HiPerDART
Development of High Performance
Diagnostic
Array Replication Technology
Grant Agreement n. 223378
18th month Publishable Summary
2010 August 31st
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1.
Publishable Summary
The aim of the HiPerDART project is to develop a higher standard clinical
microarray technology platform, by proposing a highly innovative probe
printing technology, called Supramolecular NanoStamping (SuNS).
Since in HiPerDART both assay workflow will be minimized and signal-to-noise
ratios will be improved, the technology platform promises to dramatically
improve reliability, reproducibility and costs of these medical tests. In an
industry dominated by U.S. players it is imperative to establish a strong
European presence at this early stage in the development of the clinical
market. We’ll use a complex disease as colon cancer to prove the strength of
the HiPerDART microarray technology platform. The HiPerDART consortium is
strongly convinced that including the SuNS technology into microarray
production will overcome current issue such as production speed, flexibility,
reproducibility, cost.
The objectives of the project were stated in a ten times decrease of production
time and costs, increasing the microarray feature uniformity.
To address the requirements the assay sensitivity will be increased ten times,
reducing assay workflow time by half, decreasing five-fold the hybridization
time, improving twice the stability of classifier signature and improving the
biomarker qualification for diagnosis, treatment outcome, prognosis and
personalized medicine.
Rolling Circle Amplification (RCA) is the technique which should address the
high sensitivity requirements. A basic study of the RCA reaction in terms of
characterization of the main blocks of RCA was ran to find out the ideal
conditions to develop the polymerization on a solid surface. The development
of a replica surface started by characterizing polymer brushes 3D architectures
to be functionalized on the second part of the project. The development of an
automated stamping machine for replication of template surfaces was started.
The difficult task of finding the appropriate biomarkers for colon cancer was
initiated. The coming microarray technology will allow early diagnosis required
to decrease the death impact degree of this disease.
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A variety of results has been achieved. Several important progresses have
been made for the development of LHD (Linear High Density) arrays, the most
critical being the characterization of the loop and amplicon species and the
yield of the reactions involved in the overall process. The first prototype of
LHD array allowed to define and characterize the usefulness of this format in
comparison with the traditional platforms commercially available. The most
evident advantage in using LHD array is the high sensitivity toward target
detection, up to ten times more than the traditional 60nt probes. Moreover,
this characteristic allowed to reduce by a factor of ten the amount of material
needed for sample hybridization, overcoming one of the main issues
associated with the microarray technology and allowing disease detection with
very limited sample amount. A space confinement methodology was developed
showing that a photo resists mask works very well for biomaterials
confinement by producing desired morphology of the spots and higher
uniformity of signal intensity of the array spotted inside the microwells. The
bulk replica surface was characterized showing that an hyper-branched
structure (polymer brushes) could dramatically enhance the above mentioned
sensitivity requirements. On the other hand a variety of basic tests have been
run to determine the variables involving the design of an hybridization
cartridge on the final device. On the stamping level, the design and
manufacturing of a fully automated stamping machine was done along with its
installation. A significant number of slides has been run through the machine
to assess its stability and to confirm process repeatability. As consequence of
very positive results for all the characterization tests, the equipment was
released for use. A detailed study on pressure and temperature profiles as well
as parallelism tolerance has been done allowing us to set a preliminary set of
QC procedures. A detailed study on predictors has been done leading to the
individuation of ten predicting best probes. Validation was achieved performing
a leave one out cross validation for a logistic regression model adjusted by
gender, sex, study and stage.
HiPerDART project results represent a potential positive impact in the
economic and social aspects of the microarray diagnostic capability and
performances. We do expect that the final configuration device will allow fast
and reliable prediction for colon cancer dramatically increasing the diagnostic
tools.
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