scholarly journals Copolymer Coatings for DNA Biosensors: Effect of Charges and Immobilization Chemistries on Yield, Strength and Kinetics of Hybridization

Polymers ◽  
2021 ◽  
Vol 13 (22) ◽  
pp. 3897
Author(s):  
Luka Vanjur ◽  
Thomas Carzaniga ◽  
Luca Casiraghi ◽  
Giuliano Zanchetta ◽  
Francesco Damin ◽  
...  

The physical–chemical properties of the surface of DNA microarrays and biosensors play a fundamental role in their performance, affecting the signal’s amplitude and the strength and kinetics of binding. We studied how the interaction parameters vary for hybridization of complementary 23-mer DNA, when the probe strands are immobilized on different copolymers, which coat the surface of an optical, label-free biosensor. Copolymers of N, N-dimethylacrylamide bringing either a different type or density of sites for covalent immobilization of DNA probes, or different backbone charges, were used to functionalize the surface of a Reflective Phantom Interface multispot biosensor made of a glass prism with a silicon dioxide antireflective layer. By analyzing the kinetic hybridization curves at different probe surface densities and target concentrations in solution, we found that all the tested coatings displayed a common association kinetics of about 9 × 104 M−1·s−1 at small probe density, decreasing by one order of magnitude close to the surface saturation of probes. In contrast, both the yield of hybridization and the dissociation kinetics, and hence the equilibrium constant, depend on the type of copolymer coating. Nearly doubled signal amplitudes, although equilibrium dissociation constant was as large as 4 nM, were obtained by immobilizing the probe via click chemistry, whereas amine-based immobilization combined with passivation with diamine carrying positive charges granted much slower dissociation kinetics, yielding an equilibrium dissociation constant as low as 0.5 nM. These results offer quantitative criteria for an optimal selection of surface copolymer coatings, depending on the application.

1984 ◽  
Vol 246 (5) ◽  
pp. G550-G555
Author(s):  
E. R. Seidel ◽  
L. R. Johnson

Development of the muscarinic cholinergic receptor and sensitivity of oxyntic gland mucosa to a muscarinic agonist were studied in rats of various ages. The gastric lumen of the fetal rat at the 20th day of gestation contained a statistically significant amount of basal pepsin, which increased log linearly over the first 30 days of life. Carbachol was effective in stimulating the secretion of pepsin as early as 12 h after birth. Basal acid could be measured in the gastric lumen 12 h after birth. The secretion of basal acid increased log linearly over the first 30 days of life. Carbachol was an effective secretagogue even in the fetal rat. The density of the muscarinic cholinergic receptor of the adult rat oxyntic gland mucosa was 99.3 fmol/mg protein with an apparent equilibrium dissociation constant for quinuclidinyl benzilate of 0.40 nM. The receptor was well developed even in the fetal rat, which bound 79.6 fmol/mg protein with an apparent equilibrium dissociation constant of 0.26 nM. Except for immediately after birth, receptor density was maintained between 70 and 90% of the adult level over the first 30 days of life. These results suggest that cholinergic regulation of gastric acid and pepsin secretion is probably functional by either late gestation or at least immediately after birth.


Biochemistry ◽  
2006 ◽  
Vol 45 (49) ◽  
pp. 14632-14641 ◽  
Author(s):  
Vito Graziano ◽  
William J. McGrath ◽  
Lin Yang ◽  
Walter F. Mangel

1986 ◽  
Vol 250 (6) ◽  
pp. C948-C954 ◽  
Author(s):  
L. Hopp ◽  
F. Khalil ◽  
H. Tamura ◽  
M. Kino ◽  
B. M. Searle ◽  
...  

The binding of ouabain and K+ to the Na+ pump were analyzed in serially passed cultured vascular smooth muscle cells (VSMCs) originating from spontaneously hypertensive (SH), Wistar-Kyoto (WKY), and American Wistar (W) rats. Our techniques have utilized analyses of displacement of [3H]ouabain by both unlabeled ouabain and K+ from specific binding sites on the VSMCs. We have found that each of the VSMC preparations from the three rat strains appeared to demonstrate one population of specific ouabain receptors (Na+ pumps); the number of Na+ pump units (mean +/- SE, expressed as 10(5) units/cell; number of observations indicated in parentheses) of both the SH and WKY rats was significantly lower than the number of Na+ pump units of W rat VSMCs [SH: 3.00 +/- 0.02 (231), WKY: 2.87 +/- 0.05 (245), and W: 3.62 +/- 0.04 (225)]; the equilibrium dissociation constant values (microM) for ouabain in VSMCs of SH and WKY rats were similar but were significantly higher than that of VSMCs derived from W rats [SH: 4.69 +/- 0.09 (231), WKY: 4.57 +/- 0.12 (245), and W: 3.69 +/- 0.17 (225)]; and among the VSMCs originating from the three rat strains, the apparent equilibrium dissociation constant value for K+ (mM) was the lowest in those of the SH rat [1.04 +/- 0.003 (143), compared with VSMCs of the WKY rat [1.54 +/- 0.006 (135)] and W rat [1.19 +/- 0.003 (136)]. Our previous studies have demonstrated increased passive Na+ and K+ transport rate constants of SH rat VSMCs compared with either W or WKY rat cells. These findings suggest the possibility of higher permeabilities of the SH cells.(ABSTRACT TRUNCATED AT 250 WORDS)


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