The final research area focuses on attaching biological probes (e.g. single-stranded DNA/RNA, proteins, antibodies etc) onto the magnetic tags. Two different strategies are used for the two different tag architectures:
The SU-8 polymer used to encapsulate the magnetic elements presents native epoxide groups on the surface. Nucleophiles, such as primary amines, form covalent bonds via a ring-opening reaction, and this has been demonstrated by the addition of the green fluorophore, NH2-CH3-fluorescein (AMF), as seen in figure 1.
Figure 1: a) the epoxide ring-opening mechanism with a primary amine, b) schematic showing 3 stages of a DNA hybridisation experiment, c) 20x fluorescence microscope image of 3-bit tags (on substrate) reacted with AMF.A new ion-milling fabrication procedure severely diminishes the quality of the epoxide surface, so a new chemical etch and oxidation treatment (patent pending) has been developed to counter this and produces surface carboxyl groups. These form amide bonds with primary amines, catalysed by carbodiimide chemistry, and is demonstrated with a red fluorophore, NH2-PEG-tetramethylrhodamine, shown in figure 2.
Figure 2 a) The molecular structure of our chosen fluorophore with its functionalities highlighted, b) 50x fluorescence microscope image of SU-8 backbones (on substrate) following etching, oxidation, and coupling to the fluorophore.
A thin electrodeposited layer of gold is used to cap these tags, which provides the platform for a self-assembled monolayer to form. Sulphur has a strong affinity for gold meaning hydrocarbon chains containing thiols and disulphides will anchor to the surface, exposing their functional head groups (figure 3). In our experiments the reactive N-hydroxysuccinimide group was chosen as the reactive head group, and it is vulnerable to nucleophilic substitution reactions with primary amines.
Figure 3 a) the chosen disulphide molecule (made in house) has NHS end groups, b) how the sulphur atoms of the SAM arrange themselves on a gold (111) surface c) the substitution reaction with a primary amine.
A 5' NH2 modified 26 base-pair oligonucleotide primer was attached to the functionalised array of pillars, before hybridisation with a 5' fluorescently labelled complementary strand - the results for an array of 15μm diameter tags is shown in figure 4. More details of this research can also be found in the following paper: "Hybridisation of Electrodeposited Magnetic Multilayer Micropillars." J.J. Palfreyman et al., IEEE Trans. Mag. 43 (6) 2439-2441.
Figure 4 left) schematic showing an electrodeposited 3-bit pillar tag; the above SAM is grown and reacted with an amine-modified strand of DNA, which hybridises to its fluorescently labelled complement, right) a CCD fluorescence image (taken with a Bioanalyzer) of a 64 x 64 array of 15μm diameter tags as shown in the schematic
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