DELIVERABLE
Project Acronym: Symbiotic
Grant Agreement number: 665046
Project Title: Innovative autonomous electrical biosensor synergistically assembled inside a
passive direct methanol fuel cell for screening cancer biomarkers
D1.6 – First White Paper
Revision: 1.0
Authors:
Lúcia Brandão (ISEP)
Alfredo Silva (INOVA+)
Project co-funded by the EU within the H2020 Programme
Dissemination Level
P
Public
C
Confidential, only for members of the consortium and the Commission Services
X
D1.6 – First White Paper
Revision History
Revision
Date
1.0
18/08/2016
Author
Lúcia Brandão
Alfredo Silva
Organisation
ISEP
INOVA+
Description
First version
Statement of originality:
This deliverable contains original unpublished work except where clearly indicated otherwise.
Acknowledgement of previously published material and of the work of others has been made
through appropriate citation, quotation or both.
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TABLE OF CONTENTS
Page
1. Executive Summary .................................................................................................. 4
2. Project Presentation................................................................................................. 5
3. First Period Objectives ............................................................................................. 7
4. Results Obtained and Challenges Faced ................................................................... 8
4.1 WP1 - Management and dissemination ..................................................................... 8
4.1.1 Results Obtained .............................................................................................. 8
4.1.2 Challenges Faced ............................................................................................. 9
4.2 WP2 - MIP based biosensors for cancer biomarkers.................................................. 10
4.2.1 Results Obtained ............................................................................................ 10
4.2.2 Challenges Faced ........................................................................................... 10
4.3 WP3 - Selectivity of the biosensors ........................................................................ 11
4.3.1 Results Obtained ............................................................................................ 11
4.3.2 Challenges Faced ........................................................................................... 11
4.4 WP4 - Passive DMFC development ......................................................................... 12
4.4.1 Results Obtained ............................................................................................ 12
4.4.2 Challenges Faced ........................................................................................... 13
4.5 WP5 - Final assembling of the integrated biosensor ................................................. 13
4.5.1 Results Obtained ............................................................................................ 14
4.5.2 Challenges Faced ........................................................................................... 14
5. Future Objectives ................................................................................................... 15
TABLE OF FIGURES
Figure 1 - Schematic representation of the Symbiotic solution .......................................... 5
Figure 2 – Symbiotic project website ............................................................................. 8
Figure 3 – Biosensor developed on catalyst surface ........................................................10
Figure 4 – Concept of surface immobilization on different sensor surfaces .........................11
Figure 5 – a) Scanning Electron Microscopy image of the polymerized precursor for the Pt-free
catalyst. b) Scanning Electron Microscopy image of the Pt-free catalyst material after heat
treatment. c), d) High resolution TEM images of the catalyst. ...........................................12
Figure 6 – A) and B) Overview image of the set-up used to connect the fuel cell to the
electrochromic device and C) image of the electrochromic device in the coloured state powered
by the methanol fuel cell. .............................................................................................14
CAPTION TO TABLES
Table 1 – List of organisations that are part of the Symbiotic consortium ........................... 6
Table 2 – List of dissemination activities during Period 1 .................................................. 9
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1. Executive Summary
Symbiotic is a Horizon 2020 project that aims to develop an autonomous electrochemical
biosensor that is lightweight, disposable and low cost by using an innovative approach: hosting
its bioreceptor element inside a passive direct methanol fuel cell (DMFC). This will allow to build
an electrically independent, very simple, miniaturized, autonomous electrical biosensor.
The project started in June 2015, and is scheduled to last three years. It is divided into two
periods, the first lasting for the first year of the project (June 2015 to May 2016), and the
second for the remaining two years (June 2016 to May 2018). The first period of the Symbiotic
project ended recently, concluding the first part of the work.
In this first period several long-term technical decisions were taken, various challenges were
faced, and the first results were obtained. The present document aims to describe these
challenges and results, giving a broad view of the state of the project and future expectations
for period two.
The deliverable is structured in successive sections as to follow a logic path, from a generic
presentation of the project to the future objectives. The document sections are thus organised
as following:
• Project Presentation: what the project aims to achieve, organisations involved, and work
plan overview;
• First Period Objectives: what were the consortium expectations for the first period;
• Results Obtained and Challenges Faced: a per work package broad description of the
work done, including listing of the results obtained, and challenges met;
• Future Objectives: expectations for the second part of the project.
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2. Project Presentation
Incredibly low detection limits for disease markers can be achieved with electrochemical
biosensors. However their generalized use in routine healthcare systems for screening cancer
markers is still limited by the need of both specialized power-grid expensive equipment and
technical personnel for the analysis and interpretation of the reading signal. This project aims
to overcome this gap by merging electrical biosensors to fuel cells, combining the advantages
of both areas of research in a single synergetic device. The proposed electrochemical biosensor
will be completely autonomous, operating at room temperature and using the oxygen present
in the air, thereby allowing diagnosis everywhere.
Figure 1 - Schematic representation of the Symbiotic solution
Symbiotic is a response to the Horizon 2020 call FETOPEN-1-2014, and is being funded by the
European Union under the Future and Emerging Technologies (FET) programme. Its primary
objectives are to:
1. Develop and characterize several MIP electrochemical biosensors for cancer protein
biomarkers. These electrochemical biosensors are expected to have: i) high sensitivity
to the respective biomarker; ii) good selectivity for the target biomolecule; and iii) very
low detection limit;
2. Develop and characterize passive direct methanol fuel cells (DMFC), of low cost, and
high efficiency, operating at room temperature and non-forced air flow;
3. Determine the MIP biosensors’ accuracy using human samples from healthy individuals
and patients with cancer;
4. Integrate simple and low-cost devices composed by the best performing fuel cell anodes
and the most promising electrochemical biosensors architectures;
5. Develop the method for signalling biomarker presence in the autonomous biosensor
including electrochromic materials, LEDs and thin film transistor;
6. Fabricate a prototype of the autonomous biosensor.
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The project’s consortium is made up of five organisations, with a sixth (INOVA+) providing
support:
Table 1 – List of organisations that are part of the Symbiotic consortium
Num. Short Name Country
Long Name
1
ISEP
PT
Instituto Superior de Engenharia do Porto
2
Imperial
UK
Imperial College of Science Technology and Medicine
3
UNINOVA
PT
UNINOVA - Instituto de Desenvolvimento de Novas
Tecnologias
4
VTT
FI
Teknologian Tutkimuskeskus VTT Oy
5
AU
DK
Aarhus Universitet
The Symbiotic initiative has started on June 2015, and is scheduled to last for three years,
ending on May 2018. It is divided into two periods, the first lasting for the first year of the
project (June 2015 to May 2016), and the second for the remaining two years (June 2016 to
May 2018).
The project work plan is organised around five work packages, all of which span the entire
length of the project:
•
WP1 - Management and dissemination (leader ISEP): Project’s overall management
and coordination work, dissemination, and preparation of the future results exploitation;
•
WP2 - MIP based biosensors for cancer biomarkers (leader ISEP): Development
and characterization of several molecularly imprinted polymer (MIP) based
electrochemical biosensors sensitive to cancer protein biomarkers;
•
WP3 - Selectivity of the biosensors (leader AU): Determination of the biosensors’
selectivity by using human samples, and characterize protein/MIP biochemical
interactions with regard to the observed selectivity;
•
WP4 - Passive DMFC development (leader IMPERIAL): Development, integration, and
characterization of the passive direct methanol fuel cell (DMFC) that will transduce the
biosensor signal;
•
WP5 - Final assembling of the integrated biosensor (leader UNINOVA): Creation
of the final device, integrating the fuel cells developed in the previous work packages
with a signalling interface.
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3. First Period Objectives
The first period of the project, which ended recently, in general aimed at settling the core
technical decisions still open at the start of the project, developing the base technologies, and
achieving the first results. Hence Period 1 served as a stepping stone for the second period,
when the project will produce the system proposed.
The specific objectives for Period 1 were as follows:
•
•
•
•
•
WP1 - Management and dissemination (leader: ISEP):
o
Create project communication tool, define the ethical issues involved in the work,
and further breakdown and define the work structure;
o
Create project website;
o
Create package of dissemination materials and dissemination plan;
o
Jumpstart and guide the project work during the period;
WP2 - MIP based biosensors for cancer biomarkers (leader: ISEP):
o
Determine the conditions for radical polymerization initiation;
o
Achieving the anchoring of stable polymers on catalyst surface;
WP3 - Selectivity of the biosensors (leader: AU):
o
Establish Electrochemical/Optical test sensor;
o
Study of the first types of interfering species on the system;
WP4 - Passive DMFC development (leader: Imperial):
o
Define DMFC requirements, produce first DMFC stack designs, and validate first
single-cell DMFC operating at target conditions;
o
Determine ink-jet printing surfactant compatibility with anode and cathode
electrocatalysts;
WP5 - Final assembling of the integrated biosensor (leader: UNINOVA):
o
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Definition of the display operating conditions, and creation of the final display
design.
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4. Results Obtained and Challenges Faced
4.1 WP1 - Management and dissemination
4.1.1
Results Obtained
During Period 1 the various scheduled deliverables were developed and submitted. The project
was jumpstarted on the kick-off meeting, with subsequent meetings allowing the consortium to
further decide on the technical details. Specific work included:
•
Development of the management and quality plan, including creation of the deliverables’
template, partner communication procedure, and support documents;
•
Communication with the EC, including requests for information regarding the creation of
reports, administrative issues, etc.;
•
General support for the partners, including information on filling of financial
requirements, paper publishing, etc.;
•
Organisation of the project meetings, including scheduling, setup logistics, hosting (in
the case of face-to-face and technical meetings), and follow up;
•
Verification of quality of all project deliverables and their submission on the Participant’s
Portal;
•
Detailing of the work plan for the duration of the project, including division of
responsibilities, scheduling of tasks, definition of the goals, etc. This was done primarily
during the consortium meetings, both face-to-face and web-based;
•
Implementation of the work plan including coordination of the day-to-day operations,
request for updates from the partners and feedback, monitoring of delays and remedy
solutions, etc.;
•
Development of the project website (www.symbiotic-project.eu), and its update;
Figure 2 – Symbiotic project website
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•
Development of a set of materials (flyers, poster) to support the dissemination effort;
•
Development of a dissemination plan to detail the dissemination effort;
•
Performing of several dissemination actions (see table below).
Table 2 – List of dissemination activities during Period 1
Activity Type
Title
Date
Place
Size of Countries
Audience Addressed
Event
attendance
NanoPT
1619/02/2016
Braga,
Portugal
250
Worldwide
Project
presentation
Visit to ISEP of the
Portuguese Minister of
Science and Technology Manuel Heitor
25/02/2016
Porto,
Portugal
50
Portugal
Event
attendance
iBEM
21/03/2016
Porto,
Portugal
120
Worldwide
Project
presentation
Presentation to Martin
Bachmann from The Jenner
Institute, Oxford University
21/03/2016
Porto,
Portugal
10
Portugal
Event
attendance
3rd Austrian Biomarker
Symposium
1011/03/2016
Vienna,
Austria
500
Worldwide
National journals and TV
Press releases channel “Mentes que
Brilham” (Porto Canal)
18/05/2016
Porto,
Portugal
10 000
Portugal
Event
attendance
Biosensors 2016
2527/05/2016
Gothenbur
g, Sweden
1500
Worldwide
Visit to
partners
facilities
Visits to ISEP
Oct 2015,
May 2016
Porto,
Portugal
10
Portugal
Project
Presentation
Annual Institute meeting
presentation to associates
and visiting local industry
13/01/2016
Aarhus
Denmark
250
Denmark
4.1.2
Challenges Faced
The primary challenge was to jumpstart the project, which included the EC pre-project
negotiations, organisation of the kick-off meeting, setup of the consortium communication
process, definition of the quality plan, and further detailing of the work plan. All of these tasks
were achieved during the first half of Period 1.
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4.2 WP2 - MIP based biosensors for cancer biomarkers
4.2.1
Results Obtained
Several approaches were considered for the plastic antibody assembling, namely free radical
polymerization and electropolymerization of suitable monomers on different anode catalyst
architectures.
The most promising results ended up with the demonstration of a new approach to biosensing
devices aiming their ease and simple application in routine health care systems for cancer
screening even in a population not at risk. Our method considered a new concept for the
biosensor transducing event that simultaneously allows to obtain an equipment-free, userfriendly, cheap biosensor. The use of the anode triple-phase boundary layer of a passive direct
methanol fuel cell as the biosensor transducer is proposed (Figure 3).
For that, fuel cell anode catalysts were modified with a molecularly imprinted polymer (plastic
antibody) acting as the biorecognition element of health-related protein markers (ferritin was
used as model protein) and subjected to a fuel cell environment. The anchoring of a stable
polymeric layer (to be used as the bioreceptor) on the anode catalyst surface used a simple
one-step grafting from approach through radical polymerization. Such modification indeed
shows an increase in fuel cell performance due to the proton conductivity and macroporosity
characteristics of the polymer affecting the triple-phase boundary.
catalyst
polymer
Figure 3 – Biosensor developed on catalyst surface
Finally, the response and selectivity of the bioreceptor inside the fuel cell showed a clear and
selective response from the biosensor where a concentration detection limit two orders of
magnitude lower than in a three electrodes configuration was observed. Our proposed
pioneering transducing approach thus allows a significant amplification of the electrochemical
biosensor response.
4.2.2
Challenges Faced
Main challenges involved determination of the best initiation conditions for radical
polymerization. The technical and operating conditions for electrochemical evaluation of
biosensor due to the expected conflict requirements between working with biological samples
and energy producing systems were also stressed.
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4.3 WP3 - Selectivity of the biosensors
4.3.1
Results Obtained
Exploratory experiments under fuel cell environment were performed, allowing to address
preliminarily the influence of biological fluids on DMFC performance when the plastic antibody
is inserted at the anode. Other experiments allowed to evaluate the plastic biosensor response
to a model protein (ferritin) in real biological fluids.
Furthermore, the development of complementary sensor systems to the autonomous biosensor
in order to have a better understanding of the quantitative evaluation of the final sensor
performance is also underway. For that, a test system that allow the independent quantification
of binding and identification of bound components to the sensor substrate via multiple analytical
readouts is under development. Exploration of protein mass spectrometry for evaluating MIP
performance is also on course.
The main characteristics of the complementary sensor system are:
•
Establishment of a combined electrochemical/optical access system;
•
Demonstration of optical local refractive index measurement in the electrochemical set
up;
•
Development of carbon support chemistry on the sensor surface;
•
Preliminary work on formation/immobilisation of metal nanoparticles at the carbon
support chemistry.
Figure 4 – Concept of surface immobilization on different sensor surfaces
4.3.2
Challenges Faced
The main challenges to be faced are related to a proper anchoring on the anode fuel cell catalyst
particles provided that they are uniformly distributed and showing no aggregates on the sensory
platforms under development.
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4.4 WP4 - Passive DMFC development
4.4.1
Results Obtained
The objectives defined for the period were a stepping stone to work towards the primary
objective of providing a low-cost passive DMFC which can be used as the basis for the test
sensor. These interim objectives were achieved within the period as planned, and work has
started on the next steps toward the final objective. Beyond, the proof-of-concept of the project
was successfully demonstrated in nearly passive conditions within a DMFC environment for a
biosensor targeting the model protein ferritin.
For the passive DMFC development, the following work structure was followed during the period:
•
Paper design of fuel cell system: this work involved the fuel cell operational specifications
and the fuel cell stack design;
•
Development and testing of poison tolerant cathode catalyst which can operate under
the non-standard (for fuel cells) conditions which exist within the test environment
required for this system. A new oxygen reduction was that performs in anion containing
solutions, and at pHs which will be encountered in the test system and under which
conditions the standard oxygen reduction catalyst (Platinum) performs very poorly. This
catalyst should be capable of achieving the required level of performance, as shown in
results in single cell test devices.
•
Ink development for cathode catalyst. As the morphology of the new cathode catalyst is
different, the electrode development activity included ink development (comprising ink
formulation and processing), ink deposition, characterisation and testing.
a)
b)
d)
c)
Figure 5 – a) Scanning Electron Microscopy image of the polymerized precursor for the Pt-free
catalyst. b) Scanning Electron Microscopy image of the Pt-free catalyst material after heat
treatment. c), d) High resolution TEM images of the catalyst.
•
Ink development for anode catalyst. This part of the activity was focused on the
development of electrode suitable as anode for the Direct Methanol Fuel Cell (DMFC)
used as part of the Symbiotic project. Different compositions of the ink have been
considered and these have been deposited directly on Nafion membranes and carbon
papers using printing technologies.
•
Scale up of the DMFC manufacturing process. Taking the design produced by the paper
study, trial attempts have been made to see how such the system could be produced on
the large scale.
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4.4.2
Challenges Faced
The main challenge is the design of an electrode stack that is suitable for integration with the
electrochromic display and fluid sampling. The designed stack should meet the needs of the
final product, while keeping the stack simple and scalable. Future work will continue to improve
this design, including improvements to the flow fields and improve system reliability and
robustness.
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4.5 WP5 - Final assembling of the integrated biosensor
4.5.1
Results Obtained
Taking into consideration the outputs from the different partners, the team working in WP5
established that the easier way to integrate both the fuel cell and the signalling device would
be with an electrochromic display. Simultaneously, this device would allow to adjust to the
specifications of the power output of the fuel cell and wouldn’t require any specific circuit if the
signalling was directly connected with the biosensor. Two approaches for the production of the
device were studied: one using deposition of the materials by physical techniques (such as
sputtering), and another following a solution-based approach using a hydrothermal method for
synthesis of the electrosensitive particles.
Beyond, and also as a proof of principle, a commercial DMFC was connected directly to an
electrochromic device and coloration and decolouration was observed.
Figure 6 – A) and B) Overview image of the set-up used to connect the fuel cell to the electrochromic
device and C) image of the electrochromic device in the coloured state powered by the methanol fuel cell.
4.5.2
Challenges Faced
The main challenge was on the decision of the system actuating as the signalling device. As an
alternative of the colorimetric display, it was also proposed to integrate an electrochromic
transistor. Nevertheless, such display would require a more complex circuit and higher power
consumptions and therefore was left aside for this project.
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5. Future Objectives
Future work involves the optimization of the biosensor aiming a cancer biomarker. The choice
of the most selective and sensitive biomarker is undergoing with Portuguese Institute of
Oncology. The first steps to the fully integration within a complete autonomous device will be
also undertaken.
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