Characterization of an inexpensive, nontoxic, and highly sensitive microarray substrate

Abstract

An agarose film has been proposed as an efficient substrate for producing microarrays. The original film preparation procedure was simplified significantly by grafting the agarose layer directly onto unmodified microscope glass slides instead of aminated glass slides, and the blocking procedure was replaced with a wash in 0.1x standard saline citrate (SSC) and 0.5% sodium dodecyl sulfate (SDS) without decreasing the performance of the produced microarrays. Characterization of the grafted agarose film using atomic force microscopy (AFM) and scanning electron microscopy (SEM) showed that the agarose film had a 10-fold increase in surface roughness compared to glass and that the interior of the agarose film was porous, with pore sizes between 100-500 nm. A comparison of hybridization on aldehyde-activated agarose-coated microarray slides and commercial amino-reactive microarray slides showed that aldehyde-activated agarose-coated slides had the highest signal-to-noise ratio of 850, suggesting that the aldehyde-activated agarose microarray slides are suitable in applications where analytes have a wide concentration range. By immobilizing the DNA probes using ultraviolet (UV) light, the signal-to-noise ratio was further increased to 3000 on the agarose microarray slides. The specificity of the UV cross-linked DNA probes was demonstrated using 21 and 25 bp long capture probes, enabling discrimination of target molecules differing in only one base.

Immobilisation of DNA to polymerised SU-8 photoresist

“…Thus, also unmodified probes that must be attached to SU-8 through at least one of the bases, can discriminate a mismatch hybridisation. These results corroborate previous findings that short unmodified probes, in this case immobilised to an agarose film by UV cross-linking, could discriminate a mismatch hybridisation (Dufva et al, 2004).…”

“…The hybridised density of the 60 bp probes immobilised on SU-8 was between 8 and 13 fmol/mm 2 while microarrays on commercial aldehyde coated glass had a hybridised density of 9 fmol/mm 2 . These hybridisation densities were comparable with those obtained previously on 2D microarray substrates (Dufva et al, 2004;Fixe et al, 2004aFixe et al, , 2004b. The microarray spots on SU-8 were perfectly circular with a diameter of 100 m and appeared more homogeneous than the corresponding spots on the aldehyde coated slides, which had diameters of 150 m (Fig.…”

Fabrication of high quality microarrays

“…Another choice is whether or not to fabricate the slides yourself. Home made slides are much less expensive at least in reagents costs than the commercial slides and still provide equal or better performance characteristics than the commercial slides [60,61]. Fabricating your own slides can be a cumbersome process that may actually not pay off economically despite commercial slides being expensive.…”

Microfluidic DNA microarrays in PMMA chips: streamlined fabrication via simultaneous DNA immobilization and bonding activation by brief UV exposure

“…The probes had the variant base/bases positioned as close to the center of the probe as possible, and they contained a poly(T)10-poly(C)10 tag (TC tag) in the 5' end (Dufva et al 2006) (Petersen et al 2007). The TC tag increases hybridization signal by unknown mechanisms that may include increasing the probe immobilization efficiency during UV cross-linking (Dufva et al 2004(Dufva et al , 2006Gudnason et al 2008). A melting temperature (T m )-matched probe set was designed based on criteria previously described (Petersen, Poulsen et al 2008).…”