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Dados para a Candidatura: SFRH/BPD/101419/2014 (Bolsa de Pós-Doutoramento)

Nome do Candidato: João Paulo Grosso Pacheco
Domínio Científico: Química - Eletroquímica

Dados da Candidatura:

Informações sobre a candidatura

Tipo de bolsa: Bolsa de Pós-Doutoramento
Domínio científico
Química
principal:
Local de Realização: No país

Orientador(es) como indicado(s) pelo candidato:

Orientador Cristina Maria Fernandes Delerue Alvim de Matos
REQUINTE / Instituto Superior de Engenharia do Porto

Curricula de Orientador(es):
Com base nas indicações do candidato o sistema notificou o(s) orientador(es) para se associarem à candidatura e fornecerem
um currículo.
N. de orientações BD N. de orientações BPD
Tipo Orientações BD Orientações BPD
Nome apenas financiados pela apenas financiados
Orientador neste concurso neste concurso
FCT FCT
Cristina Maria Fernandes
Orientador 7 0 3 3
Delerue Alvim de Matos
(o nome do orientador é uma ligação ao respectivo CV)

Acolhimento
Instituição(ões) que confere(m) o Grau:

Instituições de Acolhimento:
Instituicão Departamento Centro de Investigação

n/a n/a REQUIMTE

Endereço para Correspondência

Morada: Rua Prof. Carolina Freitas Soares Carvalho, 130 3º dt frt

Código-postal: 4470-480 Localidade: Maia

Pais: Portugal

Telefone: Fax: E-mail Contacto:

913699214

Procurador do Candidato
Nome: n/a

Morada: n/a
Código-postal: n/a Localidade: n/a
Pais: n/a
Telefone (casa): Telefone (emprego): Telefone (móvel):

. Fax: E-mail Contacto:

. n/a n/a

Programa de Trabalhos
Título: Construction of point-of-care molecularly imprinted electrochemical devices for cancer
biomarkers detection
Domínio Científico: Eletroquímica
Data Início: (Programa Trabalhos) 01-01-2015 Duração: (meses) 36
Data Início: (Bolsa) 01-01-2015 Duração: (meses) 36

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Periodo de Permanência no Estrageiro n/a

Execução do plano

Research will be developed at REQUIMTE Network (Chemistry and Technology), Associate Laboratory. The work will take place at the
research center GRAQ (Group Reaction and Chemical Analysis) of Instituto Superior de Engenharia do Porto (ISEP). GRAQ infrastructure of
the labs and equipment (namely electrochemical equipment) are suitable for the development of the proposed research work.

The supervisor (Cristina Delerue-Matos) has a strong background on development of advanced chromatographic methodologies (LC
and GC-MS); sample preparation by applying different extraction techniques (MAE, QuEChERS) and their application to different matrices
(water, wastewaters, soil, sediments, biota, food…); development of electrochemical methodologies: preparation of nanomaterial
(carbon nanotubes, graphene, gold nanoparticles, magnetic nanoparticles, ...) electrochemical biosensors (enzymatic, immunosensors,
molecularly imprinted, DNA sensors,…) for clinical, food control and environmental applications.

The supervisor is the scientific coordinator of the group GRAQ (www.graq.isep.ipp.pt) with more than 60 researchers including
professors, Master, PhD and Post-doctoral students. Besides working on various research projects, she is the principal researcher of
several projects and the international coordinator of a FP7 project. She is the author of about 190 publications in international journals
in ISI Web of Knowledge. She supervised more than 15 PhD students and 24 master students.

Researched at GRAQ/REQUIMTE also include the development of sensors for cancer diagnosis through to project “Nano-electrode arrays
Biosensor for Early and Decentralized Breast-Cancer Diagnosis” (PTDC/SAU-ENB/114786/2009). So this project is important, as a logical
extension of this investigation line. Furthermore, an existing collaboration with IPO (Porto) through Dr. Joaquim Abreu de Sousa (head of
Surgical Oncology and director of the breast clinic of IPO and President of the Portuguese Oncology Society) and his team will allow
discussion of the results and the selection of the samples for method validation.

These conditions will enable the successful execution of the proposed work plan

Sumário

Biomarkers have many potential applications in oncology and the development and validation of point-of-care analytical methods has an
important role in this area. This project proposes the construction of new sensitive and selective electrochemical sensors for detection
of cancer biomarkers proteins that could be used to monitor response to treatment and/or evaluation of recurrence/progression of
cancer diseases. The design of the sensors will be based on molecular imprinted polymers technology (MIPs), in order to achieve high
selectivity towards the selected cancer biomarkers. Surface imprinting (2-D) will be performed by electropolymerization of electroactive
monomers after immobilization of biomarkers proteins on commercial screen printed electrodes (SPE). With this approach it?s expected
that low-cost, easily handling and disposable sensors could be prepared, with potential to be integrated with small pocked
electrochemical devices.

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Estado da Arte:

There are a range number of biomarkers which have been identified with different types of cancer (which include several proteins)
they can be detected in the circulation (whole blood, serum, or plasma) or secretions (stool, urine, sputum, or nipple discharge), or can
be tissue-derived. Among several applications in oncology, cancer biomarkers are very useful to assess a cancer´s response treatment
and to check for recurrence. In some types of cancer, biomarkers levels may reflect the extent or stage of the disease and be useful in
predicting how well the disease will respond to treatment. A decrease or return to normal levels of biomarker may indicate that the
cancer has responded favorably to therapy [1-3]. The immunometric assays are extensively used as clinical routine methods due to
features of being fast, sensitive, and highly automatable. However, they have limitations: their general inability to differentiate
between protein variants, the possibility of being affected by interferences, and the possible long lead time and high costs for
development [4]. Therefore, the development of analytical methods with sensitivity, specificity, and robustness for these biomarkers
can play an important role in this area. Electrochemical sensors are excellent tools to use as point-of-care devices due to their excellent
sensitivity, rapid response, simplicity, low cost, easy miniaturization and integration into automatic systems [5].
Among several strategies for fabrication of sensors, molecular imprinted polymers (MIPs) are one of the most promising tools for the
design and construction of biomimetic recognition systems [6, 7]. Generally, MIPs are obtaining by bulk (3-D) polymerization, a process
by which selected functional monomers (either organic or inorganic materials) are polymerized around a target analyte (template) in the
presence of a crosslinker agent. After polymerization, the template molecule is extracted and a polymer matrix, with sites
complementary in shape, size and functionality to the imprinted molecule, is obtained [8, 9]. They show favored affinity to the template
molecule compared to other molecules [9] The advantages of using MIPs are that they are robust, more stable to chemical and thermal
conditions, easy to prepare, reusable and low cost. In principle any molecules from the smallest one to the big structures like proteins or
even cells can be subject of imprinting in different matrices [10]. However, imprinting of macromolecules (like proteins) is more
problematic and challenging, and its development has been slower, compared with a wide range of successfully applications for small
molecules [11]. Several problems have been identified, including size (poor mass-transfer and permanent entrapment), complexity,
conformational flexibility, and solubility (limited choice of solvent). Surface imprinting (2-D) as proved to be a good approached. It
produces polymers in thin films or fixed to a supporting surface and polymerize around it [12, 13]. The imprinted binding sites are
located at or close to the surface of the MIPs, which solves the problems of restricted mass transfer, enabling easy extraction and
rebinding [13]. Although there are several reports about molecular imprinted sensor for protein detection [14-17], few are developed for
cancer-related protein detection [18].

Objectivos:

Taking advantage of the host institutions infrastructures and the expertise in the development of electrochemical sensors of
GRAQ/REQUIMTE research group, and to extend the investigation in sensors for cancer biomarkers the objectives of this project are
outlined below:

• To develop highly selective and sensitive electrochemical sensors for point-of-care detection of selected cancer biomarkers proteins.
• To apply the developed sensors essentially as tools to help in the monitoring of treatment response and in detection of recurrence or
progression of cancer diseases. Thus, biomarkers recommend for that purpose for different types of cancer will be selected, such as:
Carcino-embryonic antigen (CEA), Cancer antigen 153 (CA 15-3), CA cancer antigen 19-9 (CA 19-9), Cancer antigen 125, Prostate-
Antigen (PSA).
• To get a fast, easy to use, low cost, with lower consumption of reagents and environment friendly technology. For this reason the
project will be based on electrochemical sensors.
• To contribute to the design of new molecular imprinted polymers sensors for proteins and contribute to novel approaches for achieving
better imprinting factors.
• To contribute with new information and strategies for further incorporation in miniaturization and development of commercial
electrochemical sensors.
• To increase the capacity of the research groups in response to requests in health and clinical evaluation.

Another objective is the strengthen the collaborations between the research group of the supervisor and a team of IPO (Porto) headed by
Dr. Joaquim Abreu de Sousa (Head of surgical oncology and Director of the breast clinic of IPO and President of the Portuguese Oncology
Society) (collaboration established in the FCT-financed project "Nano-electrode arrays Biosensor for Early and Decentralized Breast
Cancer Diagnosis" (ref. PTDC/SAU-ENB/114786/2009)).

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Descrição Detalhada:

Circulating soluble protein cancer biomarkers such as CEA, PSA, CA 125, CA 15-3, CA 19-9, were selected since they are recommended
for monitoring response therapy in colorectal, prostate, ovarian, breast and pancreatic cancers, respectively [1].
Point-of-care devices should be portable, simple, easy use, cost effective and in most cases disposable. In order to achieve these
requirements, commercial screen printed electrodes (SPE), with either carbon or gold working electrodes (WEs), will be used as the
transducers. The reduced size of the SPEs lowers the sample volume down to 30-40 µL, which is advantageous especially in the analysis
of biological samples. Otherwise they can be easily integrated with small pocket size electrochemical devices, which makes then
attractive to be used in point-of-care applications. The selectivity will be achieved be using molecularly imprinted technology. Surface
imprinting (2-D) directly on SPE will be accomplished through electropolymerization. Among several methods for MIP´s preparation
electropolymerization will be used, since it is very simple, and permits the easy control of the polymer thickness and morphology by
adjusting the electrochemical conditions. Otherwise it’s widely used in electrochemical sensor preparation because it enables a direct
communication between the coating and the surface of a transducer [19]. Because proteins are large molecules, it’s expected that they
could be irreversibly entrapped, and only few are at the outer surface of the MIP film. Thus, surface imprinting approach will performed
to imprinting of the target cancer markers, in two steps:
(1) Attaching the protein template to the surface a SPE, either by adsorption or covalently binding;
(2) Electropolymerization of a polymer around the proteins previously bounded to the SPE, controlling the film thicknesses to ensure
proteins are not irreversible entrapped.
Since proteins are normally eletroinactive or show reduced eletroactivity, voltammetric analysis will be conducted using [Fe(CN)6)]3
as redox probe, measuring the differences between signals before and after protein binding.

Task 1 - Attachment of cancer biomarker proteins to SPE

Initially, directly adsorption on Au surfaces will be tested, by dropping of cancer biomarker on working electrodes surface. The binding
of proteins to gold surfaces is a complex process, which generally involves transport from the solution to the interface, binding to the
surface and subsequent relaxation conformation [20]. This phenomenon was already used in preparation of electrochemical MIP sensors
for proteins [17, 18]. Different concentrations and incubation times will be studied and the adsorption will be evaluated by
electrochemical impedance spectroscopy (EIS). It’s expected that the charge transfer resistance (Rct) signal increase if adsorption of
protein occurs.
Alongside, adsorption on glassy carbon SPE (GC-SPE) modified with nanomaterials, such as Au nanoparticles (AuNP) will be evaluated.
Nanomaterials have unique features such as larger surface-to-volume ratio with makes then attractive to be used as supports for proteins
MIPs preparation, improving complete template removal, better site accessibility and reduction of mass transfer resistance. Otherwise
they are used in sensing devices to lower the detection limits. Therefore, the binding of proteins to GC-SPE modified with AuNP will also
be tested.
In case of no binding occurs both on Au-SPE and modified GC-SPE, Au surfaces will be modified with self-assembled monolayers (SAM)
such as cysteamine. These will be functionalized with aldehyde groups, such as Glutareldeheyde, which has the ability to covalently
binding to proteins. Alternatively Silica nanoparticles modified with Glutareldeheyde can also be deposited on Au surfaces and used to
binding to protein.
At the end of this task we expect to have an adequate attachment of each cancer biomarker to the SPE, for further
electropolymerisation around it.
Task 2 – Electropolymerisation optimization of selected monomers
After the task 1 will be completed, electropolymerization around the attached proteins will be performed. The quality of the imprinted
cavities and thus the ability to selective binding to the template is strongly dependent of two factors: interactions between the
monomer and the template and template removal after polymerization. Thus, several electroactive polymers (pyrrol and derivatives,
aniline an derivatives, phenol and derivatives, o-phenylenediamine…) and electrochemical conditions for electropolymerization (range of
potential, number of cycles, scan rate) will be tested for each cancer marker. The imprinting factors (IF) will be determined, by
performing binding experiments of both MIPs and control non-imprinted polymers (NIP). The obtained results will be used to select
better imprinted polymer. If the templates are excessively entrapped within the polymer matrix, it’s difficult to effective remove the
template and creating the complementary cavities. So, in this task the optimization of removal conditions will also be performed
sideways with the electropolymerization optimization. Extraction will be performed by testing several denaturation agents and/or
proteinases.

Task 3 – Sensors characterization and analytical performance evaluation

The molecular imprinted electrochemical sensors will be characterized by Fourier transform infrared spectroscopy (FTIR), scanning
electron microscopy (SEM), impedance spectroscopy (EIS) and cyclic voltammetry (CV). Square wave voltammetry (SWV) essays in the
presence of the redox probe [Fe(CN)6)3-/4- will be performed for analysis of cancer biomarkers. The analytical performance of
fabricated MIP sensors will be evaluated through calibration curves, limits of detection and quantification, repeatability and
reproducibility, and also stability tests. A major feature of MIP-sensor is the selectivity toward the imprinted template. In this task, the
signal of response to the target cancer biomarkers will be tested in the presence of higher concentrations of several selected interfering
compounds. The reproducibility of the fabrication procedure will also be tested, in order to assure that they can be used as disposable,
without significant changes in the response signals of different electrodes. So, fabrication of numerous sensors (more than 50) will be
performed and the differences in signal responses between then will be determined.

Task 4 – Application to real samples

Finally, the ability of the sensors to detection of the cancer biomarkers in real samples will be studied. Samples without any
pretreatment will be analyzed. The validation of the developed electrochemical sensor will be executed by the analysis of serum
samples, which will be obtained from IPO (Porto), and the obtained results using the developed sensors will be compared with the results
obtained by the established procedures used in IPO.

Anexos:
Timeline.pdf

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Referências:

1. Henry, N.L. and D.F. Hayes, Cancer biomarkers. Molecular Oncology, 2012. 6(2): p. 140-146.
2. Rusling, J.F., et al., Measurement of biomarker proteins for point-of-care early detection and monitoring of cancer. Analyst, 2010.
135(10): p. 2496-2511.
3. Sahab, Z.J., S.M. Semaan, and Q.-X.A. Sang, Methodology and Applications of Disease Biomarker Identification in Human Serum.
Biomarker Insights, 2007. 2(BMI-2-Sang-et-al): p. 21-43.
4. Torsetnes, S.B., et al., Multiplexing Determination of Small Cell Lung Cancer Biomarkers and Their Isovariants in Serum by
Immunocapture LC-MS/MS. Analytical Chemistry, 2014. 86(14): p. 6983-6992.
5. Kimmel, D.W., et al., Electrochemical Sensors and Biosensors. Analytical Chemistry, 2012. 84(2): p. 685-707.
6. Malitesta, C., et al., MIP sensors - the electrochemical approach. Analytical and Bioanalytical Chemistry, 2012. 402(5): p. 1827
7. Suryanarayanan, V., C.-T. Wu, and K.-C. Ho, Molecularly Imprinted Electrochemical Sensors. Electroanalysis, 2010. 22(16): p.
1811.
8. Agbenyega, J., Molecular imprinted polymers. Materials Today, 2009. 12(1-2): p. 7-7.
9. Cheong, W.J., S.H. Yang, and F. Ali, Molecular imprinted polymers for separation science: A review of reviews. Journal of Separation
Science, 2013. 36(3): p. 609-628.
10. Fundamentals of Molecular Imprinting and Sensor Applications, in Handbook of Molecular Imprinting. 2012, Pan Stanford Publishing.
p. 1-1.
11. Whitcombe, M.J., et al., The rational development of molecularly imprinted polymer-based sensors for protein detection. Chemical
Society Reviews, 2011. 40(3): p. 1547-1571.
12. Turner, N.W., et al., From 3D to 2D: a review of the molecular imprinting of proteins. Biotechnol Prog, 2006. 22(6): p. 1474-89.
13. Lv, Y., T. Tan, and F. Svec, Molecular imprinting of proteins in polymers attached to the surface of nanomaterials for selective
recognition of biomacromolecules. Biotechnology Advances, 2013. 31(8): p. 1172-1186.
14. Cai, D., et al., A molecular-imprint nanosensor for ultrasensitive detection of proteins. Nat Nanotechnol, 2010. 5(8): p. 597-601.
15. Kan, X.W., et al., Molecularly imprinted polymers based electrochemical sensor for bovine hemoglobin recognition. Sensors and
Actuators B-Chemical, 2012. 168: p. 395-401.
16. Li, L., et al., Surface molecularly imprinted polymers-based electrochemical sensor for bovine hemoglobin recognition. Analyst,
2013. 138(22): p. 6962-6968.
17. Moreira, F.T.C., et al., Protein-responsive polymers for point-of-care detection of cardiac biomarker. Sensors and Actuators B
Chemical, 2014. 196: p. 123-132.
18. Viswanathan, S., et al., Molecular imprinted nanoelectrodes for ultra sensitive detection of ovarian cancer marker. Biosensors &
Bioelectronics, 2012. 33(1): p. 179-183.
19. Sharma, P., et al., Electrochemically synthesized polymers in molecular imprinting for chemical sensing. Analytical and Bioanalytical
Chemistry, 2012. 402(10): p. 3177-3204.
20. Voros, J., The density and refractive index of adsorbing protein layers. Biophysical Journal, 2004. 87(1): p. 553-561.

Bolsas Anteriores

Ano de Conclusão: Referência: SFRH/BD/30279/2006

Instituição: FCUP Periodo: 2006-2010 (meses)

Ano de Conclusão: Referência: SFRH/BPD/73943/2010

Instituição: REQUIMTE / ISEP Periodo: 2011-2014 (meses)

Atividade profissional
Deseja manter alguma atividade profissional durante o periodo da bolsa?
NÃO

Graus Académicos (Indicação dos certificados de disciplinas realizadas)

Grau académico Doutoramento (Doctorate / PhD) Descrição: Doutoramento em Química

(nr Anos): (4 anos)

Situação: Concluido Data de conclusão: 21-12-2010

Classificação: Certificado: Doutoramento_Química_ JoaoPacheco.pdf
Suplemento: Suplemento_Doutoramento_ Percentil: Percentil_Doutoramento_ JoaoPacheco.pdf
JoaoPacheco.pdf
Pais do grau: Portugal
ECTS: 0

Grau académico Mestrado (Pré-Bolonha / Master with Descrição: Mestrado em Química

(nr Anos): Thesis / MSc with Thesis)
(2 anos)
Situação: Concluido Data de conclusão: 27-09-2006
Classificação: Certificado: Mestrado_Química_JoãoPacheco.pdf

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Suplemento: Percentil: Percentil_Mestrado_ JoaoPacheco.pdf

Pais do grau: Portugal

ECTS: 0

Grau académico Licenciatura (Pré-Bolonha ? 4 ou 5 anos) Descrição: Licenciatura em Química

(nr Anos): (4 anos)

Situação: Concluido Data de conclusão: 17-09-2004

Classificação: 15 Certificado: Licenciatura_Química_JoãoPacheco.pdf

Suplemento: Percentil: Percentil_Licenciatura_ JoaoPacheco.pdf

Pais do grau: Portugal

ECTS: 0

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