Time, min

FIGURE 5.19 Binding of 16*CF1 (complementary oligonucleotide) in a 10-nM solution to 16*B (immobilized oligonucleotide) immobilized via sulfosuccinimidyl-6-(biotinamido)hexanoate (NHS-LC-biotin) and streptavidin to a biosensor, (a) No regeneration; (b) chemical regeneration; (c) thermal regeneration.

Time, min

FIGURE 5.19 Binding of 16*CF1 (complementary oligonucleotide) in a 10-nM solution to 16*B (immobilized oligonucleotide) immobilized via sulfosuccinimidyl-6-(biotinamido)hexanoate (NHS-LC-biotin) and streptavidin to a biosensor, (a) No regeneration; (b) chemical regeneration; (c) thermal regeneration.

TABLE 5.8 Influence of Chemical and Thermal Regeneration on the Fractal Dimensions and Binding Rate Coefficients for the Binding of 16 CF1 in Solution to 16*B Immobilized on a Biosensor Surface (Abel et ai, 1996)

Analyte in solution/receptor on surface k Dr

10 nM 16*CF1/16*B immobilized 1338±31.3 1.272±0.021

via NHS-LC-biotin and streptavidin 10 nM 16*CF1/16*B immobilized 86.53±3.21 1.211±0.026

via NHS-LC-biotin and streptavidin (chemical regeneration) 10 nM 16*CF1/16*B immobilized 100.0±6.70 1.394±0.047

via NHS-LC-biotin and streptavidin (thermal regeneration)

binding of 16 CFl in solution to 16*B immobilized on the biosensor surface, using chemical and thermal regeneration, respectively. In this case, a single-fractal analysis is sufficient to adequately describe the binding kinetics. Table 5.8 shows the values of the binding rate coefficients and the fractal dimensions obtained in these cases. Note that the temperature of hybridization was 26.7°C, and for thermal regeneration the fiber-optic surface was heated to 68.5°C. Note that as one goes from the chemical to the thermal regeneration cycle, k increases by about 15.5%—from a value of 86.53 to 100—and Df increases by about 13.4%— from a value of 1.2112 to 1.3942.

Abel et al. also analyzed the binding kinetics of 10-nM 16*CFl in solution to 16 B immobilized to a biosensor surface. The only difference between this case and the previous two cases is that for the immobilization step the authors used avidin instead of streptavidin. A single-fractal analysis was employed here also to model the binding kinetics (Fig. 5.19c). The values of k and Df are given in Table 5.8. In this case, the binding rate coefficient is much higher than in the previous two cases. The fractal dimension value of 1.2722 is between the two values obtained in the previous two cases. In this case, the fractal dimension analysis provides a quantitative estimate of the degree of heterogeneity that exists on the biosensor surface for each of the DNA hybridization assays. More data is required to establish trends or predictive equations for the binding rate coefficient in terms of either the analyte concentration in solution or the "estimated" fractal dimension of the biosensor surface under reaction conditions.

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