Imaging Immobilisation

Turcu, F, Schulte, A., Hartwich, G., Schuhmaim, W. Imaging immobilised ssDNA and detecting DNA hybridisation by means of the repelling mode of scamiing electrochemical microscopy (SECM). Biosens Bioelectron 2004, 20,925-932. 

The immobilisation of proteins into inorganic mesoporous host materials has attracted considerable attention due to the potential applications in biochemical, biomedical, industrial and bio-analytical fields [1] Biocompatible supports endowed with fluorescent tracers and adequately modified for specific interactions with cellular antigens are an amenable tool for image in living cells processes that are relevant to diseases. 

In a second experiment, Cy5-labelled antiBSA antibodies were immobilised on a silanised glass slide precoated with metallic nanoislands using a polydimethylsiloxane (PDMS) flow-cell. The antibody solution was left for 1 hour to attach and then the cell was flushed with deionised water. The slide was then dried with N2. For this experiment, a portion of the slide was not coated with metallic nanoislands, in order to act as a reference. Figure 20 shows the image recorded using the fluorescence laser scanner mentioned previously. The enhancement in fluorescence emission between those areas with and without nanoislands (B and A, respectively) is again evident. For both chips, an enhancement factor of approximately 8 was recorded. There is considerable interest in the elucidation and exploitation of plasmonic effects for fluorescence-based biosensors and other applications. 

Among the many microscopy-based techniques for the study of biomolecules immobilised on surfaces, scanning probe microscopies (SPM) and especially atomic force microscopies (AFM) are arguably the most used techniques because of their molecular and sub-molecular level resolution and in situ imaging capability. Moreover, the invasiveness of AFM, which is less of a problem for the DNA molecules, is essential for another two functions, apart from the mapping of surface nanotopographies, namely the quantification and visualisation of the distribution of chemistry, hydrophobicity and local mechanical properties on surfaces and the fabrication of nanostructures. 

Fig. 25 AFM images of silicon chip siufaces a cleaned, rehydroxylated silicon surface, b self-assembled MPTS layer on silicon, c immobilised oligonucleotide probe, d surface after hybridisation to a complementary target [119]. Reprinted with permission...

Fig. 26 AFM topography (top) and LF (bottom) images of a layer of 26 bp oligonucleotide immobilised on poly-L-lysine coated surface. The nanodefects on poly-L-lysine film are indicated with a solid line and the immobilised ssDNAs are indicated in dotted circles [163]. Reprinted with permission... 

Fig. 19 AFM images of PE surface a virgin PE, b AC directly attached to PE, c AC-immobilised involving one-step spacer, d DS functionalised PE, e DS-PiFGA functionalised PE...

A critical issue in the development of an electrochemical DNA-biosensor is the sensor material and the degree of surface coverage. MAC Mode AFM images were used to characterize different procedures for immobilising nanoscale double-stranded DNA (ds-DNA) surface nanofilms on carbon electrodes, Rg. 6.1. 

The explanation of the examination to the child must be given at the appropriate level of understanding. The carers also should consent to and receive a careful explanation of any form of immobilisation that may be required to allow the examination to be completed successfully. Lead protection is always worn by the carer when supporting a child for imaging. 

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