For experimental realization of a test structure for detection of DNA used a surface nanostructured porous silicon, which offers the advantage of integration in a microfluidic system made form Belgium partners. Using a p-type silicon wafer (100) with 5-10Ωcm resistivity a porous structure was obtained by the electrochemical etching of silicon in 13% hydrofluoric acid solution and a current of 20mA/cm2. Etching process was monitored by computer registering the current-voltage parameters providing information about the uniformity porosification (fig1.). Morphological characterization revealed a pore size of 5-7nm (Fig2).
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Fig.1. Current-voltage characteristics during porosification process |
Fig.2 SEM image of nanostructured porous silicon |
In the usual microarray experiments, DNA is deposited on the commercials solid support and the interaction with a real sample is monitored using various detection methods (optical, electrical or fluorescent). This agreement was aimed at achieving an integrated microfluidic structure with a wide microarray for detecting specific types of HPV.
DNA microarray-sized performances are closely dependent on surface properties. DNA immobilization efficiency in terms of sensitivity and specificity is one of the most important steps in obtaining a microarray chip for diagnosis of HPV family of viruses. To achieve a greater efficiency in microarray-type structure was used as substrate porous silicon substrate that was chemically modified by creating a monolayer of APTES.
The DNA samples were printed using the robot and the deposition parameters were optimzed Position held spot checks of the need to follow the molecular processes in the technological flux (Fig. 3)
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Fig.3 Array configuration of specific HPV DNA
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After printing the specific DNA evidence for two types of HPV virus, the structures were incubated overnight at a constant temperature of 40C. Each type of test was conducted in four identical replicas in terms of area, method of chemical modification and the submission of DNA primers. From this stage of the process flow were made two parallel plots in the sense that each contained two identical samples of each type. The first batch was kept at 40C while the second group was exposed to electron bombardment for an hour. For this treatment was used an electron beam microscope type VEGA II LMU.
The second important step in making the remaining structure is the lock-free surface after submitting DNA samples. For those of structural blocking method is a kind of immersion time structures in solution of BSA (protein with high affinity to any surface) 1%. Both before and after this stage cleaning processes are needed to remove excess unbound biological material to the surface.
The structures are now ready to be used by any analysis that makes clinical detection of HPV virus.
To demonstrate the functionality of this structures GeneticLAB used samples from patients identified by classical methods as be infected with HPV. Samples were purified and marked fluorescent. The solution was used for the hybridization process of the microarray test structures. The results indicate that porous silicon structure made which were exposed to an electron fascicules as are having the best data and the reproductive efficiency of immobilization of the primers on the surface. This has allowed a quantitative analysis of the results. (Fig.4-5)
Fig.4. Fluorescence image after hybridization for PS /
exposed to the electron beam in SEM; |
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Fig.5. Hybridization spots fluorescent signal when primers: a) GP5-6 and b) L11-12
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The results demonstrate the functionality of the proposed structure for obtaining an accurate diagnosis related to virus infection. Thus this structure can be integrated into the microfluidic system by the project partners or be used by GeneticLab as an alternative solution for detecting HPV infection. |
