Marie Curie Fellowships

 

2010-2013: Marie Curie European Reintegration Grants (ERG) Call: FP7-PEOPLE-RG-2009

" CD-HLAsens "

The overall objective of CD-HLAsens is to develop a generic technological platform for point-of-care diagnostics capable of genomic detection with electrochemical transduction. As a model system, biosensor arrays for the screening of coeliac disease will be developed based on the concurrent detection of nucleic acids associated with genetic predisposition, i.e. HLA typing. This overall objective can be subdivided into the following sub-objectives:

1. Evaluation of novel sensor surface patterning (nanostructuring, polymer hydrogel)

2. Electrochemical detection schemes for the detection of coeliac disease susceptibility associated alleles (low resolution HLA typing)

3. On chip integration (microfluidics, temperature control)

4. On chip assay development for HLA typing in <15 minutes

5. Clinical validation and application of developed biosensor arrays to real blood samples

Such a detection strategy could be used for general population screening as well as for neonatal screening. Together with classical assay formats, novel biomolecule detection schemes based on supramolecular associations will be developed with the aim to fine tune the specificity and sensitivity of the sensors.

Scientific, technological or socio-economic benefit of the project

The advantages of the proposed biosensor system include their ease of use, their sensitivity, their inherent selectivity, their versatility (allowing ‘in-field’ use) and their cost effectiveness. The proposed innovation to be developed in this work will result in a technology platform of wide application and unquestionable socio-economic benefit, which is expected to be further exploited for other diseases thus increasing European competitiveness whilst contributing considerably to the quality of life well-being of the population. Furthermore, the tools to be developed will facilitate a cost-effective means of screening the population, which will allow its further application in developing countries where the costs of biopsy, currently used for definitive diagnosis of celiac disease, or genetic diagnostics are inhibitive.

State of the art

Coeliac disease diagnostic – HLA typing

Coeliac disease is a gluten-sensitive enteropathy that affects as much as 1% of the population and patients with coeliac disease should maintain a lifelong gluten-free diet, in order to avoid serious complications and consequences. It is essential to diagnose coeliac disease at the earliest possible conjecture. The Human Leukocyte Antigen system (HLA) is the name of the human major histocompatibility complex. This group of genes resides on chromosome 6 and encodes cell-surface antigens-presenting proteins that are involved in the immune system, autoimmunity and reproductive success. Of the encoded those of major interest are the so-called Class II antigens that have the function of presenting phagocytosed antigens from outside of the cell to T-lymphocytes. Five loci have been recognised to be related to class II antygens: HLA-DR, -DQ, -DP, -DM, and –DO. HLA typing is fundamental in histocompatibility and is always carried out previous to solid organ and bone marrow transplantation. In Coeliac disease HLA typing can find application in establishing the genetic risk (predisposition)[1; 2]. The factors linked to the Coeliac disease genetic predisposition are well known and classified as DQ2 and DQ8 HLA variation. These two variations can be detected by detecting a limited number of alleles: DQA1*0501, DQA1* 0505, DQA1*0201, DQB1*0201, DQB1* 0202 and DQB1*0302. The association between certain HLA types and coeliac disease is one of the strongest known, and the linkage peak to this region has been very high in all the genome wide linkage studies, and it is clear that understanding the exact nature of the HLA association is not only essential in unravelling the genetics of coeliac disease, but also helpful to understanding the pathogenesis on cellular level and finally, and of relevance here, in developing the use of HLA testing in a clinical situation as an adjunct to diagnosis in coeliac patients.

DNA sensing

The exceptional specificity provided by the inherent ability of complementary DNA molecules to duplex, or hybridise, by base pairing presents the biotechnologist with a simple means of detecting target sequences in a sample [3]. In recent years, the reverse hybridisation format, where the probe DNA is immobilised onto the surface of a solid support, has shown to be very effective. This approach enables the researcher to identify multiple targets once probes are immobilised in discrete locations[4; 5]. DNA biosensors use probe support materials, such as gold, platinum, glass and silicon, which provide a transduction element for the observation of events associated with hybridisation[6]. The diversity of methods is considerable and includes electrochemical systems. It is envisaged that these biosensors will provide means of inexpensive and point of care genetic analysis. In the short term, it is believed that these approaches cater for the identification of a relatively small number of targets that is required for routine diagnostics [7; 8]. Finally, current HLA typing methods do not reach the desired detection limits, are laboratory-based and often suffer from non-specific binding. These areas clearly need to be advanced in order to come to the implementation of biosensors for different applications.

Lab-on-a-chip

In the past decade, a large number of micro- and nanofluidic components, relying on a variety of working principles have been realised. Today, for most of the problems in microfluidics a multitude of solutions is available. In particular, lab-on-a-chip (LOC) or µTAS systems have been the focus of intense research [9; 10]. Sample delivery, preparation and fragmentation are key problems in this context, for which a variety of solutions have been developed [11]. Hence, a “toolbox” of considerable size already exists. In the future, the challenge to develop specific microfluidic components executing specific functions will persist, especially those that allow fast, multiplexed assays with a small sample volume. In addition to this, a major challenge is encountered in the system integration problem. In many application areas of microfluidics there is a specific demand for cheap and disposable, yet fully integrated systems allowing for multi-parameter assays of high sensitivity requiring highly precise control over assay temperature and reagent storage and distribution[7].

Multidisciplinarity

Given the nature of the research to be carried out within CD-HLAsens, the proposed work has a high degree of multidisciplinarity, focusing on biosensors, microfluidics, biotechnology, soft-hard interfaces and interactions. As such the proposal contains a high proportion of interest for various thematic priorities within the EU. For the realisation of the technology platform the sample delivery to the sensor array, the design of the sensor platform itself, the microfluidics design, dynamics and microfabrication will be realised to achieve the most suitable device. Furthermore, the detection system envisaged will consist of a microarray of electrochemical sensors. As such, surface chemistry and molecular biology for sensor derivatisation, transducer fabrication, and operation and statistical data processing will need to be investigated and brought together in order to achieve the objectives of CD-HLAsens.

Research methodology

The proposal has been divided into 5 objectives to be completed within the period required for the project. A timeline illustrating the milestones to be achieved and the interactions between the various objectives is presented in Table 1.

Objective 1 – Electrochemical HLA typing

In HLA class II genes most of the polymorphisms are in the second exon of the gene, and different alleles may only vary by one or two base pairs in their sequences. Primers will be designed for the multiplex amplification of specific alleles associated with coeliac disease. Low resolution class II typing protocols will be designed for both the DQ2 and the DQ8 heterodimers, that is, for the detection of HLA DQA1*05, DQA1*03, DQB1*02 and DQB1*0302 alleles. For example, in the case of the low resolution typing of the DQ2 heterodimer, primers and protocols will be designed that allow classification into general HLA groups. The amplicons will be detected using the genosensor arrays to be developed in Objectives 2 and 3, with immobilised probes designed against specific alleles. Thus primers will be designed for DQ2/DQ8 low resolution classification, and protocols for their multiplexed amplification elucidated. Probes of 18-20 bases will be designed for immobilisation on the genosensor arrays and hybridisation with the specific alleles amplicons. This work will be carried out in collaboration with TATAA (www.tataa.com).

Objective 2 – Surface chemistry and transduction methodology

In Objective 1, requirements and specification on the performances to be achieved by the electrochemical sensors will be elucidated. Of particular importance, the ability to differentiate between alleles that may differ by only one or two bases with sufficient sensitivity will be critical to the platform to be developed. From past experience acquired by the researcher during his previous Marie Curie fellowship, current approaches to the immobilisation of DNA probes at sensor surface are extremely limited to increase probe density and develop labeless transduction strategies.

New surface chemistries, to maximise DNA probe surface immobilisation and allele discrimination will therefore be developed. New developments in free radical living polymerisation, such as atom transfer radical polymerisation (ATRP) will be applied to the derivatisation of the sensors to integrate “DNA branches” onto polymer brushes. This approach will enable a considerable increase in DNA probe density while improving surface biocompatibility by careful selection of the polymer backbone. In a first instance, acrylamide brushes will be considered, however other known biocompatible monomers such as 2-methacryloyloxyethyl phosphorylcholine will be assessed in order to limit non-specific interaction and maximise sensitivity.

Figure 1 – (a) ATRP polymerisation of DNA-acrylamide modified probes duplex to form a “DNA-crosslinked” polymer (b). Upon de-hybridisation, the polymer will form brushes with a radically different physical and mechanical beahaviour.

A new strategy for the detection of single base differences between alleles will stem from this immobilisation strategy. By imprinting the allele to be detected within the polymer brushes and therefore carefully locking the DNA probes location within the polymer layer (Figure 1) using 2 or more DNA probes as well as mild chemical cross-linking, a 3D polymeric environment will be synthesised where only binding of the perfect match will induce large conformational changes (i.e. additional cross-linking) within the layer. A partial matching sequence might bind to the polymeric system, but would not generate sufficient conformational changes to be directly measured. However, if necessary, elucidation of the mismatch could be possible by use of appropriate base labels.

Consquently, the transduction of DNA binding to the brushes can be detected directly, i.e. without the need for labeling, by measuring the increase in impedance of the polymer as it becomes more cross-linked, i.e. stiff. Depending on the sensitivity of the system, sensitive amperometric measurements would be realised by following the recent development in electrochemiluminescence. DNA probes, locked into the polymer matrix, will be labelled electron acceptors and donors (i.e. Ferrocene-Ruthenium), which upon binding of the perfect match, would be brought into vicinity and generate or quench an optical signal (Figure 2).  

Figure 2 – Example of electrochemiluminescent detection of hybridisation events via molecular quenching, taking place at an electrode surface

Objective 3 – Electrode array development 

Size, structure and arrangement of sensors is of crucial importance to reach maximum sensitivity. Objective 3 will therefore address these requirements. The researcher has gained important insight in the microfabrication of sensors as well as their nanostructuring during his Fellowship [12; 13; 14]. This task will allow him to continue to expand his experience in the field. Of particular interest for this objective is the realisation of micro- and nano-electrode arrays, their integration and functionalisation.

Objective 4 – Microfluidic chip design and sensor integration

Objective 4 will aim at improving the designs and prototypes realised during the previous Fellowship, and integrating additional features to the microfluidic system, such as controlled temperature control and sample and reagent delivery to realise the best assay conditions in order to reduce the assay time. Additional aspects to be considered consist in sensor pre-treatment, speed of measurements and also integration of electronics and signal processing units towards the realisation of a completely integrated Laboratory-on-a-chip system.

Objective 5 – Validation of integrated device and regulatory aspects

Although all development work will be carried out using synthetic probes, amplicons and PCR products, validation of the HLA typing sensor array will be carried out using real samples made available by the host organisation via already existing partnerships in celiac disease management. Parameters to be evaluated will consist in sensitivity, cross-reactivity and elucidation of false-negative and false-positives as well as sensor robustness under storage conditions. The researcher has reached the point of maturity expected in the technological development of diagnostic devices following his Fellowship and now seeks to achieve a better understanding of regulatory aspects necessary to the concretisation of a research concept into a commercial product (e.g. CE marking, 510(K), FDA).

References:

1. Kaukinen, K., J. Partanen, M. Maki and P. Collin (2002). HLA-DQ typing in the diagnosis of celiac disease. American Journal of Gastroenterology 97(3): 695-699.

2.  Louka, A. S. and L. M. Sollid (2003). HLA in coeliac disease: Unravelling the complex genetics of a complex disorder. Tissue Antigens 61(2): 105-117.

3.  Andersen, E. S., M. Dong, M. M. Nielsen, K. Jahn, A. Lind-Thomsen, W. Mamdouh, K. V. Gothelf, F. Besenbacher and J. r. Kjems (2008). DNA Origami Design of Dolphin-Shaped Structures with Flexible Tails. ACS Nano 2(6): 1213-1218.

4.  Auburn, R. P., D. P. Kreil, L. A. Meadows, B. Fischer, S. S. Matilla and S. Russell (2005). Robotic spotting of cDNA and oligonucleotide microarrays. Trends in Biotechnology 23(7): 374-379.

5.  Barbulovic-Nad, I., M. Lucente, Y. Sun, M. J. Zhang, A. R. Wheeler and M. Bussmann (2006). Bio-microarray fabrication techniques - A review. Critical Reviews in Biotechnology 26(4): 237-259.

6.  Ju, H. X. and H. T. Zhao (2005). Electrochemical biosensors for DNA analysis. Frontiers in Bioscience 10: 37-46.

7.  Tudos, A. J., G. A. J. Besselink and R. B. M. Schasfoort (2001). Trends in miniaturized total analysis systems for point-of-care testing in clinical chemistry. Lab on a Chip 1(2): 83-95.

8.  Wang, Y., H. Xu, J. M. Zhang and G. Li (2008). Electrochemical sensors for clinic analysis. Sensors 8(4): 2043-2081.

9.  Schueller, O. J. A., S. T. Brittain and G. M. Whitesides (1999). Fabrication of glassy carbon microstructures by soft lithography. Sensors and Actuators A 72: 125-139.

10. Bruin, G. J. M. (2000). Recent developments in electrokinetically driven analysis on microfabricated devices. Electrophoresis 21(18): 3931-3951.

11.  Laser, D. J. and J. G. Santiago (2004). A review of micropumps. Journal of Micromechanics and Microengineering 14(6): R35-R64.

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2008-2010: Intra-European Fellowships (IEF) Call: FP7-PEOPLE-2007-2-1-IEF 

" RBCE-GenoDiagnoSens"

The overall objective of RBCE-GenoDiagnoSens is to exploit breakthroughs at the confluences of micro-, nano- and bio-technologies for the realisation of a low-cost minimally-invasive intelligent diagnosis system prototype using a nanotechnologybased device for the ultra-sensitive detection and analysis of RNA/DNA present in circulating tumorous cells (CTC’s) involved in breast cancer using an array of electrochemical nano-biosensors.
The advantages of the exploited electrochemical biosensors for RNA/DNA analysis are their sensitivity, their inherent selectivity, their versatility and their cost effectiveness.

State of the art

Breast cancer and diagnosis

 

Cancer cells in bone marrow samples and bronchial alveolar lavage have shown promises as prognostic indicators in e.g. early-stage breast cancer, lung cancer [1,2] and cancer cells in peripheral blood have also been demonstrated to reflect the biological characteristics of tumours including the potential for metastasis development, tumour recurrence and prognosis [3,4,5]. In a prospective, multicentre study of 177 breast cancer patients the number of circulating tumour cells were shown to be an independent predictor of progression-free survival and overall survival in patients with metastatic breast cancer [6].
Breast cancer is the most common cancer, and according to GLOBOCAN 2002 approximately 1.1 million new cases are diagnosed each year and the 5-year prevalence is approximately 4.4 million cases. The 5– year survival of breast cancer with regional spread is 76% and 10 years survival approximately 55 %. Thus, the disease has in many cases been changed into a chronic disease where the disease may be controlled by optimal clinical management and monitoring of the effect of different treatment modalities. It is therefore important to have sensitive and specific methods to monitor the status of the disease and cancer biology. The analyses of CTC’s as minimal invasive “tumour biopsy” samples provides the possibility for multiple tumour biopsies for frequent control of molecular markers of cancer cells to determine the response to the therapy. Analyses of CTC’s in breast cancer have in several studies been shown to reflect the disease status and be useful to monitor disease progression and therapy response [7,8].
Technology for sensitive and specific haematological determination of cancer cells has the potential to provide improved staging, prognosis and therapy monitoring of cancer of virtually all organs. Ashworth, who described a patient where cancer cells were detected in the blood after the patients’ death, published the first report of circulating tumour cells as early as 1869. Cancer cells were initially analysed by different light microscopic procedures of cells isolated from blood and gained large scientific and clinical interest.
However, owing to very low specificity and sensitivity in controlled studies, these methods were not considered to be of any clinical significance [9]. The introduction of novel immunological methods for immunomagnetic isolation/enrichment and characterisation in combination with highly sensitive molecular analytical techniques for detection and amplification of nucleic acids (e.g. RT-PCR), haematological spreading of cells with the characteristics of tumour cells can be detected in the bone marrow and peripheral blood of patients with cancer of virtually all organs, e.g. breast, lung, colorectal cancer, prostate, melanoma [7-11]. The current screening methods are limited by the relatively low speed and high expense of Fluorescent Activated Cell Sorting. (FACS) and the complexity and high hands-on-time of pre-enrichment methods.
The ability to detect and characterise such cells routinely at concentrations as low as 1 cell per ml of bone marrow/blood could have a profound influence on the early diagnosis, risk stratification in the adjuvant setting, early detection of relapse, and the development and evaluation of new-targeted therapies. Analysis of circulating tumour cells would be obvious for diagnosis of blood malignancies (i.e. leukaemias) and flow cytometry analysis and immunophenotyping is standard practise today. In this area a simple and rapid device such as the RBCEGenoDiagnoSens platform would have a definitive application. The analysis of CTC could be applied as an inexpensive screening tool, but probably has much stronger applications for the detection of recurrent disease, prognosis, the follow-up of therapy (e.g. the case of breast cancer proposed here). Thus, the rationale for CTC of epithelial cancer in most carcinomas is quite straightforward. According to literature, most studies have been carried out with breast cancer, colorectal cancer and prostate cancer, and in all these diseases the same clinical indication is valid, i.e. prognosis, detection of recurrent disease, follow-up of treatment and possibly also staging of the disease [6].
Addressing the health care requirement of the future of an individualised theranostic approach, the specific applications that will be demonstrated in RBCEGenoDiagnoSens will be the RNA/DNA detection of breast cancer cells (more specifically, circulating tumour cells or CTC’s). The innovation proposed in RBCEGenoDiagnoSens will result in a concrete prime deliverable of a technology platform of wide application and unquestionable potential socio-economic benefit, which could contribute considerably to the quality of life of the population and control of health care cost.

DNA Biosensors 

 

The exceptional specificity provided by the inherent ability of complementary DNA molecules to duplex, or hybridise, by base pairing presents the biotechnologist with a simple means of detecting target sequences in a sample. In recent years, the reverse or type II hybridisation format, where the probe DNA is immobilised onto the surface of a solid support, has shown to be very effective. This approach obviates the need to immobilise the sample and, more importantly, enables the researcher to identify multiple targets once probes are immobilised in discrete locations. By the application of photolithographic methods, a glass chip can be encoded with an ordered array of over 100,000 different probes. With this format, hybridisation at individual probe sites can be observed if the sample DNA is previously labelled with radioisotopes or with tags suitable for optical detection. However, the small geometries involved require large, sophisticated, sensitive and high-resolution optical equipment for hybridisation detection (e.g. Affymetrix GeneChip). As a laboratory based method for sequencing, polymorphism screening or gene expression monitoring, this represents a quantum leap in the volume of information that can be acquired in a single analysis, but are prohibitively expensive. This approach, however, thus does not lend itself to complete miniaturisation or for the provision of inexpensive genetic testing. DNA biosensors based on the detection of hybridisation events are currently being developed and offer an alternative to standard amplification product detection. These DNA biosensors use different probe support materials, such as gold, platinum, glass and silicon, which provide a transduction element for the observation of events associated with hybridisation. The diversity of methods is considerable and includes amperometric, potentiometric, quartz crystal microbalance and impedimetric systems. It is envisaged that these biosensors will provide means of inexpensive and point of care genetic analysis. In the short term, it is believed that these approaches will not compete with the high information density research-focused optical arrays, but rather cater for the identification of a relatively small number of targets that is required for routine diagnostics.
In addition, current sensors do not reach the desired detection limits, are laboratorybased and often suffer from non-specific binding. These areas clearly need to be advanced in order to come to the implementation of biosensors for different applications. 

Lab-on-a-chip

 

In the past decade, a large number of micro- and nanofluidic components, relying on a variety of working principles have been realized. Today, for most of the problems in microfluidics a multitude of solutions is available. In particular, lab-on-a-chip (LOC) or μTAS systems have been the focus of intense research. Sample delivery, preparation and fragmentation are key problems in this context, for which a variety of solutions have been developed.
The problem of fluid transport might be solved in a number of different ways including simple pressure driven flows or more exotic principles as electrowetting or thermocapillary pumping. A key requirement of microfluidic systems designed for biochemical assays is fast mixing. To address this problem a variety of solutions have been reported, among others multi-lamination, split-recombination or electrokinetic mixers. Hence, a “toolbox” of considerable size already exists. In the future, the challenge to develop specific microfluidic components executing specific functions will persist, especially those that allow fast, multiplexed assays with a small sample volume. In addition to this, a major challenge is encountered in the system integration problem. The full implementation of the advantages of microfluidic systems is due not only to the availability of components of moderate complexity, but also to the full integration of systems allowing the performance of a multitude of operations or the processing of a large number of fluidic samples in parallel. In many application areas of microfluidics there is a specific demand for cheap and disposable, yet fully integrated systems allowing for multi-parameter assays of high sensitivity. In many cases, polymers are the most suitable materials meeting such requirements. Polymer microsystems can be mass fabricated by technologies such as injection moulding and will play a major role in RBCE-GenoDiagnoSens.
The microfluidics developments undertaken will have to understand and take into account the constraints set by polymer microfabrication technology. The microsystem aims to be affordable and mass-producible by cost-effective means. Very often, such polymer components have to be integrated with active elements on silicon substrates, such as sensors. Such integration of silicon and non-silicon technologies still constitutes a major challenge, especially when in the field of microfluidics for (bio)sensor technologies. 

Multidisciplinarity

Given the nature of the research to be carried out within RBCE-GenoDiagnoSens, the proposed work has a high degree of multidisciplinarity by focusing on nanobiosensors, microfluidics, immunology, biotechnology, soft-hard interfaces and interactions for the development of intelligent diagnosis instruments for future healthcare. As such the proposal contains a high proportion of interest for various thematic priorities within the EU.
For the realisation of such technology platform several disciplines are to be brought together. To achieve the initial sample pre-processing and delivery to the nanosensor array as well as the design of the sensor platform itself, microfluidics design, dynamics and microfabrication studies will be taken into account to realise the most suitable device. In addition, the detection system envisaged will consist in a microarray of electrochemical sensor. By derivatising each sensor with a different functionality, i.e. cancer biomarkers elucidated from the fields of immunology and biotechnology, a
bundle of genetic information will be extracted and processed to provide a profile of the cancerous state. As such, surface chemistry and molecular biology for sensor derivatisation, transducer operation and statistical data processing will need to be investigated and brought together in order to achieve RBCE-GenoDiagnoSens. 

Research methodology

The proposal has been divided into 5 objectives to be completed within the 24 months period required for the project. A timeline illustrating the milestones to be achieved and the interactions between the various objectives is presented in Table 1.

Objective 1 – Development of DNA electrochemical biosensor surface chemistry 

Surface chemistry will be at the heart of the proposed technology. Optimum immobilisation of DNA probes at the sensor surface is of prime importance. A number of approaches have been reported in the literature and new approaches will be investigated. We envisage using “thiolated” DNA probes, readily available from commercial sources. Thiols are known to self-assemble from solution at metal surfaces such as gold and platinum. Alkanethiols bearing different functional end groups such as acids, amines or alkane functionality are routinely used within the biosensor and surface chemistry communities to tune surface properties to their needs.
The main challenge resides in the optimisation of the immobilisation of thiolated-DNA probes. Although thiolated-DNA readily self assembles at gold surfaces, the length of the DNA probes usually limits the quality of the monolayer obtained. Therefore, short alkanethiols are often included in order to re-arrange the DNA monolayer into an ordered nanostructure.

Initial plans for optimisation will look at (i) the immobilisation of thiolated DNA on gold electrode followed by backfilling with a second alkanethiol, (ii) direct co-immobilisation of DNA in the presence of the backfilling alkanethiol. The effect of the ratios of one to the other will be measured. In addition, the nature of the backfilling alkanethiol will be investigated. New approaches to limit the effect of non-specific binding at the sensor surface will be investigated. For example, compounds such as alkanethiols incorporating poly(ethylene glycol) units present great promise, and new monomers based on phosphorylcholine moieties have been reported to considerably limit sensor fouling. Electrochemical techniques such as amperometry, voltammetry and electrochemical impedance spectroscopy are the techniques of choice to characterise the prepared surface. In addition, the host institution is well equipped in state-of-the-art molecular interaction instrumentation such as surface plasmon resonance (Biacore), electrochemical quartz crystal microbalance, as well as atomic force microscopes and confocal microscopes, excellent additional tools for the characterisation of the prepared sensors.
From previous extensive experience in surface chemistry and coupling techniques applied to the field of biomimetic and synthetic polymers, the researcher already possesses a strong theoritical and practical understanding of the challenges ahead, whilst support and training in the new field of DNA handling and biosensing will be provided by the host institution.

Objective 2 – Biotechnology and ssDNA preparation

As initial targets, known breast cancer markers, referred to as BRCA1 and consisting of Exon13, Exon16, Exon20, Exon21 and Exon22 will be used and the corresponding DNA probes obtained from commercial sources. However, the complementary DNA sequences will be produced in-house using techniques such as polymerase chain reaction (PCR) and multiplex ligation-dependant probe amplification (MLPA). The host institution is a renown institute in the field and the team possesses extensive experience and facilities. It is expected that the researcher will be actively exposed to these preparative techniques and gain a practical professional training in this area. The resulting double stranded (ds) DNA will be denatured to produce the complementary single stranded (ss) DNA. Means to isolate the ssDNA will be investigated. DNA amplification on solid support could simplify this step and will also be looked at. Finally, novel breast cancer biomarkers currently under intensive research, should be made available via the host institution’s contacts and tested appropriately. 

Objective 3 – New ultra-sensitive electrochemical DNA hybridisation detection

An extremely sensitive chip-based sensor allowing the qualitative and quantitative detection of amplified DNA will be developed following two novel detection amplification approaches. In the first approach, molecular beacons labelled with an electrochemical redox probe, for example ferrocene, will be immobilised on gold electrode surfaces via thiol coupling. In the absence of target, the redox label is in close contact with the electrode surface, allowing electron transfer and signal generation. In the presence of target, the beacon opens up, spatially moving the redox label away from the electrode surface, resulting in a quantitative reduction in signal. The system thus represents a washless, reagentless genosensor of high sensitivity and genericity in its design.

Figure 1 Schematic of electrochemical molecular beacon

The second approach will look at the application of target sensitive liposomes. Hundreds to thousands of enzyme or electrochemical probe molecules can be encapsulated within these liposomes, thus providing a much enhanced signal when compared to the conventional one enzyme/one probe label per reporter DNA. 

Figure 2: Schematic of target sensitive genoliposome filled with ALP reporting enzymes

Objective 4 – Design and realisation of first generation of microfluidic device and

microelectrode array

While objective 1, 2 and 3 represent the core of the technological platform and will be initially realised on available gold microelectrodes, considerations into microfabrication will be taken into account. Previous experience of the researcher as well as facilities available at the host institution (mask aligner, photolithographic set-up, spin-coater and developer) make possible the development of a first prototype of microfluidics for sample pre-processing and delivery to the sensor platform. In addition, industrial and academic contacts available via the host institution will expose the researcher more widely to microfabrication technologies (photolithography, injection moulding, silicon etching...) and the challenges to take into consideration when working towards a mass fabricated commercial micro-device.

Objective 5 – Integration and proof of concept of prototype

This final objective will bring together the four previous objectives and will demonstrate the operation of the DNA based electrochemical microsensor platform. Although, this objective will be run in parallel with the other objectives, as presented in Table 1.
It is important for the researcher to reach a point of maturity in his understanding of the research and development process of a fully functional diagnostic device. Similarly, it is as interesting for the host institution to have developed a technological platform available for future applications and research lines within the group.  As described in Objective 4, several microfluidics and microelectrode design will be created and the optimised versions brought together to realise the proof of concept of the operation of the electrochemical detection platform.

References:

1 - Ahrendt SA, Chow JT, Xu LH et al. Molecular detection of tumor cells in bronchoalveolar lavage fluid of patients with early stage lung cancer. J Natl Cancer Inst. 1999;91:332-339.

2- Braun S, Pantel K, Muller P et al. Cyto-keratin-positive cells in the bone marrow and survival of patients with stage I, II and III breast cancer. N. Engl J Med 2000;342:525-533.

3- Wharton RQ, Jonas SK, Glover C et al. Increased detection of circulating tumor cells in the blood of colorectal carcinoma patients using two reverse transcriptase-PCR assays and multiple blood samples. Clin Cancer Res. 1999;5:4158-4163.

4- Smith BM, Slade MJ, Englisg J et al. Response of circulating tumor cells to systemic therapy in patients with metastatic breast cancer: comparison of quantitative polymerase chain reaction and immunocytochemical techniques. J Clin Oncol. 2000;18:1432-1439. 

5-Uchikura K, Takao S, Nakajo A, et al. Intraoperative molecular detection of circulating tumor cells by reverse transcription-polymerase chain reaction in patients with biliary-panceratic cancer is associated with hematogenous metastasis. Ann Surg Oncol. 2002;9:364-370. 

6- Christofanilli M, Budd GT, Ellis MJ, et al. Circulating tumor cells, disease progression, and survival in metastatic breast cancer. N Engl J Med. 2004;351:781-791. 
7- Christopherson WM. Cancer cells in the peripheral blood: A second look. Acta Cytol. 1965;86:169-174.
8- Ylikoski A, Karp M, Pettersson K, Lilja H, Lovgren T. Simultaneous quantification of human glandular kallikrein 2 and prostate-specific-antigen mRNAs in peripheral blood from prostate cancer patients. J Mol Diagn. 2001;3:111-122. 

9- Smith B, Selby P, Southgate J, Pittman K, Bradley C, Blair GE. Detection of melanoma cells in peripheral blood by means of reverse transcriptase and polymerase chain reaction. Lancet 1991;338:1227-1229 

10- Silva JM, Dominguez G, Silva J et al. Detection of epithelial messenger RNA in the plasma of breast cancer patients is associated with poor prognosis tumor characteristics. Clin Cancer Res. 2001;7:2821-2825. 

11- Pantel K, Izbicki J, Passlick B et al. Frequency and prognostic significance of isolated tumor cells  in bone marrow of patients with non-small-cell lung cancer without overt metastases. Lancet;347:649-653.

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