Exploring the diffusion of DNA strands into nanoporous structures for establishing a universal electrochemical biosensor

Abstract

The development of universal electrochemical sensing platforms with high sensitivity and specificity is of great significance for advancing practical disease diagnostic methods and devices. Exploring the structural properties of electrode materials and their interaction with biomolecules is essential to developing novel and distinctive analytical approaches. Here, we innovatively investigated the effect of DNA length and configuration on DNA molecule transfer into the nanostructure of a nanoporous gold (NPG) electrode. The NPG electrode can not only distinguish and quantify short DNA strands but can also prevent the diffusion of long DNA, thereby minimizing or eliminating background interference. Leveraging these findings, we developed a universal DNA-based NPG electrochemical biosensing platform for the detection of different types of biomolecules. As a proof-of-concept, this sensing platform was integrated with nuclease-assisted target-recycling recognition and amplification reactions to achieve sensitive and specific detection of single-stranded DNA, microRNA-21, and carcino-embryonic antigen, with detection limits of 4.09, 27.4, and 0.28 fM, respectively. The demonstrated universality, sensitivity, specificity, and capability for analyzing complex samples ensure a comprehensive and robust detection approach for nucleic acid-based molecular diagnosis.

Scheme 1. (A) Illustration of the response current variation with DNA length on the NPG sensing platform. (B) Schematic diagram of the working principle of the DNA-based NPG electrochemical biosensor for the universal detection of DNA, miRNA, and protein, which is composed of three modules: (a) nuclease-assisted target-recycling recognition and amplification module, (b) NPG platform-based signal molecule enrichment module, and (c) signal conversion module.

Fig. 1. (A) The DPV response current of NPG/AuE over time in Tris–HCl buffer solution containing 1 μM ssDNA(5)-MB, ssDNA(6)-MB, ssDNA(8)-MB, ssDNA(10)-MB, and ssDNA(22)-MB, respectively. (B) The DPV response current of NPG/AuE over time in Tris–HCl buffer solution containing 1 μM DNA with different configurations, ssDNA(22)-MB, G4-MB, HP-MB, and dsDNA-MB.

Fig. 2. (A) Schematic diagram of the sensitive detection of Exo III. (B) The DPV response of the NPG/AuE sensing platform towards different concentrations of Exo III (0, 0.0001, 0.001, 0.01, 0.1, and 1 U μL⁻¹, respectively). (C) The corresponding linear relationship between response current and the logarithm of Exo III concentration.

Fig. 3. (A) Schematic diagram of the sensitive detection of csDNA. (B) PAGE verification of the csDNA detection principle: (M) DNA marker (20, 40, 60, 80, 100, 120, 140, 160, 180, 200, 300, 400, 500 bp), (a) ssDNA(22), (b) csDNA, (c) ssDNA(22) + csDNA, (d) ssDNA(22) + Exo III, (e) ssDNA(22) + csDNA + Exo III. The concentration of ssDNA(22), csDNA, and Exo III are 1 μM, 0.5 μM, and 1 U μL⁻¹, respectively. (C) The DPV response of the NPG/AuE sensing platform towards different concentrations of csDNA (0, 10 fM, 100 fM, 1 pM, 10 pM, 100 pM, 1 nM, 10 nM and 100 nM, respectively). (D) The linear relationship between response current and the logarithm of csDNA concentration.

Fig. 4. (A) Schematic diagram of the sensitive detection of miRNA-21. (B) PAGE verification of the miRNA-21 detection principle: (a) ssDNA(22), (b) miRNA-21, (c) ssDNA(22) + miRNA-21, (d) ssDNA(22) + DSN, (e) ssDNA(22) + miRNA-21 + DSN, (M) DNA marker (20, 40, 60, 80, 100, 120, 140, 160, 180, 200, 300, 400, 500 bp). The concentrations of ssDNA(22), miRNA-21, and DSN are 1 μM, 1 μM, and 0.005 U μL⁻¹. (C) The DPV response of the NPG/AuE sensing platform towards different concentrations of miRNA-21 (0, 100 fM, 1 pM, 10 pM, 100 pM, 1 nM, 10 nM, and 100 nM, respectively). (D) The linear relationship between response current and the logarithm of miRNA-21 concentration.

Fig. 5. (A) Schematic diagram of the sensitive detection of CEA. (B) PAGE verification of the CEA detection principle: (M) DNA marker (20, 40, 60, 80, 100, 120, 140, 160, 180, 200, 300, 400, 500 bp), (a) Apt, (b) Apt + CEA, (c) Apt + RecJ_f, (d) Apt + CEA + RecJ_f. The concentrations of Apt, CEA, and RecJ_f. are 200 nM, 100 nM, and 0.005 U μL⁻¹, respectively. (C) The DPV response of the NPG/AuE sensing platform towards different concentrations of CEA (0, 1 fM, 10 fM, 100 fM, 1 pM, and 10 pM, respectively). (D) The linear relationship between response current and the logarithm of CEA concentration.

Fig. 6. (A) The CEA levels in real serum samples determined by the proposed strategy and commercial ECL. ECL results are clinical outcomes provided by the hospital. (B) The relative levels of miRNA-21 in real serum samples determined by the proposed strategy and commercial qRT-PCR assay.