Control of Probe Density at DNA Biosensor Surfaces Using Poly(l-lysine) with Appended Reactive Groups

Abstract

Biosensors and materials for biomedical applications generally require chemical functionalization to bestow their surfaces with desired properties, such as specific molecular recognition and antifouling properties. The use of modified poly(l-lysine) (PLL) polymers with appended oligo(ethylene glycol) (OEG) and thiol-reactive maleimide (Mal) moieties (PLL-OEG-Mal) offers control over the presentation of functional groups. These reactive groups can readily be conjugated to, for example, probes for DNA detection. Here we demonstrate the reliable conjugation of thiol-functionalized peptide nucleic acid (PNA) probes onto predeposited layers of PLL-OEG-Mal and the control over their surface density in the preceding synthetic step of the PLL modification with Mal groups. By monitoring the quartz crystal microbalance (QCM) frequency shifts of the binding of complementary DNA versus the density of Mal moieties grafted to the PLL, a linear relationship between probe density and PLL grafting density was found. Cyclic voltammetry experiments using Methylene Blue-functionalized DNA were performed to establish the absolute probe density values at the biosensor surfaces. These data provided a density of 1.2 × 10¹² probes per cm² per % of grafted Mal, thus confirming the validity of the density control in the synthetic PLL modification step without the need of further surface characterization.

Figure 1

(a) Scheme showing the control of the probe density, during the synthesis of PLL-OEG-Mal polymers, which provides the targeted probe density and cDNA binding at the substrate upon immobilization of the modified PLL. x and y indicate the percentages of oligo(ethylene glycol) and maleimide grafted to the PLL side chains, respectively. (b) Synthesis of PLL-OEG-Mal. PLL is reacted with desired relative ratios of Mal-OEG₄-NHS (y = 0–9.1%) and methyl-OEG₄-NHS ester (x = 15.9–29.1%) to give the final modified PLL with the desired degrees of functionalization.

Figure 2

(a) Typical QCM-D measurement of the full process of PLL-OEG(30.3)-Mal(1.8) adsorption, PNA coupling and cDNA binding on a gold substrate. Both the main frequency (Δf, blue) and the dissipation (ΔD, red) are displayed. Example of five normalized Δf for the (b) PNA-thiol probe and (c) cDNA absorption steps using different PLL polymers with increasing maleimide densities (Mal = 0.0–9.1%). The dashed lines refer to the positions used to calculate the Δf of the corresponding step. In all the multistep adsorption experiments the concentrations were 0.3 mg/mL for the modified PLL solutions, and 1 μM for both the PNA-thiol and cDNA solutions. PBS washing steps at pH 7.2 (gray bars) were placed between adsorption steps. The fifth overtone was used for both the frequency and the dissipation.

Figure 3

(a) QCM-D frequency shift (fifth overtone) of the PLL-OEG-Mal deposition step versus the total degree of functionalization of the modified PLL, quantified by ¹H NMR. (b) QCM-D frequency shift of the cDNA binding step versus the fraction of Mal grafted to the PLL polymer, quantified by ¹H NMR. All experiments were performed using 0.3 mg/mL of modified PLL, 1 μM PNA thiol solution (activated by TCEP), and 1 μM cDNA solution in PBS at pH 7.2. PLL-OEG-Mal polymers with different degrees of functionalization were used (Mal = 0.0–9.1%, OEG 15.9–29.1%). Data points represent individual measurements.

Figure 4

Surface density of cDNA-MB detected from CV experiments using PLL-OEG(24.9) PLL-OEG(28.1)-Mal(3.1), PLL-OEG(29.1)-Mal(5.5), and PLL-OEG(19.4)-Mal(9.1) polymers. Calculations were done by means of eq 2 (Experimental Section). All gold substrates were prepared using 0.3 mg/mL of modified PLL, 1 μM PNA thiol solution (activated by TCEP), and 1 μM MB-modified cDNA solution in PBS at pH 7.2. Fresh solutions of 0.1 M NaClO₄ were used as electrolyte. Data points and given single standard deviations are based on 3–4 measurements each.