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Highly sensitive and specific low-cost lab-on-a-chip system for Lyme disease diagnosis

Final Report Summary - HILYSENS (Highly sensitive and specific low-cost lab-on-a-chip system for Lyme disease diagnosis)

Executive summary:

The overall objective of HILYSENS was to develop an innovative diagnostic tool that could fill in the gap between clinical research and the diagnostics market in Lyme Disease. The core of the project was the development of a lab-on-a-chip microfluidic device that permits a very sensitive detection of anti-Borrelia (the causative bacteria of Lyme disease) immunoglobulins (Ig) in human blood serum. To achieve the high sensitivity required to detect the low protein amounts, a micro-fluidics lab-on-a-chip approach is used to trap the immunoglobulins through the binding to the antigens in a very small localised region. Fluorescence detection was achieved by quantum dots (QD) technology due to its excellent properties in terms of enhanced signal to background detection and performance in multiplexed assays. A highly-sensitive fluorescence read-out system for fast and automatic detection of the multiple fluorescent signals generated by the lab-on-a-chip multi-antigen assay together with user friendly software was developed to enable unambiguous interpretation of test results. New promising antigens shown to elicit specific serological responses in acute and chronic Borrelia infections in experimental clinical studies have been further validated in this project and arrayed into the chip detection chamber to generate a multi-antigen HILYSENS microfluidic tool.

Project Context and Objectives:

The scientific and technical objectives, were as follows:

1.Development of a novel lab-on-chip diagnostic test cartridge (WP2, milestones 2 and 3) with the following features:
-All functions integrated in the chip without external connections (inlet reservoirs, mixing valves, detection area and fluids pumping).
-Fast and flexible MeV Ion lithography techniques was used and further optimized to achieve the required properties (wall smoothness and wettability) of the micro-fluidic structure and to design high aspect ratio channels to allow strong capillary forces.
-Capillarity pumping structures for the transport of sample and reagents to the detection chamber. The pumping structure was dimensioned to achieve a flow rate (up to 0.4μl/min) allowing a testing time target of less than 20 minutes and allowing to pump the whole tested volume through the detection chamber.
-Detection chamber, where the human immunoglobulins against Borrelia antigens has been trapped by the immobilized antigens, with an immobilisation capacity of more than 1000 protein molecules. The design is compatible with the proposed read-out system, and include the required wave-guide structures.
-Multi-antigen assay: multiplex detection of different immunoglobulins in the same sample was achieved by coating different areas within the detection chamber with up to 15 different antigenic proteins.
2.Validation of highly specific antigenic proteins as biomarkers for Lyme Disease diagnostic. Specific antigenic peptides and proteins previously reported in small experimental clinical studies to differentiate between acute, chronic infections and autoimmune response associated with LD has been validated in at least 50 samples from LD patients provided by SME BCA.
3.Production of antigenic recombinant proteins and peptides. Those antigenic proteins and peptides that proved useful for diagnostic purposes and are not commercially available has been produced, at small-scale for the lab-on-a-chip prototypes, in WP3.
4.Development of procedures for the immobilization of the antigens into the detection chamber and the coating of QDs with antibodies against human sera to be incorporated as reagents in the chip.
5.Development of a new fluorescent read-out device for the lab-on-a-chip test cartridge able to detect and quantify the QD signals from the different antigen spots in the detection chamber. Preliminary specifications for the reader have been defined.
6.Development of a signal processing and evaluation algorithm together with a user-friendly software interface for simple and unambiguous interpretation of results (WP5, milestone 5).
7.Testing of the HILYSENS tool in clinical environment. The tool has been tested in 50 human samples provided by small and medium-sized entreprises (SME)-Hospital BCA. Careful correlation has been established between the clinical diagnostic (based on standardised symptoms list) and the laboratory findings. Key features of the HILYSENS tools including sensitivity, specificity, reproducibility and detection limits were defined (WP6, Milestone6).

Other objectives essential as well to ensure the successful achievement of the project goal (i.e the exploitation of the results by the consortium small and medium-sized entreprises (SME)) included:
8.Dissemination of the diagnostic product to the potential market audience including SMEs from the diagnostic sector, technology and business investors, end-user clinicians and clinical laboratories. In addition project results and knowledge were disseminated to patients' associations and health authorities in order to improve widespread Lyme Disease awareness.
9.Transfer of the project results to the consortium through training activities planned to promote the uptake of the results by small and medium-sized entreprises (SME) partners.
10.Ensure the protection of Intellectual Property Rights and the exploitation of the project results through a well-thought preliminary business plan showing the roadmap to market.
11.Ensure a smooth functioning of the Consortium, optimising resources and efforts and guarantee that all EC requirements in terms of communication and reporting were met.

Project Results:

The project started gathering feedback from potential end-users and defining system specifications with the contribution of all partners (WP1). In WP2 (Development of the lab-on-a-chip device -LOC-), work focused first on the fabrication of the fluidic channels of the LOC and testing of the flow. The lab-on-a-chip design rules were established within the first year of the project. In the second half of the project several procedures suitable for mass-production of the LOCs were tested, and 100 chips were produced by UV-embossing with a nickel master. In WP3, two non-commercially available antigenic Borrelia proteins were recombinantely produced and purified. Furthermore, several Borrelia strains were grown to use as whole lysate antigens for testing. A set of antigens aimed to detect Borrelia acute, chronic and autoimmune-related infections were tested by ELISA in human sera samples. Based on the results, 16 antigens were selected for the LOC. In WP4, functionalisation of Quantum Dots with anti-IgG and anti-IgM antibodies was carried out and the process for the immobilization of the antigens on the chip surface was developed. Finally, a fluorescence reader was designed in WP5, able to acquire the red and green-signal from the LOC with a good sensitivity and to allow signal quantification. Regarding dissemination actions and materials, the project web-site was constructed and is online and updated since January 2011 and diverse promotional material such as poster, leaflets, etc. were designed and produced to be used in dissemination actions. The Consortium was highly active attending over 20 events where the project results were disseminated mainly to the scientific community but also to the industry and the general public.

Main results achieved:
-A lab-on-a-chip with capillary force driven operations was designed by ion-beam lithography. Since this technique was eventually not reliable enough for full prototype mass production, the LOCs were fabricated using UV-embossing with a nickel master.
-Antigens for detection of Borrelia infections were produced and tested in retrospective human samples from infected patients. 16 different antigens were selected for the LOC.
-A fluorescence reader was built to record and automatically extract and process the fluorescence information from the LOC. The reader presents the results on a user friendly web-based interface without the need of a skilled user.
-Project website and dissemination material (poster, leaflets, slides and public abstract) have been generated and used in a great number of dissemination actions.
-An Exploitation Agreement has been achieved by the participant SMEs.

Small and medium-sized entreprises (SME) were properly trained in order to ensure an affective knowledge transfer.

-Project coordinator has ensured the optimal functioning of the Consortium, the optimisation of resources and efforts and timely reporting and delivery of results. Communication among Consortium members has been very fluent and all partners have actively participated.

Potential Impact:

The expected final project results are as follows:
1.The HILYSENS lab-on-a-chip containing all reagents
2.The fluorescence reader
3.The process to produce parent and prodegeny stamps for Nanoimprint lithography (NIL) with high aspect ratio.

Socio-economic impact:
To ensure that the expected impact on the patients is achieved, any new-developed diagnostic technology must not only solve the technical problems of current methodology but also ensure that such technology will be successfully taken up by "lead-users" (technology manufacturers) and "end-users" (clinical laboratories in this case). Regarding the latter, a recent study on product usage of new molecular diagnostic tests for infectious diseases conducted in 11 European countries identified the following factors restraining a more widespread use of new molecular techniques in infectious pathogen testing:
-the current prices charged per test (mentioned in the top three of answers by 9/11 surveyed countries)
-the lack of standardisation
-the fact that the applied techniques are demanding in both the skills required of the user and the laboratory space in which the tests are carried out.

HILYSENS fully addresses the last two concerns by providing a compact and robust lab-on-chip system, designed to work with small volumes of complex fluids and without the need of expert operators. This tool together with a portable reader and user-friendly software will enable more precise, accurate and reproducible testing. Making it possible to become the standard tool for the disease diagnosis.

The use of microfluidics represents a dramatic reduction in reagents costs compared to standard laboratory techniques. Moreover HILYSENS goes further beyond to ensure that the expected impact on patient's health will be achieved by solving, though technological advance, a capital drawback of current lab-on-a-chip devices: their high production costs. HILYSENS will be the first lab-on-a-chip device that utilises the capabilities of MeV ion beam lithography to the full, for making an entire lab-on-a-chip device and lay the ground for subsequent flexible low-cost mass production.The project results will also contribute to the market growth in medical biosensor devices. The global market for biosensors and other bioelectronics is projected to grow from 4,035 million EUROS in 2004 to 5,424 million EUROSin 2009, at an AAGR (average annual growth rate) of about 6.3% . United States and Europe dominate the global market for medical biosensors, collectively capturing 69.73% share estimated in 2008 . HILYSENS new technology will contribute to the rapid increase in the lab-on-a-chip European market growth and will increase the competitiveness among the key worldwide players engaged in developing biosensor technology.

Wider economical impact on patients and Health Systems
As stated in the Communication from the Commission to the Council on "Putting knowledge into practice: A broad-based innovation strategy for the EU" there is a clear public interest in helping the emergence of market solutions that would provide answers to citizens concerns, as it is the case of HILYSENS. If not treated in the early stages Lyme Disease can progress to chronic stages of more serious symptoms even interfering with patients daily activities and causing inability to attend school or work . All of these make the long-term cost of Lyme Disease to families, school systems and health care systems astounding. According to a study published in 1993 in Contingencies, an actuarial trade publication, the cost to society for Lyme Disease was about one billion dollars per year. The average treatment and diagnosis and lost wages related to Lyme Disease was 40,750 EUROS per year per patient. Cases have more than doubled since then, so today's costs are probably 660 million EUROS or more annually. Another European study reported the total annual cost for Lyme disease is estimated to be significant at 364,000 EUROS (range 52,000-675,000 EUROS) when projected to the whole of Scotland. The cost analysis takes account of the direct costs of consultation, laboratory tests, antibiotic treatment and management of any sequelae, as well as indirect costs of the loss of healthy time through illness.

Sensitive and reliable patient diagnosis provided by HILYSENS device will optimise resources available to medical practitioners, heavily reducing the current costs of the disease, increasing profitability and most importantly, quality of care. HILYSENS will help to medical practitioners to plan and guide therapeutic interventions. Targeted and personalised treatment will effectively tackle childhood and adult LD, minimising the debilitating impact of the current mistreatments, reducing the length of time that patients are sick and in general improving the quality of life of patients.

List of Websites:

http://www.hilysens.eu