Au Nanoparticle-Based Amplified DNA Detection on Poly-l-lysine Monolayer-Functionalized Electrodes

Abstract

Affinity sensing of nucleic acids is among the most investigated areas in biosensing due to the growing importance of DNA diagnostics in healthcare research and clinical applications. Here, we report a simple electrochemical DNA detection layer, based on poly-l-lysine (PLL), in combination with gold nanoparticles (AuNPs) as a signal amplifier. The layer shows excellent reduction of non-specific binding and thereby high contrast between amplified and non-amplified signals with functionalized AuNPs; the relative change in current was 10-fold compared to the non-amplified signal. The present work may provide a general method for the detection of tumor markers based on electrochemical DNA sensing.

Keywords: AuNPs; DNA sensing; electrochemical biosensor; poly-l-lysine; signal amplification.

Scheme 1

Concept of a sandwich-type DNA assay that combines PLL chemistry to provide antifouling properties and DNA probe density control with Au nanoparticle signal amplification: (i) self-assembly of the PLL pre-functionalized with reactive and OEG moieties, (ii) immobilization of capture probes (HS-DNA, red) onto a PLL-OEG-Mal-functionalized surface, (iii) their subsequent interaction with the target DNA (tDNA, blue) having dual recognition sites for both capture and reporter probes, and (iv) AuNPs tagged with reporter probes (rDNA, AuNPs, green) for hybridization to the target.

Figure 1

QCM-D sensograms of: the assembly of PLL-OEG₂₂-Mal_4.6 (0.25 mg/mL in PBS, pH 7.4); thiol-probe DNA (HS-DNA, 1 µM in PBS); target DNA (tDNA, 1 µM in PBS) and reporter probe DNA (rDNA, 1 µM in PBS), reporter DNA with AuNPs (rDNA-AuNPs, 0.66 nM in PBS), or non-reporter DNA (nrDNA-AuNPs, 0.7 nM in PBS) as a control experiment.

Figure 2

Cyclic voltammograms of gold substrates before and after coating with: monolayer of PLL-OEG₂₂-Mal_4.6 and HS-DNA, tDNA, and then followed by rDNA, rDNA-AuNPs or nrDNA-AuNPs. (a) Stages of preparation the sensing layer, adsorption of PLL, immobilization of the HS-DNA probe and the hybridization with the tDNA, and (b) studying the hybridization process between t- DNA and rDNA, rDNA-AuNPs, and nrDNA-AuNPs. All measurements were performed in 100 mM K₂SO₄ with 1 mM [Fe(CN)₆]^3−/4− vs Ag/AgCl as a reference electrode (scan rate 100 mV s⁻¹).

Figure 3

Nyquist plots of the EIS data of: (a) Hybridization steps of the t-DNA, followed by rDNA or the amplification step with the rDNA-AuNPs (0.66 nM), and a control experiment with non-complementary nrDNA-AuNPs (0.7 nM); (b) bare gold substrate before and after the subsequent adsorptions of PLL-OEG₂₂-Mal_4,6, HS-DNA probe, and 1 nM tDNA. All the experiments were performed in 100 mM K₂SO₄ with 1 mM [Fe(CN)₆]^3−/4− at a scan rate of 100 mVs⁻¹.

Figure 4

The charge transfer resistance (R_ct) and double-layer capacitance (C_dl) obtained by fitting the EIS data (Figure 3) to a Randles equivalent circuit (shown in the inset of Figure 3a) for each functionalization step. Standard deviations (see also Table S2) are based on three individual measurements performed on different samples, for each functionalized substrate.

Figure 5

Representative chronocoulometry curves for electrodes unmodified (“Tris buffer”) or modified with HS-DNA (1 µM) in 10 mM Tris buffer (pH = 7.4) with 50 µM RuHex before and after hybridization with tDNA (1 µM), rDNA (1 µM) and rDNA-AuNPs (0.66 nM) as amplification response. Likewise, the control measurement with nrDNA-AuNPs (0.7 nM).