A dual DNA biosensor based on two redox couples with a hydrogel sensing platform functionalized with carboxyl groups and gold nanoparticles

• Dual-target electrical detection of DNA labeled with redox probes.
• Hydrogel pNIPA is a perfect three-dimensional matrix for DNA immobilization.
• LA ICP MS and EQCM quantitative characteristic of the efficiency of hybridization process.
• The pNIPA platforms can easily be made reusable.

Poly(N-isopropylacrylamide-co-acrylic acid) hydrogels containing COOH groups and gold nanoparticles were used to propose a new dual-target hybridization assay that involves a dual hybridization event. The target DNA strands were labeled with two different redox probes: ferrocene and anthraquinone derivatives. The targets were simultaneously detected by obtaining two well separated voltammetric peaks. Such good stability of the biosensor can be attributed to the strong covalent bond between Au nanoparticles and the thiolated probe DNA. Also the bond between carboxyl group of the gel and amine-terminated capture-probe DNA is strong in pH ca. 7. The sensor response increased linearly with the logarithm of the target DNA concentration in the range from: 2 × 10−12 to 1 × 10−6 M and 2 × 10−13 to 2 × 10−6 M for target DNA labeled by antraquinone and ferrocene derivative respectively. The determined detection limit was approx. 8 × 10−13 M and 7 × 10−14 M for target DNA labeled with antraquinone and with ferrocene, respectively, which corresponded to the presence of ∼2 × 108 copies of DNA in the solution (0.5 mL). The proposed biosensor can be used for the detection of DNA at the fM level. This achievement is very important for biosensors that use DNA labeled with redox probes, as here we have shown that the sensitivity improved by at least two orders of magnitude compared to the traditional biosensors.

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