centrifuging for 10 minutes at 13 000rpm, taking the supernatant and centrifuging that for 45 minutes at 45 000 rpm. The supernatant was disposed of and the sediment re-dispersed in fresh 10mM phosphate buffer 0.1 M NaCl pH 7.0. This was repeated 3 times and after the final wash the solution was adjusted to hybridisation buffer conditions, 0.3 M NaCl 10 mM phosphate buffer pH 7.0. Gold nanoparticles were then ready for hybridisation to complementary DNA sequences immobilised on silicon substrates (as shown in Figure 3.16).
3.8
Fabrication Development
Fabrication of DNA samples on silicon produces very good patterns for successful samples, but is often a low yield process in comparison to fabricating planar metal structures due to the large number of processing steps involved and the fragility of biological samples. There are several stages where slight variations of the conditions can prevent patterning from being successful.
To reduce the rate of re-oxidisation of the silicon surface and to avoid exposure of the alkene to light samples were kept in a dark nitrogen environment prior to the UV illumination patterning. Samples were also patterned in a nitrogen environment because of the long exposure time. This was found to successfully increase the yield of fabrication.
Water or moisture absorption by the alkene prior to patterning greatly reduced its function- ality. A number of methods were used to minimise exposure to moisture whilst defrosting the alkene by drying it out with nitrogen, and absorbing condensation that formed around the glass vial. It was also flushed through with nitrogen and sealed prior to re-freezing. The alkene powder was weighed and added to the DCM solvent and sealed in a glass vial as quickly as possible, whilst the silicon substrates were being cleaned in fuming nitric acid. The UANHS alkene solution was then spun on to the samples immediately after removal of the oxide layer with HF acid. The alkene solution worked best when used within 2 hours. If another batch of samples was attempted using an alkene solution older than this, the patterning tended to fail or have a lower yield.
Some problems were identified with the UV illumination of the alkene and this was resolved using the following method. Initial patterning for the first diffraction gratings fabricated had used an Excimer laser (248nmwavelength). Whilst illumination times were very short
(due to the high intensity of laser light), the beam quality was poor and diffraction grating lines appeared quite “grainy”. The early gratings had a 10 micron line width, and 20 micron period. This was resolved by placing the mask directly on top of the sample, with gravity providing adequate force to ensure close, parallel contact between the mask face and the alkene coated silicon surface. Initial masks had been fabricated from chrome on fused silica, but in order to ensure an inert surface gold was used for direct contact masks. Masks were cleaned with isopropanol between batches to keep them clean. These steps removed the diffraction fringes from the DNA grating patterns, but the laser-speckle effect remained due to the coherent nature of the laser beam with poor spatial beam quality.
It was desirable to be able to use a UV lamp for patterning for several reasons. Firstly, to improve the line quality of the DNA grating by allowing homogeneous illumination with an incoherent UV source. An Oriel Instruments 500 W mercury xenon Hg(Xe) lamp was used for UV illumination of the UANHS alkene. The exposure time and lamp intensity were varied to find the optimum conditions for patterning.
When illumination conditions had been optimised fabrication was proving to be inconsistent with the UV lamp. If the grating pattern was faint but visible, it indicated alkene attachment (otherwise there would be no pattern), but no DNA conjugation. Since patterning was more consistent with the monochromatic laser emission at 248nm, a filter was tried to cut out the high energy, short wavelength emission from the HgXe lamp, which could have been causing damage to the functional group of the alkene and preventing DNA conjugation. The lamp spectrum was very broad from∼180−2500 nm. An interference filter centred on 253.7nm
wavelength with a 10 nm FWHM transmission was used to limit the wavelength range to that required to initiate the photoreaction (around 250nm). The illumination intensity and time were then optimised for the new illumination conditions. Optimum fabrication was achieved when an intensity of 1.0 mW/cm2 was incident on the mask at 254 nmwavelength
(measured with a UV power meter set to 250 nm), for a time of 6 minutes. This improved the fabrication yield and consistency of patterning using the UV lamp and therefore these conditions were adopted for the UV patterning of the alkene as diffraction gratings on silicon.
Early grating patterns had used a 20 µm period design with a 50 : 50 linewidth:separation ratio. It was observed that bubbles often formed under the glass coverslip during DNA conjugation, which created areas of the grating where DNA did not attach. This problem was resolved by altering the linewidth:separation ratio to 1 : 3 and using a larger period, enabling gas to escape and not build up. The design of the new grating structure which was adopted is described in detail in Chapter 4.