Non-Faradaic Impedimetric Detection of Heavy Metal Ions via a Hybrid Nanoparticle-DNAzyme Biosensor

Abstract

Due to rapid industrialization, novel water-quality monitoring techniques for the detection of highly toxic and hazardous heavy metal ions are essential. Herein, a hybrid noble nanoparticle/DNAzyme electrochemical biosensor is proposed for the simultaneous and label-free detection of Pb²⁺ and Cr³⁺ in aqueous solutions. The sensor is based on the combination of a two-dimensional naked-platinum nanoparticle film and DNAzymes, whose double-helix configuration disassembles into smaller fragments in the presence of target-specific heavy metal ions. The electrochemical behavior of the fabricated sensor was investigated with non-faradaic electrochemical impedance spectroscopy (EIS), resulting in the successful detection of Pb²⁺ and Cr³⁺ well below their maximum permitted levels in tap water. So far, there has been no report on the successful detection of heavy metal ions utilizing the non-faradaic electrochemical impedance spectroscopy technique based on advanced nanomaterials paired with DNAzymes. This is also one of the few reports on the successful detection of chromium (III) via a sensor incorporating DNAzymes.

Keywords: DNAzymes; EIS; biosensor; chromium; heavy metal; impedance; lab on chip; lead; nanoparticles; non-faradaic.

Figure 1

Schematic representation of the immobilization process for thiol-modified DNAzymes. (i) Si/SiO₂ substrates have been patterned with Au interdigitated electrodes and have been used for the (ii) Pt NPs deposition step via the magnetron sputtering technique. (iii) The ssDNA substrate probes were immobilized on the sensors’ surface via drop-casting. MCH was employed (iv) in order to convey a blocking effect with a dual role; remove any non-specifically bound catalytic strands from the surface and act as an interaction barrier between single DNA strands. The final step of the process (v) involved the hybridization of the DNAzyme sequences with the immobilized substrate strands.

Figure 2

(a) Optical image of the interdigitated electrodes of a single sensor, with an inter-finger spacing of 10 μm. (b) SEM image of a single sensor on the margin of the gold electrode. Platinum nanoparticles having a mean diameter of 5 nm have been deposited via DC magnetron sputtering. (c) SEM overview image of a sensor prior to NP deposition. The inset shows the magnified picture of the IDEs, where the inter-finger spacing is equal to 10 μm.

Figure 3

(a) Cross section (schematic) of the sensing device where the platinum (Pt) nanoparticle layer deposited on the electrodes (IDEs) can be seen. (b) Schematic representation of the thiol-modified DNAzyme functionalization distributed on top of the two-dimensional platinum (Pt) nanoparticle (NP) film. The Pt NP film offers a wide range of inter-nanoparticle gaps (noted as “d”) that can be under 1 nm and well over 2 nm.

Figure 4

X-ray photoelectron spectroscopy (XPS) spectra results of a Si/SiO₂ sample (black line), a Si/SiO₂/Pt NPs sample (red line), and a Si/SiO₂/Pt NPs sample modified with thiol DNAzymes (blue line).

Figure 5

EIS responses of the devices at different concentrations of the two HMIs. The Bode plots of sensors functionalized with Pb-specific DNAzyme upon exposure to increasing concentrations of Pb²⁺ are presented for the entire frequency range in (a) and between 500 and 1800 Hz (b), while those functionalized with Cr-specific DNAzyme upon exposure to increasing concentrations of Cr³⁺ are presented for the entire frequency range in (c) and between 500 and 700 Hz in (d).

Figure 6

Equivalent circuit of the proposed device.

Figure 7

Fitting between experimental and the simulation data, according to the equivalent circuit model of Figure 3. Continuous and dashed lines represent experimental and simulated data, respectively.

Figure 8

Impedimetric biosensor calibration-curves obtained at 500 Hz for the two HMIS: (a) Pb²⁺ and (c) Cr³⁺. Enlarged graphs for these curves are presented in (b,d) for the two metal ions, respectively. Biosensor response and control experiments are represented by black squares and red closed discs for either Pb²⁺ or Cr³⁺ and cyan diamonds for Cd²⁺.

Figure 9

Resistive biosensor calibration curves obtained at 500 Hz for the two HMIS: (a) Pb²⁺ and (c) Cr³⁺. Enlarged graphs for these curves are presented in (b,d) for the two metal ions, respectively. Biosensor response and control experiments are represented by black squares and red closed discs for either Pb²⁺ or Cr³⁺, respectively, and cyan diamonds for Cd²⁺.

Figure 10

Effect of storage conditions on the performance of the (a) impedimetric and (b) resistive biosensors. Two storage conditions were assessed: one at 4 °C and the other at room temperature, over a period of up to 6 weeks post-fabrication. The vertical axis displays the reduced signal from the sensors’ responses, which is normalized to the initial response measured immediately after fabrication in “week 0” for 5 nM of Pb²⁺. The error bars indicate the standard deviation derived from measurements of five distinct sensors at each time interval.

Figure 11

The sensors’ response obtained at 500 Hz for Pb²⁺ and Cr³⁺ impedimetric detection in real samples.

Figure 12

The sensors’ response obtained for Pb²⁺ and Cr³⁺ resistive detection in real samples.

Figure 13

The DNAzymes consist of the enzyme strand (es), the substrate strand (ss), and the cleavage site; upon recognition of a heavy metal ion (HMI) target, the substrate strand is cleaved [32].