PDPH-PAA

The maximum response of the sensor to DNA-conjugated PDPH-PAA (2800 RU Fig. 9A(I)) was equivalent to 2.8 x 105pgcm 2. The amount of ssDNA covalently bound to the polymer was 10 nmol mg x, as mentioned above. Thus, the amount of ssDNA on the gold substrate was equivalent to 2.8 pmol cm 2. 

SPR testing demonstrated the affinity of DNA-conjugated PDPH-PAA for fully matched DNA. Immobilization of DNA-conjugated PDPH-PAA was carried out at pH 5.5 for 1 h by SPR machine. The hybridization was carried... 

Fig. 9 SPR response to DNA-conjugated PAA-based polymers on a sensor chip. Samples were DNA-conjugated PDPH-PAA (A) lacking EA and (B) containing ethanolamine sidechains at (curves I) pH 5.5 or (curves II) pH 8.0...

The hybridization selectivity and efficiency of DNA-conjugated PDPH-PAA were investigated with 1 pg mL 1 of injected sample DNA. The response to fully matched DNA (89 RU) was equivalent to 0.92 pmol cm 2, which indicates a hybridization efficiency of 33%, based on the amount of probe DNA on the surface (2.8 pmol cm 2). This hybridization efficiency was lower than that of DNA-conjugated TA-polyallylamine however, for DNA-conjugated TA- polyallylamine and PDPH-PAA, the maximum response when 1 pgmL-1 of unmatched DNA sample was injected decreased from 94 RU to 21 RU. These results suggest that less nonspecific DNA adsorption occurs with DNA-conjugated PDPH-PAA due to the electrostatic repulsion between the car-... 

Fig. 10 SPR responses of DNA-conjugated polymers to fully matched and unmatched DNA. The sensor consisted of (A) DNA-conjugated TA-polyallylamine immobilized on the gold substrate or (B) DNA-conjugated PDPH-PAA immobilized on the substrate. Curves I and III fully matched DNA curves II and IV unmatched DNA. DNA-conjugated polymer was immobilized on substrate for 24h. SPR responses were normalized with respect to maximum signal observed for fully matched DNA...

DNA-conjugated polymers were prepared from polyallylamine or polyacrylic acid (PAA) modified with ssDNA as a probe, and thioctic acid (TA) or 3-(pyridyldithio)propionyl hydrazide (PDPH) for self-assembled immobilization. The oligonucleotides sequences of the probes and of the fully matched and unmatched DNAs were S -TCC-TCT-TCA-TCC-TGC-TGC-TAT-GCC-TCA-TCT-T-R, 5 -AGA-TGA-GGC-ATA-GCA-GCA-GGA-TGA-AGA-GGA-3, ... 

The carboxy group of PAA (MW 1 080 000) is covalently modified with amino-linked ssDNA as probe and PDPH for self-assembly immobilization. The chemical structure of the polymer unit is shown in Fig. 8. The amounts of ssDNA and PDPH in the polymer were determined by absorption measurements to be equivalent to 1/1500 [molecule/monomerunit] and 1/93 [molecule/monomerunit],respectively. 

Using PAA as the starting point, a self-assembly DNA-conjugated polymer was prepared for DNA chip fabrication. The amounts of ssDNA and PDPH in the polymer were determined by absorption measurements to be equivalent to 1/714 [molecule/monomerunit] and 1/46 [molecule/monomerunit], respectively. A 20-mer ssDNA as P-1 DNA and PDPH for self-assembled immobilization were covalently attached to PAA as side chains. After self-assembled immobilization of the DNA-conjugated polymer on the gold surface of a sensor, the P-1 DNA chain was hybridized to a 34-mer ssDNA as P-2 DNA, which had a sequence fully matched to the desired target DNA. Analysis of the first hybridization (between P-1 and P-2 DNA) and of the second hybridization (between P-2 and the target DNA) was done by fluorescence measurements. 

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