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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 X

C Confidential, only for members of the consortium and the Commission Services


D1.6_V1.0 © Symbiotic of 15

Revision History

Revision Date Author Organisation Description

1.0 18/08/2016 Lúcia Brandão

Alfredo Silva

ISEP

INOVA+ 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This deliverable contains original unpublished work except where clearly indicated otherwise.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.



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



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.



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.



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.



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 Definition of the display operating conditions, and creation of the final display

design.



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

http://www.symbiotic-project.eu/



• 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

Audience

Countries

Addressed

Event

attendance NanoPT

16-

19/02/2016

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,


Event

attendance

3rd Austrian Biomarker

Symposium

10-

11/03/2016

Vienna,

Austria 500 Worldwide

Press releases

National journals and TV

channel “Mentes que

Brilham” (Porto Canal)

18/05/2016 Porto,

Portugal 10 000 Portugal

Event

attendance Biosensors 2016

25-

27/05/2016

Gothenbur

g, Sweden 1500 Worldwide

Visit to

partners

facilities

Visits to ISEP Oct 2015,

May 2016

Porto,


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.



4.2 WP2 - MIP based biosensors for cancer biomarkers


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, user-

friendly, 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.

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.


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.

catalyst

polymer



4.3 WP3 - Selectivity of the biosensors


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


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.



4.4 WP4 - Passive DMFC development


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.

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.

a) b)

c) d)




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.



4.5 WP5 - Final assembling of the integrated biosensor


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.


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.



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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