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ELISHA Résumé de rapport

Project ID: 505485
Financé au titre de: FP6-NMP

Final Report Summary - ELISHA (Electronic Immuno-Interfaces and Surface Nanobiotechnology: A Heterodoxical Approach)

The project was designed to improve knowledge in molecular interfaces and enable manufacture of new affinity sensors, by understanding the rules for controlled manufacture at the nanoscale. Interrogating antibodies on nanostructured surfaces will help to produce practical immunosensor formats that work as simply the most successful commercial biosensors and which can be manufactured at high quality and low cost. These were the main aims of ELISHA.

To provide significant breakthroughs in science often requires a change of orthodox thinking, hence the addition of heterodoxy in the full title of the project to indicate a departure from existing knowledge and traditional electrochemical thinking. Practically the project focused on three areas.

When an antibody recognises and transiently binds to its antigen there is a small electrode current produced in the supporting nanostructured matrix which can be measured and which is concentration-dependant with respect to the antigen detected. The project investigated the nature and origin of the signal transduction mechanism observed in these nanostructured affinity-reagent based biosensors.

Using the knowledge obtained, immunosensor model prototypes were designed and optimised to give simple, novel, low-cost and reliable affinity sensors for a number of important clinical, environmental and related model analytes. These included cancer markers as a representative protein target (prostate-specific antigen (PSA)), fluoroquinoline antibiotics as a representative hapten target (small molecule types) and other proteins.

The manufacture of electrochemical, label-free immunosensors was also complimented by the development of dedicated electronics to provide specific interrogation protocols to deal with any non-specific binding events and to amplify the affinity event occurring.

The project was structured into 7 main sections, 1) Production of Antibodies; 2) Transducer Manufacture 3) Sensor Fabrication; 4) Immobilisation and Signal Generation; 5) Electronics Development; 6) Non-Specific Binding and Data Processing and 7) Prototype Instrumentation.

All of these were completed by around month 24 with some intermittent work as new batches of transducers were needed and when more supplies of antibodies needed to be purified. The addition of molecular tags to antibodies also progressed throughout the project to allow new immobilisation methods to be explored.

Sensor fabrication, immobilisation methods and signal generation were the main project focus in the first half of the project. The production of immunosensors that exhibited the correct level of sensitivity, low non-specific binding and which are practical to manufacture were the main focal points.

At its conclusion, the project had identified several possible routes for immunosensor fabrication, all of which gave responses to the specific analytes that matched the antibody specificity. A good example is the cancer marker, prostatic specific antigen or PSA. This is used as a screen for prostatic cancer and is analysed within blood samples. The graph overleaf shows that PSA can be easily detected at levels much lower than is present in blood.

In addition to this, two other protein analytes have been demonstrated to work using the ELISHA fabrication protocols. S-100 is a biomarker for trauma, such as stroke or heart attack and the antibody to this was used in an immunosensor format to give detection down to Haemoglobin is the main protein present in red blood cells. It is often used as a measure of micro-haemoglobinuria, or very low levels being passed in the urine.

Again the ELISHA technology enabled immunosensors to be made that detect haemoglobin down to 5 nanomolar. This generic capability to obtain specific responses for many different antibodies has provided the evidence that the ELISHA method is a platform technology that can be developed into many areas where antibodies are used as diagnostic tools.

The speciality electronics designed for the instrumentation to interrogate and report the responses of the immunosensors are unique to the ELISHA project. This enables very low responses to be detected directly when coupled with the nanostructured immunosensor matrices, so the antibody recognising its antigen gives a measurable signal. The portable prototype device was constructed in the final year of the project for use at point-of-care or in field analysis and incorporates algorithms to enable rapid data processing and techniques to remove any signal due to non-specific binding to give specific antibody responses for the target analytes.

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Paul Anthony MILLNER
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