Assessment and Validation of a Device for Measuring Hydrogen Peroxide in Exhaled Breath Condensate in Healthy Volunteers
Conditions: Proof Of Concept Study
Sponsor: Imperial College London

 
 
 
 
 
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IRAS Project ID 289549,  
Protocol, version 1, Date 15/09/2020 
 
 
 
Assessment and validation of a device for 
measuring hydrogen peroxide in exhaled 
breath condensate in Healthy Volunteers 
 
 
IRAS Project ID: 289549 
Study Management Group 
Principal Investigator: Dr Omar Usmani, National Heart & Lung Institute, Imperial College 
 
Co-investigator: Dr Danny O’Hare, Department of Bioengineering, Imperial College 
 
Sponsor:  Imperial College London is the main research Sponsor for this study.  For further 
information regarding the sponsorship conditions, please contact the Head of Research 
Governance and Integrity at r.nicholson@imperial.ac.uk . 
 
Funder:  Innovate UK Smart Grants: October 2019 
 
 
This protocol describes the Assessment and validation of a device for measuring hydrogen 
peroxide in exhaled breath condensate and provides information about procedures for 
entering participants.  Every care was taken in its drafting, but corrections or amendments may 
be necessary. These will be circulated to investigators in the study.  Problems relating to this 
study should be referred, in the first instance, to the Principal Investigator.  
 
This study will adhere to the principles outlined in the UK Policy Frame Work for Health and 
Social Care Research It will be conducted in compliance with the protocol, Data Protection 
Act 2018 and General Data Protection Regulations (Europe) and other regulatory 
requirements as appropriate.  
 

 
 
 
 
 
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TABLE OF CONTENTS 
 
1. INTRODUCTION 
4 
1.1 
Background 
4 
1.2 
Study Rationale 
6 
2. STUDY OBJECTIVES 2 
6 
3. STUDY DESIGN 
6 
4. PARTICIPANT RECRUITMENT 
11 
4.1 
Pre-recruitment evaluations 
11 
4.2 
Inclusion Criteria 
11 
4.3 
Exclusion Criteria 
11 
4.4 
Withdrawal Criteria 
12 
5. ADVERSE EVENTS 
12 
5.1 DEFINITIONS 
12 
5.2. 
Reporting Procedures 
12 
6. ASSESMENT AND FOLLOW UP 
13 
7. REGULATORY ISSUES 
13 
7.1 
Ethics approval 
13 
7.2 
Consent 
13 
7.3 
Confidentiality 
13 
7.4 
Indemnity 
13 
7.5 
Sponsor 
13 
7.6 
Funding 
13 
7.7 
Audits 
14 
8. STUDY MANAGEMENT 
14 
9. PUBLICATION POLICY 
14 
10. REFERENCES 
14 
 
 
 
 

 
 
 
 
 
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TITLE 
Assessment and validation of a device for measuring hydrogen 
peroxide in exhaled breath condensate in Healthy Volunteers 
DESIGN 
Assessment of accuracy and precision of a novel instrument for collecting 
exhaled breath condensate (EBC) and analysing the levels of hydrogen 
peroxide. 
AIMS 
(i) 
Assess consistency in three successive measurements at 
monthly intervals in individual healthy volunteers. 
 
(ii) 
Qualitative evaluation of the usability of the device and the 
collection protocols from volunteers and from clinical staff 
operating the device. 
(iii) 
To collect usability feedback, in terms of how easy to use the 
device and to hold to breathe through the device. 
OUTCOME MEASURES 
(i) 
Precision and accuracy of the EBC device in healthy subjects 
POPULATION 
Healthy volunteers 
DURATION 
12 months  
 
 
 
 

 
 
 
 
 
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1. INTRODUCTION  
 
1.1 
Background 
Current assessments of lung inflammation for asthmatics and chronic obstructive pulmonary disease 
(COPD) are mainly based on patient symptoms and simple spirometry tests. Unfortunately, these 
features are not directly correlated to eosinophilic lung inflammation. Consequently, clinicians cannot 
always predict the degree of lower airway inflammation in COPD and asthmatic patients; hence it can 
be difficult to provide appropriate treatment. [1, 2] 
 
More than 70% of COPD patients are underdiagnosed [3] and the 2-year mortality rate for severe 
COPD patients is about 50% because of exacerbation, which on average occurs 2-4 times per year [4]. 
Early diagnostics for COPD could not only reduce direct healthcare costs but also reduce the 
probability of exacerbation and mortality [5] and lead to improved patient care. COPD is a progressive 
disease with potential acute exacerbations; therefore, long-term monitoring must be in place to track 
patient condition and such monitoring requires out-patient or GP surgery visits [6].  
 
Exhaled Breath Condensate (EBC) serves as simple and non-invasive sampling which gives access to 
lung chemistry and which can potentially inform clinical decision-making. Significantly higher 
hydrogen peroxide (H2O2) concentration in EBC is found in COPD patients, (typically 0.46 M 
compared to healthy control 0.2 M[7-9]). Even higher H2O2 concentrations are reported for patients 
with exacerbation, 0.6 M in comparison with stable COPD patients and corticosteroid-treated 
patients. [10, 11] Due to the limited range between stable COPD patients and COPD exacerbation, an 
exceptionally sensitive sensor is required for reliable diagnosis. 
Most previous approaches to EBC collection have prioritised the collecting of more condensate in a 
shorter time and this has led to the use of a lower temperature condensation surface. However, 
previous EBC collector designs have not considered how EBC sample composition depends on 
temperature and flow rate, due to widely varying absolute values and temperature dependences of 
the Henry’s law constants for different analytes and the enthalpy of condensation.  A failure to 
consider quantitatively the impact of collection conditions on EBC composition has led to large 
reported variations between different researchers for same health condition[7-15] which has impeded 
progress in this field.  Here we report an approach to EBC collection aimed at optimising target analyte 
concentration by exploiting the differential thermodynamic and kinetic parameters involved. This 
model-based approach is qualitatively borne out by experimental data which we have collected by 
blowing compressed air through Dreschel bottles of normal saline in a water bath. 
The published literature largely attributes variations in EBC analyte concentration to difference in 
patient physiological conditions. However, Loyola et al. [16] demonstrated different acetone 
concentrations in EBC samples at difference condensation temperatures. Zamuruyev et al. also 
showed how the amount of analyte collected varies with condensation temperature.[17] 
Ferric ferrocyanide (Prussian blue (PB)) modified electrodes are well-established for both H2O2 and 
oxygen sensing [18].  Karyakin et al. [19] demonstrated Prussian blue H2O2 catalysis exhibits a 

 
 
 
 
 
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significantly higher kinetic constant compared to a platinum electrochemical sensor and that PB is 
comparable to horseradish peroxidase. PB modified sensors were found to have good linear range 
between 0.1 M to 100 M, with sensitivity of 0.6 A M-1c m-2 and detection limit of 0.1 M. [20] 
H2O2 electrochemical sensor sensitivity and stability commonly involve the use of nanoparticles of PB 
[21], sometimes combined with polymer reagent blends [22, 23], or conducting polymers [24-26].  
We 
prepared 
an 
easy-to-use 
water 
dispersible 
poly(3,4-ethylenedioxythiophene)-
poly(styrenesulfonate) potassium ferric ferrocyanide composite, cross linked with ethylene glycol and 
divinyl sulfone (PEDOT:PSS-PB-EG-DVS) for electrochemical H2O2 and oxygen detection purposes. The 
calibration plot shows a good linear response which easily covers the concentration ranges reported 
for EBC (0.1 M to 25.6 M) and which has a detection limit of 103 nM.  We have manufactured 
disposable biosensors based on this technology. 
Performance in artificial samples has been compared to the laboratory-based fluorometric analysis 
using Amplex Red, which is well-established as the gold standard for hydrogen peroxide determination 
in low concentration biological samples and show good agreement. 
 
 
Figure 1: Schematic of the catalytic biosensor mechanism 
B TEM image of the electrode modifier C Reproducible i-
V behaviour of the composite D condenser mechanism E 
Device schematic F disposable biosensor strip G 
Generation of test specimens for laboratory evaluation 
Figure 2: A Biosensor coulometry data measured with 
PEDOT:PSS-PB-EG-DVS 
modified 
sensor 
against 
fluorometry data measured with HRP and Amplex Red. 
The colour strips illustrate reported physiological H2O2 in 
EBC in various patient groups [7-9, 11]. The inset 
demonstrates the calibration standard for HRP and 
Amplex Red against H2O2 concentration. B. Bland Altman 
analysis comparing electrochemical sensor results and 
the Amplex Red assay 
Figure 2 
Figure 1 

 
 
 
 
 
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1.2 
Study Rationale 
Following a successful pilot study to evaluate the accuracy and precision of the EBC device, its biosensor 
element and the algorithm for corrections arising from variations in temperature and flow the device 
will be evaluated in real breath samples in healthy volunteers. This will take place at the Clinical 
Research Facility (CRF) at the Royal Brompton Hospital. Real biological specimens are complex and 
there is the potential for losses of precision and accuracy.  This can only be evaluated by comparison 
with established methodology.  Secondly, whilst many reports can correctly classify COPD patients, 
there are substantial variations in absolute levels between researchers.  We have shown with artificial 
samples that correcting for variations in heat and mass transport can substantial reduce these 
variations.  Collecting replicate samples from healthy volunteers will allow us to evaluate this approach 
in real samples. 
 
 
 
2. STUDY OBJECTIVES 2 
The questions to answer in 1st evaluation: 
 
1. 
Volume collectable in 3-5 minutes 
2. 
Concentration of hydrogen peroxide in healthy volunteer 
3. 
Variability between 3 samples collected in 1 day 
4. 
Variability between 3 tests collected in 1st month, 2nd month and 3rd month. 
5. 
Usefulness of standardisation method, data including breath temperature entering and leaving 
the device as well as breath flow rate would be collected in-situ (sensors already on device, no 
naked wires). 
Usability feedback, in terms of how easy to use, to hold and to breathe through the device 
 
The outcomes of this study will inform the design of a clinical evaluation. 
3. STUDY DESIGN 
 
 
We aim to recruit 20 healthy volunteers from our database of volunteers at the royal Brompton 
Hospital and also by advertising.  Our purpose with this study is to evaluate the analytical 
performance of the device and the data processing and consistency of measurement over 3 
measurements a month apart. 
 
I participants  wish to take part in the study they will be invited to the Clinical Research Facility (CRF) 
at the Royal Brompton Hospital where you will meet the research team face to face. They will explain 
the study to the participant, and they  will have the opportunity to ask any questions related to the 
study. They will then be asked to read and sign a consent form if they would like to participate. 
 
Volunteers will be sent the participant information by email, or in person, prior to any appointment. 

 
 
 
 
 
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The participant will then be screened to see if they are suitable for the study. The Screening 
procedure will take approximately one hour and involve the researcher going through a list of 
questions related to your health and any medications you are currently taking. 
 
 
A hard copy of the participant information sheet will be provided at the appointment and participants 
will be encouraged to ask questions. 
 
Participants will then be presented with a consent form. 
 
Venue:  This will be performed in the Clinical Respiratory Facility located at the Royal 
Brompton Hospital.  
 
Sample collection protocol 
After the consent form has been signed, the breath collection protocol can then begin. 
The handheld breath collection apparatus will be switched on by the researcher and allowed 
to achieve the correct temperature (1-2 minutes). A sterilised disposable plastic mouthpiece 
will be fitted. The participant will be asked to hold the breath collection device and breath 
normally into the plastic mouthpiece for up to 5 minutes (Figure 3). 
The researcher will take the instrument and remove the condensed breath sample, place it in 
a numbered vial and remove it for analysis in the laboratory. Ideally, three samples per 
participant will be collected, but the participant may withdraw consent at any point. 
All samples will be pseudonymised to protect participant confidentiality. 
The individual results of the analysis will be made available to the participants. If requested. 
The flow chart of the sample collection is shown in Figure 3 (A&B) below, C & D shows the 
collection device. 
 
Three successive measurements will be made at monthly intervals in each individual healthy 
volunteer requiring them to visit the Hospital on three occasions.  
 
In addition, participants will be asked to fill in a short questionnaire on visit 1 related to the 
use of portable medical devices.  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
 
 
 
 
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Figure 3 
 
 
 
 
 
 
 
 

 
 
 
 
 
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Figure 4
 
 
STUDY TESTS 
Apparatus during sample collection: 1 pipette, a bag of pipette tips, numbered Eppendorf 
vials, sterilised mouthpiece, and exhaled breath condensate (EBC) collector, 75%(v/v) 
aqueous ethanol solution for sterilisation of the mouthpiece and apparatus. 
 
The following process is repeated 3 times in total to collect 3 EBC samples per patient: 
1. 
A researcher will control device operation and sample collection. 
2. 
The researcher switches on the device and waits until the Peltier temperature 
reaches 20°C, typically 1-3 minutes 
3. 
Install the sterilised PET mouthpiece. 
4. 
Volunteer holds the device and breathes through the PET mouthpiece normally for a 
maximum of 5 minutes. 
5. 
After EBC condensation, the volunteer is to pass the device to the researcher.  
6. 
The researcher transfers the EBC sample with pipette to vials for further analysis. 
7. 
The EBC collector is wiped with 75% aqueous ethanol to remove remaining EBC. 
 
Samples will be taken the Biosensor Laboratory (Bessemer 105) for analysis according to the following 
protocol:  

 
 
 
 
 
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Protocol for EBC analysis – electrochemical sensor and Amplex red. To take place in 
Bessemer 105, Biosensor Laboratory, researchers only. 
 
All samples will be analysed. No samples will be retained. 
 
1. 
Potassium phosphate monobasic, potassium phosphate dibasic, and potassium 
chloride salts are added to 200 µl of EBC to make pH 7.4 0.1 M phosphate buffer with 0.1 M 
potassium chloride. 
2. 
30 µl of buffered EBC is dropped onto electrochemical sensor for hydrogen peroxide 
analysis. 
3. 
48 µl of buffered EBC is withdrawn into 96-well plates with addition of 1 ul 5 U/ml 
horse radish peroxidase (HRP) and 1µl 2.5mM Amplex red solution. The 96-well plates are 
than incubated for 30 minutes at room temperature with platform shaker before 
measurement with fluorometer. The excitation and absorption wavelength of the 
fluorometer are set to 535 and 587 nm respectively. 
4. 
The electrochemical analysis results are compared with the Amplex red assay.  Paired 
t tests, correlation, regression and Bland-Altman analysis will be used for comparative 
purposes, following standard practise in analytical sciences. 
 
PET mouthpiece sterilisation: 
1. 
Mouthpiece is wiped with 75% ethanol before use and safely disposed of following 
use. 
Cleaning of the Equipment & Transport of Sample 
 
We will be following the Local Imperial College Bioengineering departmental protocol for 
transporting exhaled breath samples and disinfection of equipment in the document of BioSOP026: 
 
Transport and receipt of Materials 
a) Human tissue must be transported in UN3373 compliant packaging.  This packaging consists 
of three components. 
1. A primary receptacle: the tube, vial or other container that is in direct contact with the 
specimen. 
2. A secondary packaging that fully encapsulates the primary receptacle.  Adsorbent 
material should be placed inside the secondary packaging in case the primary receptacle 
leaks. 
3. An outer packaging for shipping or transit. 
 
Designated couriers should transport the tissue to Imperial College.  For example, CitySprint can 
courier human tissue 
 
Cleaning and decontamination 
Following all procedures work surfaces (which should be in spill trays or biological safety cabinets) 
must be cleaned with 1% Virkon solution followed by soap and water, and finally 70% ethanol.  
Whenever practicable, small articles that have been contaminated with human tissue or body fluids 
should be submerged in disinfectant at the appropriate working dilution for several hours before 
washing. Other contaminated surfaces (e.g floor, cupboards and walls) should be washed down with 
disinfectant. Bench surfaces should be washed down with disinfectant at the end of every 

 
 
 
 
 
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experimental session. A discard bin containing freshly prepared disinfectant should be within easy 
reach on each working bench and should be clearly labelled with the type, strength and usage of the 
disinfectant. 
 
 
Duration of Study  
Overall, the study will last 12 months from advertising to the end of data collection. 
 
 
4. PARTICIPANT RECRUITMENT 
 
4.1 
Pre-recruitment evaluations 
Volunteers expressing an interest will be contacted directly by the research team by email, phone, or 
in person and invited to the screening meeting. A copy of the participant information leaflet and study 
outline will be sent to the volunteer by email or post or in person. 
4.2 
Inclusion Criteria 
 
Healthy subjects:  
non-smokers  
Healthy individuals, free of significant disease. 
 
•Given written informed consent prior to participation in the study including all of its 
procedures.  
•Comply with the requirements and restrictions listed in the consent form.  
•Male or female subject aged 18-65 years at screening.  
•Able to complete the study and all measurements.  
•Able to read, comprehend, and write at a sufficient level to complete study related 
materials. 
 
 
 
4.3 
Exclusion Criteria 
 
Exclusion criteria for all subjects:   
Subjects will not be eligible if any of the following apply: -  
 
•A history of recreational drug use or allergy which in the opinion of the investigators contra-
indicates their participation.  
•Participation within 3 months in any other study testing a new molecular entity or drug or 
involving invasive procedures.  
•Those, in the opinion of the investigator, who may prove non-compliant with study 
procedures.   

 
 
 
 
 
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•History of an upper or lower respiratory infection (including coryza) within 3 weeks of baseline 
assessments (assessments and entry could be deferred. 
4.4 
Withdrawal Criteria 
Participants can withdraw at any point of the study without providing a reason for this during the 
study. In order to do this, they should contact the recruiting researcher in person, or by phone or 
email. Data collected up to point of study withdraw will still be included in result analysis.. Participants 
may stop the sample collection at any point without providing a reason.  They can do this by 
informing the researcher. The EBC device will be held by the participants during sample collection. 
 
5. ADVERSE EVENTS 
5.1 Definitions   
Adverse Event (AE): any untoward occurrence in a participant.   
 
Serious Adverse Event (SAE): any untoward and unexpected occurrence or effect that: 
• 
Results in death 
• 
Is life-threatening – refers to an event in which the subject was at risk of death at the time 
of the event; it does not refer to an event which hypothetically might have caused death 
if it were more severe 
 
• 
Requires hospitalisation 
• 
Results in persistent or significant disability or incapacity 
 
 
5.2. Reporting Procedures 
All adverse events should be reported.  Any questions concerning adverse event reporting should be 
directed to the Principal Investigator in the first instance.   
 
5.2.1 Non serious AEs 
All such events, whether expected or not, should be recorded.   
 
5.2.2 Serious AEs 
An SAE form should be completed and emailed to the Principal Investigator within 24 hours. 
 
All SAEs should be reported to the  London - Surrey Research Ethics Committee where in the opinion 
of the Principal Investigator, the event was: 
• 
‘related’, i.e. resulted from the administration of any of the research procedures; and 
• 
‘unexpected’, i.e. an event that is not listed in the protocol as an expected occurrence 
 
 
Local investigators should report any SAEs as required by their Local Research Ethics Committee, 
Sponsor and/or Research & Development Office. 
 
 

 
 
 
 
 
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6. ASSESMENT AND FOLLOW UP 
There is no planned follow up. Participants will  only be informed of the results obtained from their 
samples if requested. It is unlikely that there will be any incidental findings however should there be 
any the PI will inform the participant. 
 
7. REGULATORY ISSUES 
7.1 
Ethics approval 
The Study Coordination Centre has obtained approval from the London – Surrey Research Ethics 
Committee (REC) and Health Research Authority (HRA). The study must also receive confirmation of 
capacity and capability from each participating NHS Trust before accepting participants into the study 
or any research activity is carried out. The study will be conducted in accordance with the 
recommendations for physicians involved in research on human subjects adopted by the 18th World 
Medical Assembly, Helsinki 1964 and later revisions. 
 
7.2 
Consent  
Consent to enter the study must be sought from each participant only after a full explanation has been 
given, an information leaflet offered and time allowed for consideration.  Signed participant consent 
should be obtained.  The right of the participant to refuse to participate without giving reasons must 
be respected.  All participants are free to withdraw at any time. 
 
7.3 
Confidentiality 
The Principal Investigator will preserve the confidentiality of participants taking part in the study and 
fulfil transparency requirements under the General Data Protection Regulation for health and care 
research. Data and all appropriate documentation will be stored for a minimum of 10 years after the 
completion of the study, including the follow-up period.   
 
7.4 
Indemnity 
Imperial College London holds negligent harm insurance policies which apply to this study. 
 
7.5 
Sponsor 
Imperial College London will act as the main Sponsor for this study 
 
7.6 
Funding 
The study is funded by a grant from Innovate UK. Participants will be reimbursed £30 per visit. 

 
 
 
 
 
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7.7 
Audits  
The study may be subject to inspection and audit by Imperial College London under their remit as 
sponsor and other regulatory bodies to ensure adherence to GCP and the UK Policy Framework for 
Health and Social Care Research. 
 
 
8. STUDY MANAGEMENT 
The day-to-day management of the study will be co-ordinated through Dr Omar Usmani   
 
9. PUBLICATION POLICY 
 
The methods and data from the evaluation of the EBC device, biosensor and algorithm will be 
presented at conferences to analytical sciences specialists. Data collected in this study will be 
presented at seminars and meetings with potential clinical collaborators.   
 
The results of this study will be published in a peer-review analytical sciences journal to enable wider 
engagement of researchers and ensure dissemination of findings. 
 
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A. Greening, L. P. Ho, J. M. Hohfeld, Q. Jobsis, S. A. Kharitonov, F. Kelly, D. Laskowski, C. Lehmann, A. Lindstrom, S. Loukides, 
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Smith, W. R. Steinhaeusser, W. G. Teague, K. Toren, G. Vass, J. Vaughan, C. Vogelberg, H. Wirtz, and A. E. T. F. E. Breath, 

 
 
 
 
 
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