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

Abstract

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.

Keywords: DNA hybridization; DNA hybridization kinetics; copolymers; label-free detection.

Figure 1

RPI sensor surface coated by DMA-based copolymers and spotted with DNA probes. (a) Process scheme for RPI biosensor fabrication. (b) RPI image of a glass prism spotted with single-strand DNA with surface density 0.4 ng/mm². Spot diameter is about 150 μm and spot-to-spot distance is 400 μm. (c) Cartoon of the copolymer hydrogel on the sensor surface. Single strands of 23-mer DNA (red) are immobilized at the 5′ terminal on the surface (shaded silver grey) of a 3D copolymer layer (gray with lines) coating the SiO₂ layer (blue). (d) Functional groups for covalent immobilization of DNA probe strands on copolymers. (e) Schematic of the four variants of DMA-based copolymers. Colored symbols represent the functional groups of panel (d).

Figure 2

Analysis of RPI kinetic curves for DNA hybridization on MCP4 copolymer. (a) Binding curves for spots with three different DNA spotting concentrations: 1, 3 and 10 μM shown in grey, red and blue colour, respectively. Vertical dashed lines represent the additions of target DNA strand to concentrations of 0.5, 2.5, 12.5 and 62 nM. (b) Equilibrium asymptotic amplitudes obtained from exponential fits to binding curves in panel a. Lines represent fits with Equation (3). (c) Saturation values of target surface density as a function of probe surface density. The dashed line represents the hybridization of all probe strands, i.e., yield of 100%. The continuous black line is a linear fit, from which the yield was calculated to be ~60%. (d) Observed kinetic rates obtained from the exponential fits of the hybridization curves are reported in panel A. Lines represent linear fits with Equation (4). The data points at the largest concentration are excluded from the fit applying a procedure described in [13]. In panel b, c and d, the color indicates the spotting concentration as in panel a.

Figure 3

Hybridization yield for different copolymer coatings. The saturation amount of captured target strands per area is plotted as a function of the surface number density of the probe strands. The black continuous line is of a linear fit to the black, red and blue points. The green line displays a linear fit to the green, open points. The dashed line represents S_T = S_P, i.e., 100% yield.

Figure 4

Association rate constant k_on as a function of DNA probe density S_P. The black curve represents an exponential fit to all data k_on(S_P) with Equation (5).

Figure 5

Equilibrium constant for dissociation $$K_d$$ The data points represent $$K_d=k_{off}/k_{on}$$ for DNA hybridization on different copolymers. The black continuous and dashed curves are fit with Equation (6) to red–green and black–blue points, respectively. Inset: average value and standard deviation for the dissociation rate constant $$k_{off}$$. The colors of points and curves of the main panel correspond to the copolymers as indicated in the inset.