3.2. Electrochemical Methods

The electrochemical methods belong to the most sensitive analytical methods, and therefore their privileged role in the detection of mutagenic or epigenetic OG formation is well motivated owing to its low yet significant occurrence in vivo. Over the years, several sophisticated procedures have been developed to monitor and quantify samples containing the nucleobase OG, its precursor nucleoside 8-oxo-7,8-dihydro-2′-deoxyguanosine (o⁸dGuo, in the literature frequently abbreviated as 8-OHdG as well) or even its presence in ONs or GQs.

Hao et al. [26] have prepared a nanocomposite using reduced graphene oxide (rGO) and ZnO nanoparticles (ZnO@rGO) via an in situ reduction of graphene oxide (GO) with Zn powder and coated glassy carbon electrode (GCE) (Figure 1). The ensuing system (ZnO@rGO/GCE), characterized by scanning electron microscopy (SEM) and transmission electron microscopy (TEM), gave significantly enhanced oxidation signals of o⁸dGuo, compared with unmodified GCE and GO-modified GCE (GO/GCE), as verified by cyclic voltammetry (CV). With the enzyme uricase, the interference of uric acid (UA) was effectively eliminated and accurate sensing of o⁸dGuo was attained. The linear range of 5 to 5000 nM for the detection of o⁸dGuo using ZnO@rGO/GCE has been achieved using differential pulse voltammetry (DPV). The limit of detection (LOD) was 1.25 nM.

ZnO nanoparticles (ZnO@rGO) for the detection of o⁸dGuo. Reprinted with permission from Reference [26], © 2018, Elsevier.

rGO has also been combined with another metal oxide, dysprosium oxide (Dy₂O₃) nanoparticles, for the determination of o⁸dGuo in human blood and urine samples by Manavalan et al. [27]. Dy₂O₃@rGO was synthesized by a microwave-assisted synthetic route to improve the moderate electrical conductivity; furthermore, the electrocatalytic ability was synergically improved. The electrochemical and interfacial properties were examined by electrochemical impedance spectroscopy (EIS). Under optimum conditions, the electrocatalytic performances of Dy₂O₃@rGO-modified electrode and control electrodes were analyzed by CV. The Dy₂O₃@rGO-affixed conventional screen-printed carbon electrode (SPCE) was found to exhibit tremendous electrocatalytic capability toward o⁸dGuo oxidation. The amperometric detection of o⁸dGuo worked in the linear range of 50 nM to 315.3 μM, with a LOD of 1.02 nM. The initial sensor response current was still retained after 3000 s. The storage stability was monitored every day for 10 successive days, and no significant deterioration was observed. The method worked well in real human urine and blood serum samples and the results were validated by the HPLC method as well.

Guo et al. [28] have developed an OG sensor based on the multi-walled carbon nanotubes (MWCNTs)-modified GCE characterized by SEM and EIS methods (Figure 2). The linear ranges were 56.3 nM to 6.08 µM and 6.08 µM to 16.4 µM respectively, with the LOD of 18.8 nM (S/N = 3) in CV measurements. UA and dopamine (DA) commonly coexist with o⁸dGuo in human metabolism and their electrochemical oxidation potentials are close, but with this method, their mixture gave three clear and well-separated oxidation peaks at 0.43 V (o⁸dGuo), 0.36 V (UA) and 0.22 V (DA), respectively.

Glassy carbon electrode (GCE)/multi-walled carbon nanotubes (MWCNT) sensor for the determination of o⁸dGuo. Reprinted with permission from Reference [28]. © 2016, Elsevier.

Quantitative measurement of o⁸dGuo concentration was analyzed by linear sweep voltammetry (LSV) as well, by Shang et al. [29]. First, a porous carbon nanotube was obtained from SWCNT using KMnO₄ as the etching agent, dropping the aqueous PSWCNT onto a polished GCE surface. The obtained PSWCNT/GCE-based sensor, characterized by TEM, X-ray diffraction and nitrogen adsorption/desorption isotherm, showed outstanding electrochemical performance for o⁸dGuo in the measurement ranges 2.99 nM to 3.061 μM and 3.061 μM to 87.25 μM respectively, with an LOD at 1.0 nM. The effects of temperature and time on DNA damage have also been investigated and important dynamical parameters and a kinetic equation with reaction rate constant (k = 2.090 min⁻¹) and the apparent activation energy (E_a = 30.64 kJ·mol⁻¹) for the OG oxidation have been obtained (Figure 3).

The application of porous single-walled carbon nanotube (PSWCNT) for the determination of o⁸dGuo. Reprinted with permission from Reference [29]. © 2018, Elsevier.

In an earlier study, Mugweru et al. [30] assembled films containing the metallopolymers (Os(bpy)₂(PVP)₁₀Cl)⁺ and (Ru(bpy)₂(PVP)₁₀Cl)⁺ (bpy = 2,2’-bipyridine; PVP = poly(vinylpyridines)), layer by layer, on pyrolytic graphite electrodes to obtain sensors that selectively detect oxidized DNA. Assembly of films was monitored with a quartz crystal microbalance (QCM). The two metallopolymers behaved electrochemically independent of each other in the films. This combination provided a catalytic Os square wave voltammetry (SWV) peak that is mainly selective for OG and the detection of other oxidized nucleobases from the Ru peak. The method is applicable to measurements on DNA in solution or DNA incorporated into films. Using the Os SWV peak, a single oxidized nucleobase out of 6000 nucleotides was detected. A related Os-PVP polymer with higher oxidation potential can generate electrochemiluminescence (ECL) with oligonucleotides containing OG in thin films, providing an alternative method to detect oxidative stress [31]. This purely voltammetric approach is complementary to the ECL method.

Cao et al. [32] prepared graphite nanosheets (GN) in a very simple liquid-phase exfoliation of graphite in N,N-dimethylacetamide (DMAc). Ultra-small (less than 5 nm) Cu-based metal organic framework (HKUST-1) nanoparticles were in situ anchored on the surface of GNs with a high degree of dispersion (Figure 4). The synthesized hybrids of graphite nanosheets (HKUST-1/GN) decorated with HKUST-1 nanoparticles, characterized by powder X-ray diffractometry (XRD), thermogravimetric analysis (TGA), SEM and TEM, showed excellent electrochemical sensing performance towards the DNA damage biomarker o⁸dGuo. The DPV concentration measurement was characterized with a fast detection speed (~240 s), wide linear window (10 nM–1 µM), high sensitivity (346,857 µA mM⁻¹ cm⁻²), low LOD (~2.5 nM) and good reproducibility.

The schematic representation of metal organic framework nanoparticles for the measurement of o⁸dGuo. Reprinted with permission from Reference [32]. © 2019, Elsevier.

An electrochemical method, developed by Zheng et al. [33], combines o⁸dGuo-specific aptamer (apt) with metal ion-dependent DNAzymes and exonuclease to achieve high sensitivity with a LOD of 6.82 pM and linearity from 0.01 nM to 7.0 μM applying SWV. The electrochemical sensing platform used a gold electrode modified with substrate DNA of DNAzyme, labelled with methylene blue (MB) redox probe as a working electrode. The state of the surface of the gold electrode was checked employing EIS. During the SWV analysis, reduction of the electrochemical signal is measured thanks to the release of the MB during the cleavage of the substrate DNA by the DNAzyme part of the o⁸dGuo–apt–DNAzyme complex. In addition, CV was used to verify the experimental principle. The modified gold electrode is stable for a week at 4 °C (Figure 5).

A o⁸dGuo-specific aptamer construct. Reprinted with permission from Reference [33]. © 20019, Elsevier.

Jia et al. [34] have developed a highly sensitive and selective electrochemical aptasensor by using o⁸dGuo-specific aptamer hybridized with the capture of DNA immobilized on a gold electrode with a sticky tail left, which initiated the hybridization chain reaction (HCR) through an auxiliary DNA (Aux). The formation of extended double-stranded DNA (dsDNA) structure intercalated a more electroactive species ((Ru(NH₃)₆)³⁺), therefore the high sensitivity of the electrochemical method was further increased thanks to the HCR. In the presence of o⁸dGuo, the aptamer will form a GQ structure that stops the HCR, leading to detection signal decrease. By monitoring the change in the current of the (Ru(NH₃)₆)³⁺ ion, the concentration of o⁸dGuo can be indirectly determined in the sample. The strategy was characterized by EIS measurements. Thus, the resistance continuously increased when the electrode was incubated with capture DNA, 6-mercapto-1-hexanol (MCH), aptamer and Aux. The resistance of aptamer/MCH/capture/electrode heavily decreased in the presence of o⁸dGuo. In DPV, the electrochemical signal was mainly implemented by (Ru(NH₃)₆)³⁺ immobilized on the long-nicked DNA polymers. An impressively wide linear response ranging from 10 pM to 100 μM and LOD of 2.5 pM has been attained (S/N = 3). The method was successfully applied in human urine samples (0.56 nM concentration of o⁸dGuo was determined) and interference with UA has been eliminated. The aptasensor was stable for 10 days at 4 °C. Despite the vulnerability of these electrodes, they constitute one of the most specific methods for the quantitation of o⁸dGuo lesions (Figure 6).

An aptamer-based electrochemical biosensor with hybridization chain reaction (HCR) signal amplification for the detection of o⁸dGuo. Reprinted with permission from Reference [34]. © 2018, Elsevier.

Lv et al. [35] have developed a multiple-mechanism-driven electrochemiluminescent (ECL) biosensor that utilizes competitive catalytic and steric hindrance effects by assembling hemin/GQ on carbon nitride nanosheets (CNNS). A hairpin probe, containing a recognition sequence for the aptamer probe and a “caged” GQ sequence, was conjugated to a hybrid of CNNS and gold nanoparticles (AuNPs; CNNS–AuNPs) on GCE. After the targeted o⁸dGuo was captured by aptamer probe (o⁸dGuo-apt), it opened the hairpin structure by hybridizing to it. During the treatment with exonucleases, o⁸dGuo-apts were protected against them by o⁸dGuo and they were released after their complementary sequences were digested. Recycling of o⁸dGuo-apt led to the continuous opening of the hairpin probe and the generation of “active” GQ structures. Finally, by addition of hemin, the liberated GQs were folded into a supramolecular hemin/GQ, that electrocatalytically reduced the H₂O₂; as a result, its quenching effect was decreased on the ECL of CNNS–AuNPs. A linear dependence of the sensor on the o⁸dGuo concentration over the range from 10⁻¹⁵ to 10⁻¹² M with a correlation coefficient of 0.998 was obtained. The LOD was calculated to be 38.8 aM (!), which is much lower than those for the best previously reported biosensors. In human serum spiked with o⁸dGuo, the proposed competitive dual-mechanism-driven biosensor had good accuracy and high precision. The test of the binding specificity of the method indicated that the aptameric recognition function was retained. Many optimizations are required for greater simplicity, lower cost and for instrumentation use (Figure 7).

Electrochemiluminescent (ECL) biosensor developed by Lv et al. [35]. Reprinted with permission from Reference [35]. © 2019, American Chemical Society.