Application of polymer-embedded proteins to fabrication of DNA array.
A plasma-polymerized film (PPF) of hexamethyldisiloxane [HMDS; (CH(3))(3)SiOSi(CH(3))(3)] was used to immobilize streptavidin on a glass substrate. Another layer of HMDS-PPF was also applied to the protein, which was first adsorbed to an underlayer of the same kind of film. As the result, the streptavidin was "embedded" between the two layers of HMDS, whereby biotinylated molecules could be efficiently captured. The second layer of approximately 30 to 45 A PPF was sufficient to allow the binding of biotinylated molecules, whereas thicknesses of >90 A significantly hindered the streptavidin-biotin interactions. Fluorescence analysis revealed that the absence of an HMDS plasma-polymer (HMDS-PP) layer on either side of the streptavidin film resulted in a decrease in biotin binding. This immobilization technique was used to bind biotinylated oligonucleotides in sequence-specific DNA-DNA interactions. The hydrophobic properties of the plasma-polymerized HMDS thin film acted to minimize nonspecific DNA binding to the glass substrate. A DNA array was fabricated using this procedure and showed greatly decreased nonspecific DNA binding compared with a poly-L-lysine coated substrate.
Miyachi
H
H
Research Center for Advanced Science and Technology, University of Tokyo, Japan.
Hiratsuka
A
A
Ikebukuro
K
K
Yano
K
K
Muguruma
H
H
Karube
I
I
D000327:Adsorption; D001427:Bacterial Toxins / Q000235:genetics; D001710:Biotin / Q000378:metabolism; D019921:Biotinylation; D004247:DNA / Q000235:genetics / Q000378:metabolism*; D005453:Fluorescence; D005898:Glass; D009693:Nucleic Acid Hybridization / Q000379:methods; D020411:Oligonucleotide Array Sequence Analysis / Q000295:instrumentation / Q000379:methods*; D015345:Oligonucleotide Probes / Q000235:genetics / Q000378:metabolism; D011107:Polylysine / Q000378:metabolism; D011108:Polymers / Q000737:chemistry* / Q000378:metabolism*; D011485:Protein Binding; D012680:Sensitivity and Specificity; D022641:Shiga Toxin 2; D012833:Siloxanes / Q000737:chemistry / Q000378:metabolism; D019809:Streptavidin / Q000378:metabolism; D013499:Surface Properties
D001427:Bacterial Toxins; D015345:Oligonucleotide Probes; D011108:Polymers; D022641:Shiga Toxin 2; D012833:Siloxanes; D011107:Polylysine; D001710:Biotin; D004247:DNA; D019809:Streptavidin; C015917:hexamethyldisiloxane