INTRODUCTION (i) Single molecule biochemical assay : There is an ever-increasing need to study bio-macromolecules in greater individual detail, given

the importance of their complex structures and compositions in the series of physical and electro-chemical reactions that control biological systems operation. Bulk testing may reveal the gross distribution and nature of bio-chemical species (like proteins, enzymes, nucleic acids) involved, but remain vague about identifying isolated functional contributions or metamorphoses. The chances of more subtle mutations and aberrations going unnoticed being on the higher side, critical research on deviant molecules could be seriously hampered; anomalies in unexpected quarters after all are known to clinch solutions to major biomedical challenges. This is where single molecule techniques have stepped in to shed light on cellular processes in synthetic environments closely imitating native ones. Advanced manipulation of photonic, magnetic and hydrodynamic forces has made separation and arresting of molecules a very targeted and reproducible method. For the benefit of penetrative in-vitro as well as lab-on-a-chip kind of assays, single molecules essentially require that they be anchored to the substrate at fixed contact points, without any sacrifice of their inherent properties by liability of their close interaction with that substrate. This also distinguishes them from traditional ensemble tests, wherein homogenous phenomena get recorded at the price of others not so easy to detect. Though statistically rather inconclusive, single molecule procedures can be gradually finetuned to yield several ranks more holistic results. Synchronised parallel imaging analysis of these independent molecules becomes hugely efficient.

(ii)

DNA CURTAIN RODS : Extended DNA strands are difficult to encapsulate in lipid vesicle bi-layers that are usually used to form the protective shield against the substrate the reasons are associated with the lower viscosity between the supported layers, consequent unhindered diffusion of the DNA, and that the strands tend to get folded or balled up in the absence of strong fixation. Addition of binding proteins to the bilayer does serve in creating definite tethering sites, but lacks in perfect relative arrangement of the contact points, with the strands getting aligned haphazardly. The same distortion was what used to persist in much earlier

techniques like combing (using a receding air-water meniscus) or in employing aluminium electrodes. Superceding the above is the method of curtain rods. Curtain rods are micro/nano-scale etched or lithographically structured barriers on the substrate, that ensure lipid-headed DNA strands are non-intrusively pinned, aligned, oriented and elongated. The DNA molecules then resemble freely dangling curtains, hooked by one end of the chain, via the bi-layer, and in opposition to the hydrodynamic force. Lithographically constructed metal rods offer precise surface roughness and optimally patterned topography, signifying the marked advantage of fewer contact points, least collateral disturbance by the substrate in protein adsorption, and quality visual imaging.

Fig.1. DNA CURTAIN RODS IN ACTION IN A HYDRODYNAMIC MEDIUM - Ref. [1]

During aqueous flow, as illustrated in Fig.1., the loosely dispersed DNA molecules are pushed onto the substrate. As they rush through the medium, attached to the lipid-bilayer, one end of each strand gets anchored to the perpendicular rod-like hurdles positioned strategically. When the flow is paused, the accumulation of the strands into curtains is broken, and they would begin diffusing away randomly, until the force is re-applied. The guideways parallel to the flow keep the strands directed toward the rods, and restrict them from sliding off the edges. In this project, we have experimented with a new recipe and design for the curtain rod fabrication, while trying to acquire the best dosage for exposure in Electron Beam lithography of the rod patterns.

THEORY (i) Electron Beam Lithography (EBL) : As a popular automated high-resolution method used to create accurate nanometer sized patterns on surfaces, EBL overrides many other alternatives in that, it is non-contact and needs no mask pattern, leading to intact designs. It differs from optical lithography, in focussing an electron beam on the target resist, instead of a light beam. Particular demands of EBL include special electron sensitive resists ( like the PMMA positive resist that has been used here), charge dissipating surface layers (metal or conductive polymers) and lots of time. Besides avoidable set-backs in resolution, EBL patterns inevitably suffer from the deteriorating effects of secondary electrons in the substrate. Most of the overexposure arises from these low energy secondary electrons that are freed by the incident beam, and flit about for a few microns across the resist. Pattern-generating softwares (we have used Raith 150) enable flexible customisation, following which exposure takes place in 16 bit writing, by blanking the beam (Raster scanning) between the various structures(dots, lines, areas). The integrated SEM is also run by the built-in Raith programme, that can determine the write-field, the step size, area dosage and other parameters based on SEM magnification and beam current.

(ii)

Pfeiffer e-beam Metal Evaporator : The Pfeiffer Classic 500-L has been used to perform electron beam evaporation and deposition of Chromium metal layer on the developed resist pattern. It is basically a Physical Vapour Deposition (PVD) process where material from a solid source is vapourised and allowed to condense on the target substrate. In the Pfeiffer, an electron beam heats the piece of source metal placed in a boat in a vacuum chamber, bringing it to a boil. Upon cooling the system with water circulating in heat-exchangers, the metal vapours condense and settle down on all available surfaces, thus coating the target in a film of desired thickness. The thickness profile correlates with the chamber pressure and rates of evaporation condensation.

(iii)

Scanning Electron Microscopy (SEM) : In this nano-scale imaging technique capable of excellent magnified detail, an electron probe strikes the conducting surface of a specimen, being focussed and sharpened by a system of electromagnetic lenses plus apertures. W-filaments, high-brightness LaB6 cathodes or field-emission cathodes are commonly used as electron sources. Apart from the secondary and backscattered electrons of interest to us in topographical and textural visualisation, the primary beam

produces characteristic X-rays, Auger electrons and transmitted electrons, all of which are detected by a scintillator system. The amplified signal is sent to a screen for display of the imaged features. Ideal focus, signal-to-noise ratio, depth-of-field, beam energy and beam diameter are achieved by means of trade-offs among input parameters such as accelerating voltage, beam current, working distance and aperture width. Lens astigmatism that convolutes the image in the X or Y planes is corrected for by a stigmator coil.

IMAGING AND ANALYSIS The sample was viewed under the Dark-field Optical Microscope and the Scanning Electron Microscope at successive stages of patterning of the curtain rods. (i) Dark-field microscopy : Immediately after development of the resist, and later post-Cr-lift-off, the sample was optically imaged to check for and compare the pattern outcome from the three categories of EBL dosages low, middle and high.

Examples from high dose EBL exposure (after Cr lift-off)

Examples from middle dose EBL exposure (after Cr lift-off)

It looks quite obvious that milder dosages have not worked too well for either design. Scattered Cr residues are visible too, indicating flaws in lift-off and cleaning. Both the guideways and the rods have hardly been created.

Examples from low dose EBL exposure (after Cr lift-off) In the lowest dosage range, under-exposure seems to have played havoc, eventually resulting in poorly developed patterns and leaving gaping spaces from where Cr has been lifted off, when it was not supposed to. (ii) SEM : The preliminary inferences about the dosage have been further verified through these SEM images. Proximity effect has blunted the tips of the zigzag rods.

Under high dose

Under mid-range dose

Under low dose

CONCLUSIONS We have attempted to enhance the role of nanoscale curtain rods as tangible anchors that can help organise elongated single molecules of DNA into swinging curtains on a suitable substrate. Modifications in the fabrication recipe were in terms of a. fused Silica replacement for the Silica substrate b. substituting the Cr metallic charge dissipating layer with the conducting polymer Espacer. While affirming that the polymer E-spacer can be competently utilised as a chargedissipating agent, the experiment has shown EBL to have produced the most promising patterns in the appropriate dosage bracket of 170-200 or more. At lower dosages, underexposure has been dominant, causing subsequent malformations during development and lift-off. Lift-off took longer than usual, with multiple trials of heating and sonification. A possible factor could be the adhesion properties of the glassy substrate. The dark-field microscopy and SEM images testify that unwanted Cr residues have been left over around the pattern; on the other hand, the metal has been lifted off from regions where it should have in fact defined the rods and guideways. Proximity effects can be observed around the zigzagging tips, which seem to have gotten smoothened down. The new structures will hopefully perform as expected or better, can be studied conveniently on the fused glass substrate and the relative efficiency of the 2 designs can be investigated too. REFERENCES [1] DNA Curtains and Nanoscale Curtain Rods: High Throughput Tools for Single Molecule Imaging, Teresa Fazio et.al, Langmuir Vol.24, 2008 [2] Organised arrays of Individual DNA Molecules Tethered to Supported Lipid Bilayers, Annette Graneli et al., Langmuir, Vol.22, 2006. [3] Laboratory instructions EBL, Advanced Processing of Nanostructures [4] Lecture notes, Advanced Processing of Nanostructures [5] E-beam metal evaporation on Google