Rezultaty
The magnetic sensor platform comprised an electronically addressable microarray of spin valve (magnetic) sensors. Capture DNA (i.e. short oligonucleotides probes with specific sequences) immobilised on top of the sensors hybridise to complementary sequences on target DNA labelled with paramegnetic beads, thereby immobilisiing the beads on top of the sensor. In the presence of an external magnetic field, the magnetic field from the paramagnetic beads induces a change in the magnetoresistance through the sensor, enabling the immobilisation of complementary DNA to a specific sensor to be detected as a change in voltage.
The sensor platform can enable detection of a single 1 micron paramagnetic bead, however, sensing has also been successful with 250nm beads. Application of this technology to detection of PCR amplified DNA has been demonstrated, including differential hybridisation across different sensors with sequences designed to identify a mutaion in the CFTR gene.
IP associated with this technology has been the basis for two patent applications, and some of the researrch results have already been published in peer-reviewed journals.
The key innovative feature of this technology is the combined use of magnetic fields and magnetic sensors, to localise a magnetic bead labelled target analyte adjacent to capture molecules and to detect the presence of a magnetic bead labelled target molecule using the integrated magnetic sensors respectively. Therefore, in addition to accelerating the hybridisation / conjugation reaction, the fact the sensors are integrated in this biochip platform, means that there is no need for additional sensing instrumentaion. It is therefore highly compatible with point of care applications.
Three cell line comparisons were selected for analysis of gene expression changes which could be associated with CF, including a comparison of nasal epithelial cells from CF patients with those from non-CF persons. The analyses were performed using the MWG 40k human microarray.
Genes have already been identified which appear to be consistently up/down regulated in CF tissues, compared to non-CF tissues. Further validation of these results has been implemented based on analysis of the protein products of the genes showing alterred expression, and by realtime-PCR analysis of the genes.
However, as yet these results are based on a limited number of analyses across a limited number of tissues. The possibility to increase the sample size and to validate these results across a much greater sample number would likely lead to a greater understanding of CF, and potentially to the validation of gene expression changes / proteins which can be used for CF early diagnosis or even therapy.
Based on the fact that current commercially available CF screening products typically detect upto a maximum of only 25 CFTR mutations, the CF-CHIP SNP chip sought to take advantage of the highly parallel opportunities of microarray-based SNP analysis.
Therefore, instead of adopting the approach of selecting a panel of mutaions appropriate to a specific region, a panel of 100 mutations was selected which would have global application. The 100 CFTR-SNP microarray platform is based on the primer extension approach, and has been validated where possible using real patient samples, and in cases of extremely rare mutations where no patient samples were accessible, using synthetic oligonucleotides to simulate the mutations.
The technology has performed to 99% accuracy, and provides an alternative to current commercially available platforms for detection of CFTR mutaions as a means of early diagnosis of CF, or for screening for CF carriers.
This labelling technique facilities multiplexed analysis even when the detection platform available is not capable of distinguishing between different label types, such as in the case of a magnetic sensor platform or a monochrome optical detection system. The principle of the approach is that the molecule linking the label to the target analyte can be selectively cleaved, so that when a mixed population of analytes are analysed in a multiplex reaction, the identity of analytes bound to capture molecules can be identified by sequentially cleaving the target analytes, using specific cleavage reactions that release only one analyte at time, thereby enabling the number of molecules of that analyte bound to the capture molecules to be calculated by extrapolation.
This technology can be considered as an enabling technology for a broad range of applications. It is intended to publish the results in peer a high profile peer-reviewed journal to ensure that this solution is available to accelerate the development of new sensing technologies, which are limited, by not being able to distinguish different types of analyte.
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