Figure 5.17: Diffraction pattern (stitched images) of 20µm DNA grating with hybridised 50 nm
gold particles.
and a sample with hybridised 50nm gold nanoparticles, can be seen most clearly on a log- arithmic scale of the normalised diffraction order intensities, as a function of angle for the DNA grating and the DNA grating with gold (see Figure 5.16). The period was calculated to be 20.0 µm from the angular position of the diffraction orders, as can be seen in Figure 5.17.
5.3
Diffraction Intensity Data Collection
Although 5 DNA samples had been tested and all produced only a weak diffraction grating (diffraction efficiency Im=1/Im=0 ≈ 0.5 %, see Figure 5.15) and then another 3 samples this
time hybridised with gold nanoparticles for measurement all showed much higher diffraction efficiency ratios; similar results were not obtained for subsequent fabrication batches. After image-based diffraction measurement was used in initial proof-of-concept experiments, a more complex diffraction experiment was developed to more accurately measure the intensity of diffraction orders.
The initial experiments had measured the diffraction order intensities at a fixed point within a grating using a power meter. The diffraction pattern was measured in a region of the grating where the diffraction pattern was most intense prior to the hybridisation of gold nanopar- ticles. The sample was then aligned to approximately the same region and the position was adjusted to maximise the diffraction pattern intensity, obtained with gold nanoparticles attached to the grating.
As more diffraction experiments were carried out it became evident that there was a large change in diffraction order intensities as a function of position within the grating; something that had not been characteristic of the solid chrome on silica test patterns. This variation originated from the variation in surface profile, linewidth, step height and density of DNA
coverage (modifying the reflectivity of the surface). Another source of variation was that the DNA samples had to be measured in a liquid buffer environment to preserve their functionality and ensure that gold nanoparticles remained hybridised to the grating. The next development steps implemented were to improve the DNA grating consistency, optimise the grating design and improve the data capture methodology.
Sample fabrication was improved to give a more consistent surface coverage, linewidth and step height, as detailed in Chapter 3. Bubble formation had been a problem during DNA conjugation, leaving regions of the grating with limited or no DNA coverage. To improve the surface coverage it was desirable to alter the design of the grating to reduce gas build up and allow the gas to escape by increasing the separation of the DNA lines compared to their width. At the same time it was necessary to develop a diffraction grating structure that would produce a diffraction pattern with order intensities less sensitive to variations in linewidth.
It was possible to calculate theoretical intensities for diffraction orders produced by a grating with a linewidth ofaand separationb. However, the equations that govern these calculations were derived for normal incidence. The derivation needed to be extended to modify the equation to produce correct values for the angular positions of the diffraction orders and their intensities. This was done as described in Chapter 4, additionally taking in to account the fact that both the grating lines and the intermediate regions contribute to the diffraction pattern. A new diffraction grating structure was chosen with a 40µm period and a 10 µm
linewidth, giving a 1 : 3 ratio of a : b. This grating structure (with overall dimensions of 3mm×6mm) is referred to from here onwards.
A new approach to characterising the sample and measuring diffraction patterns was devel- oped to yield more consistent results. Scanning of the sample and averaging of the diffraction order intensities over the whole grating was achieved by translating the sample in the path of the laser beam and recording the power measurement at each point within a scan matrix (see Figure 5.18). Motorised translation stages were computer controlled using a program written using the National InstrumentsTM software LabVIEWTM. The sample was mounted in a trough containing a liquid buffer mounted on a small rotation stage on top of thex−y
translation stages. The sample was rotated to ensure that the translation scan was parallel with the edges of the sample. The sample was translated so that the centre of the scan region and the centre of the grating were co-aligned.
5.3 Diffraction Intensity Data Collection 138 used to record voltages and communicate with the computer. The detector was aligned to the position of the zeroth order (m = 0) and a scan was executed. The program translated the sample in the path of the laser beam, and at each point in the scan, the diffraction intensity was averaged over 500 consecutive readings, recorded to an array and then moved to the next position. At the end of the scan the detector was aligned to the next diffraction order, the sample returned to the origin and a new scan commenced. This was repeated for diffraction orders m= 0 to m= 5.
Figure 5.18: Automated scan and intensity measurement used to translate the grating in the path of the beam and collect data for the whole grating, illustrated schematically over a sample image.
After the grating had been characterised for the inherent diffraction intensities (for orders
m= 0−5) from the DNA lines on the silicon, gold nanoparticles were hybridised featuring complementary oligonucleotide sequences to those immobilised on the grating. The proto- cols for hybridisation are described in Chapter 3. The samples were rinsed to remove any un-hybridised gold and then re-tested to characterise diffraction with the hybridised gold nanoparticles. For many samples tested the average change in diffraction with the addition of gold nanoparticles yielded less change than that caused by positional variations. Even a point-by-point comparison yielded inconsistent changes.
promising and a substantial change in diffraction intensities had been expected. However, in contrast to photolithographically fabricated metal patterns, DNA gratings were inherently more variable. Measuring the diffraction pattern was unexpectedly sensitive to positional changes. The initial assumption, that gratings which had been successful in DNA conjugation would exhibit very similar diffraction patterns, proved false.