIA based systems enables
quantification of H2O2 in a variety of aquatic samples and semisolid samples with high sensitivity (LODs and LOQs are
shown in Table 1), linear calibration characteristics and a time resolution of ~4 min between measurements and ~6
minutes between samples. However, these system parameters can easily be modified e.g. by optimizing parameters such
as the chemiluminescent reagent concentration, pumping speed of the system, tubing lengths and diameter, or choice of
MDPs with different geometries and membrane characteristics. This system can now be used in a variety of
environmental and biomedical samples.
As mentioned in the introduction, H2O2 can be found in natural waters. The formation of H2O2 in natural waters
can be due to e.g. photochemical reactions of dissolved organic substances in the water [20]. However, background
concentrations of H2O2 in other media, have to the best of our knowledge, not been studied in detail, especially when it
comes to media used in the laboratory, such as DI water, MilliQ water, different seawater samples (ASW, FSW, F2
medium) or growth media used in microbiology such as LB-broth. To our knowledge, effects of water treatments such
as autoclaving on the potential formation of background levels of H2O2 in liquid media have also not been studied. In
order to determine such potential effects of media treatment and composition on H2O2 concentration a sensor system is
required, which ideally allows continuous measurements in a broad variety of media. In liquid samples, the FIA system
is well established as it allows continuous monitoring and can overcome pH interferences, which are often problematic
for irreversible luminescent probes [6]. By combining the FIA system with a MDP we alleviated a major limitation of
the FIA system, which is the need of pure samples. Small particulates such as cell or mineral aggregations or biofilms in
the sample can block the narrow tubes of the FIA system, posing a need for sample cleaning or filtration steps. This
extra treatment can be avoided by the MDP-FIA system, as the MDP functions as a filter to substances larger than the
specific molecular cut-off of the microdialysis membrane. Thus no aggregates can get into the sampling tube, while
H2O2 enters the perfusion solution in the MDP via diffusion across the microdialysis membrane. This can also reduce
12

the recovery of other potentially interfering species. In addition, it also allows measurements in semi-sold media, such
as agar to measure directly in e.g. cell cultures.
The basic mechanisms described in literature to be responsible for H2O2 decay in natural waters are enzymatic
removal (biological) and abiotic chemical reduction [28]. The O-O bond itself is relatively weak and prone to homolysis
caused by heating, radiolysis, photolysis or the presence of redox metals, such as iron or copper [2]. Both of these redox
metals are for example present in seawater and are even enriched in F2 medium. Higher temperatures are also
frequently employed, especially in medical applications. This can lead to an accelerated degradation of the H2O2
calibration solutions over time and needs to be taken into consideration as well when calibrating and measuring in
different media.
4.1.
Calibration
As long as the PMT in the FIA system is not saturated, the system sensitivity can be tuned by modifying the
chemiluminescent reagent (AE) concentration, pump speed and MDP membrane length enabling a broad calibration
range (here tested for concentrations between 0 – 1000 nM (Figure S8: B), 0-10 µM (Figure S8: A) and 1-100 µM).
Lower AE concentrations allow a broader operational working range (Figure 4), whereas the use of higher AE
concentrations can be used to measure H2O2 in the nM to µM ranges (Table 1). However, the increase in AE
concentration increases the baseline as well. In theory, if the used solutions were completely H2O2 free, the baseline
value should stay the same, but the FIA system is able to detect very low concentrations (in the pM range [19]) and thus
the signal output can be influenced by residual amounts of H2O2. When catalase was added to the MilliQ or DI water (3
mg L-1) used for the preparation of all solutions, the H2O2 background levels should be close to zero. This is however
not true for the MDP perfusion-fluid which did not contain catalase to avoid degradation of the H2O2 diffusing into the
perfusion fluid from the sample across the microdialysis membrane. It is also not true for the calibration standards,
which contain the baseline H2O2 concentration of the specific medium. Such effects can be compensated for in the
calibration procedure, by measuring in apparent zero H2O2 solutions before and after addition of catalase. This is
necessary, as many real life systems can degrade H2O2 by releasing e.g. catalase, which then reacts with the background
H2O2 as well as shown in our measurements in growth medium with bacteria (Figure 7). Therefore, it is important to
know both the blank value and the real zero value in many applications when measuring H2O2.
4.1.1. Salt precipitation
Another issue, which needs to be considered is salt precipitation within the measuring system. At the working
pH of ~11.3, seawater as well as F2 medium showed a white precipitate, most likely Mg(OH)2 that is known to
precipitate in seawater [49]. However, sodium carbonate can also form precipitates with other ions, e.g. Cu(II), Fe(II) or
Fe(III). According to literature [1], the washing loop is sufficient to avoid excessive formation of the precipitate when
working with seawater. However, as the combination with the MDP increased the loading time interval more precipitate
formed, and the wash loop proved to be insufficient. Therefore, the pH buffer tube as well as the AE tube were flushed
with HCl instead during the loading step. 2-4 minutes prior to switching to the inject mode, the system was flushed
again with the buffer and the AE solution to allow the baseline value to stabilize. This also has the benefit of less
consumption of the AE solution.
4.1.2. Syringe pump speed
Different syringe pump speeds were tested with a 2 mm long microdialysis probe membrane. However, the
analyte recovery rate also depends on the MDP membrane length [54] and can thus be modulated by choosing MDPs
with different dialysis membrane characteristics. With the MDPs having a 2 mm long membrane, pump speeds <50 µL
min-1 gave sufficient measuring signals for H2O2 without introducing excessive loading times of the FIA sample loop
(Table S1). For this experiment (Figure 3B), relatively high H2O2 concentrations were chosen (100 µM and 50 µM), but
it is apparent that highest signal differences could be detected at the lowest syringe pump speed, thus resulting in a
lower H2O2 detection limit. Overall, for our applications we chose a pump speed of 25 µL min-1, which correlates to an
initial ~6 minute cycle to fill all tubes (this however, strongly depends on the tube length and diameter). After initial
sampling, the following measuring cycles only depend on the time required to fill the sample loop, which for our set-up
involved a loading time of ~4 minutes (20 cm long tubes, ID: 0.75 mm). This means there is a slight delay in the
system, but the values can be correlated to the specific sampling interval if all the tube lengths and IDs are known, or
experimentally tested.
4.2.
Medium effects and cross-sensitivity studies
We observed that the chemiluminescent signal and thus the apparent H2O2 level in different media can vary
widely (Table 1, Figure 4). These effects were weak when comparing e.g. DI water to MQ water, while e.g. a strong
13

decrease in the detected signal was observed when switching from DI water to F2 medium or LB medium (Figure 4).
These effects were strongest when using direct sample injection in the FIA system, where lower signals could e.g. be
compensated for by increasing the AE reagent concentration. When using the combined MDP-FIA approach much less
media effects on the chemiluminescent signal were observed and the same AE concentration could be used for all tested
media in the MDP-FIA set-up. In the direct injection measuring mode, catalase treated medium was used as the carrier
solution, which was directly mixed with the AE reagent and the buffer solution, and this resulted in more exposure to
potentially interfering species. With the MDP-FIA set-up, the probe perfusion fluid is DI water (not the catalase treated
medium), and the membrane of the MDP together with the chosen perfusion speed reduces the recovery of not only
H2O2 but also of potentially interfering species. In addition, the external carrier in the MDP-FIA set-up is catalase
treated DI water, which means that no raw sample medium is injected into the system. This also alleviates precipitation
from seawater or potentially interfering ions, which are no longer directly injected into the system.
Depending on the sample medium, the observed changes in chemiluminescent intensity can have different
reasons such as higher baseline concentrations of H2O2 in the medium, a strong change in pH or salinity, or a cross-
sensitivity to another species. The effect of changing from DI water to other media is shown in Figure 4A (FIA) and
Figure 4B (MDP-FIA) using identical settings and concentrations. Overall, some of the media effects can be attributed
to precipitating salts from the artificial seawater and F2 medium, which affect the signal and can cause back pressure to
build up in the system [49], or affect the scattering / absorption of the emitted photons in the AE reaction. F2 medium
also has a high metal content. Cross-sensitivities of the AE reagent to other species (apart from Fe(II)) have not been
reported in literature but were tested nonetheless to elucidate the underlying causes of these media effects. Changes in
the sample pH only resulted in minor changes (Figure S2) and are usually buffered by the carbonate buffer. Changes in
the salinity (NaCl concentration) influenced the slope of the H2O2 calibration curve relatively strongly (Figure S3). Salt
concentrations should thus be kept stable and the experimental NaCl concentration has to be used for the calibration.
Regarding transition metals (Figure S4), Cu(II) and Mn(II) showed no interference with the system, neither in
the presence nor in the absence of H2O2. Fe(III) showed no interference in the absence of H2O2, however, increased the
signal in the presence of 50 µM H2O2 by almost 40%. Fe(II) interfered with the system in the absence of H2O2 as well
as in the presence (50 – 60% of the signal peak integral of 50 µM H2O2).
The cross sensitivity to Fe(II) is compensated for in literature by the addition of FerroZineTM binding the Fe(II)
before it can react with the AE solution[48]. Ideally, the FerroZineTM would be directly added to the MDP perfusion -
fluid reservoir in the syringe pump. However, we wanted to avoid FerroZineTM diffusion out of the MDP membrane
into the sample. The addition of FerroZineTM to the carbonate buffer solution would be too late, as by then the reaction
with the AE reagent would have already taken place. Therefore, adding FerroZineTM to the AE solution was the best
choice.
When trying to compensate for the Fe(II) cross-sensitivity by adding 250 nM FerroZine, an increase of
approximately 120% in the signal peak integral for 50 µM H2O2 was observed in the absence of Fe(II). This increase
might indicate that FerroZineTM acts as catalyst in the reaction of H2O2 with the AE, via an unknown mechanism. In the
absence of H2O2 but the presence of 200 µM Fe(II), an increase in the DI water measurement (45% of the signal peak
integral of 50 µM H2O2 in the presence of FerroZineTM) and catalase treated MQ water (40% of the signal peak integral
of 50 µM H2O2 in the presence of FerroZineTM) was observed. 200 µM Fe(II) in the presence of 50 µM H2O2 and
FerrZoineTM caused a drop in the apparent H2O2 signal peak integral down to 30% of the Fe(II)-free measurement. We
conclude that FerroZineTM is not suitable to overcome the Fe(II) cross-sensitivity in the AE-based H2O2 detection in our
setup. However, it might be used to enhance signal intensities for low H2O2 concentrations. Other complexing agents
like e.g. EDTA may be suitable for removing the Fe(II) cross-sensitivity, but remain to be tested.
Different biomolecules such as urea, L-cysteine, glutathione and tert-butyl peroxide were also tested for their
interference on the AE-based H2O2 detection (Figure S5). Tert-butyl peroxide as well as urea showed no significant
interference, neither in the absence nor in the presence of H2O2. A minor decrease (~96%) of the signal peak integral for
50 µM H2O2 was observed in the presence of urea. However, a reaction of urea with H2O2 is more likely than the
interference with the chemiluminescent reaction. L-cysteine and glutathione caused the chemiluminescent signal to drop
below the baseline in the absence as well as the presence of H2O2. This drop indicates a degradation of H2O2 in the
presence of these two antioxidants and a complete removal of the background H2O2 rather than a cross-sensitivity. High
amounts of L-cysteine or glutathione can thus strongly affect measurements of H2O2 concentration.
Another investigated species was hypochlorous acid (Figure S6). In the absence of H2O2, no change in the
system was detected (besides a slight increase in the measured baseline after adding 1 mM). However, in the presence
of H2O2 an increase in the signal of ~126% was observed. As hypochlorous acid showed no reaction in the absence of
H2O2, we conclude that the interfering species probably is singlet oxygen (1O2) resulting from the reaction of
hypochlorous acid with H2O2 [63]. However, as 1O2 is very short lived (µs range, dependent on temperature and solvent
[64]) the observed interference could be alleviated by the introduction of the MDP in the sampling step (Figure S6) and
might even be further reduced by a reduction of the MDP perfusion speed.
14

Peroxynitrite (ONOO-) was investigated as well for its potential ability to interfere with the AE-based H2O2
measurement (Figure S7). ONOO- levels were chosen according to literature where it is described in µM concentrations
(between ~55 µM in serum and 2.8 µM in saliva [65]). The ONOO- stock concentration was determined right before
the experiment according to the product information by the provider using a spectrophotometer (max = 302 nM;  =
1670 M-1 cm-1). The experiment showed strong cross-sensitivity to peroxynitrite, as the signal peak integral in DI water
exceeded the 50 µM H2O2 peak integral by almost 92 % in the presence of peroxynitrite; even coupled to the MDP,
where the increase in the peak integral was even higher. Peroxynitrite is supplied in a solution of 0.3 M NaOH, where it
has according to the provider a half-life, at room temperature of 5 hours; whereas at pH 7.4 it is supposed to have a half-
life of a few seconds [66]. Thus, the cross-sensitivity to peroxynitrite was investigated in PBS buffer at a pH of 7.4 as
well. There was an even stronger cross-sensitivity visible when using PBS (the signal for 50 µM H2O2 concentration in
PBS was exceeded by almost 300%). We speculate, whether the cross-sensitivity is caused by peroxynitrite or one of its
degradation products. Peroxynitrite degrades at acidic conditions to ●NO2 and HO●, and regarding the observed
interferences when measuring in the presence of Fe(II) and Fe(III) together with H2O2 , the strong apparent interference
might be caused by the hydroxyl radical instead of peroxynitrite. According to the manufacturer, ONOONa was
produced via H2O2, so a certain background concentration of H2O2 cannot be ruled out.
In general, higher baselines can be an indication for H2O2 present in the blank (e.g. LB broth; Figure 7), which
can be compensated for by the addition of catalase. If the baseline decreases below the initial blank value after addition
of catalase, the difference can be assumed to be due to the background H2O2 concentration for the given
medium/solution.
We conclude that irrespective of the various mechanisms causing such media effects, careful calibration and
optimization for a given application can alleviate the effects and resulted in highly linear calibration curves for H2O2.
However, there are various cross-sensitivities which need to be taken in consideration before starting an experiment.
The system as described can be used for many applications, but due to the found cross-sensitivities there can be certain
applications were it cannot be used as a reliable specific quantification of H2O2 alone, but rather for quantification of
ROS or oxidative stress indicator.
4.3.
Enzyme Reaction.
The MDP was used in combination with an optical O2 sensor to follow the reaction dynamics of glucose oxidase
(GOX) and catalase in a PBS buffered glucose solution (Figure 5). Upon addition of GOX, glucose and O2 reacted
producing H2O2, as quantified by the combined MDP-FIA system. Upon addition of catalase the formed H2O2 was
degraded according to the reaction Scheme 1.
Scheme 1: Enzymatic reaction of GOX with glucose in the presence of O2 producing H2O2, which is subsequently
degraded into O2 and water upon addition of catalase.
This resulted in a temporary increase in O2 concentration. However, as more O2 was degraded than produced O2
depletion was observed. The addition of GOX as well as catalase was repeated to demonstrate the capability of the
MDP-FIA system to monitor dynamic H2O2 concentration changes. In the first addition step, the decrease in O2 was
higher than the increase in H2O2 probably due to the time delay between the FIA system response (6 minute cycle time)
and the immediate response of the O2 sensor. When looking at the O2 signal 6 minutes prior to the addition of catalase
the overall decrease in O2 concentration was 63 µM, while the corresponding increase in H2O2 concentration was 57
µM, which was approximately in accordance with the reaction scheme stoichiometry. The combined MDP-FIA system
is thus well suited to monitor dynamic H2O2 changes in solutions due to enzymatic reactions or other (bio)chemical
processes. Such measurements are highly relevant in both environmental and biomedical sciences.
4.4.
H2O2 Dynamics in the Sea Anemone Exaiptasia pallida.
In a second application, we assessed the H2O2 production of the anemone Exaiptasia pallida (strain H2) under a
global change relevant stress scenario [67], i.e., increased seawater temperature (32°C) and high irradiance (1500 mol
photons m-2 s-1). Exaiptaisa pallida is commonly used in ecophysiological and biomolecular research as a model
organism for cnidarian-coral symbioses [68], and technical advances in the assessment of quantitative H2O2 production
of such organisms under oxidative stress are in strong demand. Besides the very first successful detection of light-stress
induced H2O2 formation in Exaiptasia, we could also show that the positioning of the MDP has a direct effect on the
15

signal quality. When the MDP was positioned >1 cm from the animal, significant fluctuation in the signal was observed
and could be explained by the distance of the MDP to the anemone in connection to the weak mixing of the seawater
via an air stone, which hampers an even build - up of H2O2 in the seawater surrounding the sea anemone. Additionally,
bubbles could get trapped on the MDP membrane affecting H2O2 diffusion through the membrane. When positioning
the MDP in closer contact with the anemone tissue (within the more stagnant water, i.e., the diffusive boundary layer
surrounding the animal) more stable measuring signals were observed with a quasi-linear increase in signal over time
once the MDP was poditioned. The combined MDP-FIA system thus enables spatially resolved measurements of H2O2
in open or closed systems containing living organisms, and this opens for a wide range of applications in the biological
sciences, where O2 respirometry is a core technique, which can now be easily combined with quantitative measurements
of H2O2.
4.5.
H2O2 Levels in LB medium.
In a third application, the bacterium Pseudomonas aeruginosa was grown in diluted LB-broth medium. The
autoclaved medium exhibited a high H2O2 background level (~17-18 µM in the diluted sample) causing a high baseline
signal in the FIA. After addition of the bacteria, a drop in the signal intensity was observed due to catalase activity of
the bacteria leading to H2O2 consumption. Such signal behavior was observed with direct FIA measurements of H2O2 as
well as with FIA–MDP based measurements and measurements with an electrochemical H2O2 sensor (Figure S2). Such
high H2O2 background levels were also confirmed by adding catalase to the reaction mixture (MDP-FIA) or to a LB
medium blank, which was measured subsequently to finishing the experiment (DI). This illustrates the importance of
careful assessment of background H2O2 levels in sample media, typically assumed to contain no H2O2. The autoclaved
LB medium contained almost 20 µM H2O2, while non-autoclaved LB medium at room temperature showed a lower
background concentration of ~ 4.9 µM H2O2 (Figure 4). This shows that the treatment of the sample medium can have
an influence on background H2O2 concentrations which needs to be evaluated before setting up an experiment. In
addition, it indicates that media used e.g. for isolation and cultivation of bacteria may impose some oxidative stress if
they contain such high H2O2 background levels. This result was rather unexpected and should be analyzed in more
detail in the future. With proper calibration and quantification of background levels of H2O2 in apparent blank controls,
the combined MDP-FIA system is an ideal instrument for such studies.
5. Conclusion
Coupling MDPs to a FIA system bypasses laborious sample cleaning and pre-treatment steps as well as
consumption of the sample. It is possible to measure dynamic changes in H2O2 directly in complex sample solutions for
extended time periods and without special sample treatment. It was possible to determine concentrations down to the
sub µM range with good linear calibration curves. The set-up is highly tunable as the measurement range can be
modified to enable broad calibration ranges (e.g. 1 – 100 µM) or highly sensitive measurements over a smaller
operational range depending on the used MDP, the syringe pump speed, chemiluminescent reagent concentrations, PMT
settings or measurement temperature. Variation of the chemiluminescent (AE) reagent concentration allows a tuning of
sensitivity and dynamic range of the H2O2 measurements that can compensate e.g. for a lower recovery when sampling
H2O2 via the MDP membrane and/or at increased perfusion velocities of the carrier solution through the MDP. This
tunability makes the MDP-FIA method a potent technique for H2O2 determination in many different samples and
settings.
In this study we calibrated the combined MDP-FIA system within different H2O2 concentration ranges (0- 1000
nM, 0-10 µM and 0 – 100 µM). The LODs depend on the measurement medium and the chosen system settings and can
thus be optimized for different media and applications. We conducted a broad cross-sensitivity study to elucidate
observed media-effects, including sample pH, salinity, transition metals such as Fe(II), Fe(III), Mn(II) and Cu(II), as
well as relevant biomolecules such as urea, L-cysteine and glutathione. We tested other peroxides and ROS such as tert-
butyl peroxide, peroxynitrite, hypochlorous acid and singlet oxygen and found interfering species, which to the best of
our knowledge have not yet been described in literature.
We successfully applied the MDP-FIA system to quantify the dynamics of H2O2 in solutions subject to enzyme
reactions; we monitored H2O2 development in the sea anemone Exaiptasia pallida under light stress, and quantified
high background levels of H2O2 in autoclaved growth medium, which was consumed by the addition of bacteria. We
also successfully used it to measure H2O2 concentrations in agar plates; thus semi-solid media. The combined MDP-FIA
based system thus has a broad applicability for extracellular, near-real time H2O2 concentration measurements in both
natural and defined samples. The method is minimally invasive, adjustable to different media and concentration ranges,
and significantly reduces media effects in comparison to direct FIA-based measurements. While we have focused on
H2O2 measurements in this study, the system can also be used for chemiluminescent reaction-based quantification of
other chemical species [69–71] including ROS such as superoxide [72–74], where suitable chemiluminescent detection
schemes have been described.
16

Acknowledgements
We thank Whitney King (Waterville Analytical) for his technical assistance and extensive advice on the use of the
FeLume / FIA system. Sofie L. Jakobsen is thanked for technical assistance setting up the flow injection system. Kasper
E. Brodersen is thanked for fruitful discussions. This study was supported by a Sapere-Aude Advanced grant from the
Danish Research Council for Independent Research | Natural Sciences (MK), a grant by the Danish Research Council
for Independent Research | Technical and Production Sciences (MM, KK, MK, PØJ) and an EU Marie Curie Fellowship
(VS).
Author Contributions
MM, KK and MK outlined the paper. Experiments were planned and conducted by MM, KK, VS and PØJ. Data
analysis was done by MM, KK and VS. MM wrote the method paper with editorial input from VS, KK, PØJ and MK.
All authors contributed to the final paper.
Conflict of Interest
The authors declare no conflict of interest. The founding sponsors had no role in the design of the study; in the
collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results.
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21

Highlights:





Successful combination of a FIA system with microdialysis probes for H2O2 detection.
Near real time, chemiluminescence detection of H2O2 dynamics in different media.
Extracellular H2O2 dynamics in liquid and semisolid, untreated samples.
System tunable to different calibration ranges (0–1000 nM; 0–10 µM; 1-100 µM).
Cross-sensitivity study of potentially interfering species and the chemiluminescent reaction.
22