Introduction

A biosensor is defined as “a self-contained integrated device, which is capable of providing specific quantitative or semi-quantitative analytical information using a biological recognition element (biochemical receptor) which is retained in direct spatial contact with a transduction element [1].” In a typical configuration of DNA biosensor, single-stranded probe DNA sequences are immobilized on the surface of a transducer as a recognition layer, which then captures target DNA sequences in sample solution via hybridization; this event is ultimately reflected by a detectable signal that is generated from the recognition layer and then recognized by the transducer [2]. Apart from being capable to provide genetic sequencing information, DNA sensors have been contributing to the detection of gene mutation (as diagnostic markers), identifying the differences in gene expression levels in healthy and diseased cells. In a boarder conception, scope of a DNA sensor may also include: (a) being capable to determine/detect other analytes with aptamer probes, e.g. proteins or metal ions; (b) investigate the interaction between DNA and small molecules of interest, e.g. drugs, organic pollutants, etc.; (c) detect in vitro DNA damage/genotoxicity at a molecular level, e.g. oxidative DNA lesion, DNA adduct formation, etc.. In general, the abovementioned information can be reported in terms of optical [2–4], mass-based [5–7], electrical [8, 9], and electrochemical signals [2, 10–17]. In comparison, electrochemical DNA sensor offers considerably obvious advantages, including rapid response, simple configuration, low-cost device, low-power requirement, and the ease to be integrated with microelectromechanical systems. Therefore, it becomes the most suitable platform for developing a biochip or lab-on-a-chip analysis, which is qualified for high-throughput, point-of-care, real-time and on-site detection.

The signal of electrochemical DNA sensor can be read out in a label-free manner by directly detecting the electro-actively redox activity from DNA bases and sugar moieties [10, 18, 19]; however, such reduction or oxidation reaction is usually irreversible and occurs at extremely negative or positive potential, which is out of the electrochemical window of H2O, introducing an intervention of electrolysis. As an alternative, indirectly detecting the signal generated from an electro-active label becomes more convenient, for the redox potentials of such labels are relatively decent. These labels could attach to DNA by either covalent or non-covalent means. Ferrocence derivatives are a representative group of redox small molecules for covalent labeling [20, 21]. More recently, a ferrocene-tagged “hairpin” type DNA probe was developed for a reagentless detection of a specific DNA sequence [22], which lately was named as an “E-DNA” platform [16]. Employing enzymes as covalent labels has attracted overwhelming attentions, yielding the lowest detection limit of DNA to date (1 femtomolar) [23–25] due to the signal amplification mechanism via enzyme-catalytic reaction. Another group of frequently used covalent labels is nano-materials, by which a catalytic signal can also be obtained because of their unique electronic properties [26, 27]. More importantly, they are of comparatively higher stability under experiment conditions than that of enzyme-labels.

Given no labeling procedure is needed, non-covalent labels are more adaptable to use as hybridization indicators, i.g. their binding constants with double-stranded (ds-) DNA are considerably higher than that with single-stranded (ss-) DNA. Electrostatic binding, groove binding and intercalative binding (intercalation) are three types of non-covalent interaction between such labels and DNA. For instance, Co(bpy) 2+3 (bpy = 2,2′-bipyridine), Ru(NH3) 2+6 , and Ru(bpy) 2+3 are considered as the first type of DNA binder that electrostatically binds to the phosphate backbone [28–30]. Interestingly, Λ-Ru(phen) 3+3 (phen = phenanthroline), is favored to be in groove binding mode, while its Δ-enantiomer is preferred to be in intercalation mode [31]. DNA intercalator is defined as “small organic molecules that unwind DNA in order to π-stack between two base pairs in DNA structure [31, 32].” Methylene blue (MB) and anthraquionone are two model intercalator-labels in electrochemical sensing of DNA [11, 12]. Meanwhile, DNA intercalators could also involve transition metal complexes, namely metallointercalators [31, 32]. For instance, Ru(bpy)2(dppz)2+ (dppz = dipyrido[3,2-a:2′,3′-c]phenazine, denoted as Ru-dppz) binds to DNA avidly in intercalation mode with a binding constant of 106~107 M−1 [33–36]. As shown in Fig. 1, the dppz ligand acts like a new base pair and accesses from the major groove, inducing distortion of DNA structure. The distorting force is not sufficient to make DNA bases be ejected but lead to phosphate backbone angle being open, so that the major groove at the binding site is widened. Note that there have also been some evidences to support a minor groove association between DNA and Ru-dppz [37, 38]. In addition, Takenaka’s group synthesized a threading intercalator ferrocenylnaphthalene diimide with a binding constant of 1.3 × 105 M−1[39]; Maruyama’s group reported a metal complex Os(DA-bpy)2(dppz)2+ (DA-bpy = 4,4′-diamino-2,2′-bipyridine) with a binding constant of 3.7 × 107 M−1 [40]. For the high DNA-binding affinity, detection limits of 1 fmol and 160 pg mL−1 in electrochemical DNA sensing were achieved respectively.

Fig. 1
figure 1

Metallointercalator Ru-dppz, binding to DNA in intercalation mode

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Using of covalent labels firmly attached to DNA allows electrochemical measurements to be carried out in a label-free buffer solution, thereby minimizing background signal. Non-surprisingly, this is the same case once intercalator-labels with high DNA-binding affinity are used. However, the main drawback of intercalator-labels is the lack of catalytic capability as in case of enzyme- or nanomaterial-labels. As a result, how to amplify the electrochemical signal of intercalator-labels becomes crucial for enduring this limitation of sensitivity. Thorp’s group reported a novel electrocatalytic detection methodology that employs dissolved Ru(bpy) 2+3 as a redox mediator for the catalytic oxidation of DNA guanines (as shown in Eqs. 1 and 2), by which an impressive detection limit of 40 pg mm−2 was achieved due to the facile electrode kinetics of the mediator and fast second-order reaction between the metal complex and guanine [30, 41]. The approach was further developed by Rusling and Forster by immobilizing the mediator on the electrode surface [42–44].

$$ {\hbox{Ru}}\left( {\hbox{bpy}} \right)_3^{{2 + }} + {\hbox{e}} \to {\hbox{Ru}}\left( {\hbox{bpy}} \right)_3^{{3 + }} $$
(1)
$$ {\hbox{Ru}}\left( {\hbox{bpy}} \right)_3^{{3 + }} + {\hbox{guanine}}\left( {\hbox{G}} \right) \to {\hbox{Ru}}\left( {\hbox{bpy}} \right)_3^{{2 + }} + {{\hbox{G}}^{ + }} $$
(2)

With an electron acceptor/donor system, Barton’s group developed a unique electrochemical DNA sensor for the detection of DNA base mismatches through a “DNA-mediated charge transport” mechanism [45], as shown in Fig. 2. On the right, for an electrode is modified with well-matched duplex DNA, current flows through the well-stacked DNA to reduce methylene blue (MB+) intercalated near the top of the film to leucomethylene blue (LB, electron acceptor). As similar with the catalytic mechanism in Eqs. (1) and (2), the reduction of MB+ is catalytically amplified by ferricyanide (electron donor). On the left, in the case of a DNA film containing mismatched duplexes (marked in red), current flow through the DNA duplex is attenuated, MB+ can not be reduced, and the catalytic signal is lost. Such elegantly-designed “intercalator-labels/electron donor” detection formats have been adapted/developed by other research groups [46, 47].

Fig. 2
figure 2

Illustration of an electrochemical DNA sensor for the detection of DNA base mismatches with “DNA-mediated charge transport” mechanism. Adapted from [12]

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To this end, our group has developed a signal-amplifying system relied on the measurement of electrocatalytic current of ruthenium(II) polypyridine derivative on indium tin oxide (ITO) electrodes in an oxalate-containing electrolyte [48, 49], as shown by Eqs. (3) and (4). Oxalate serves as an artificial electron donor to regenerate Ru(II) and chemically amplifies its electrochemical oxidation current. Direct electrochemical oxidation of oxalate on ITO electrode is inhibited, leading to low background current. With this signal-amplified system of “ITO electrode/ruthenium intercalator-label/oxalate,” 100-fold improvement in sensitivity over the non-amplifying detection was achieved in our previous reports [50–52].

$$ {\hbox{R}}{{\hbox{u}}^{{{2} + }}} + {\hbox{e}} \to {\hbox{R}}{{\hbox{u}}^{{{3} + }}} $$
(3)
$$ {\hbox{R}}{{\hbox{u}}^{{{3} + }}} + {\hbox{oxalate}} \to {\hbox{R}}{{\hbox{u}}^{{{2} + }}} + * {\hbox{oxalate}} $$
(4)

Photoelectrochemical (PEC) detection owns potentially higher sensitivity than electrochemical detection, which is beneficial from a lower background current due to the separation of its signal generation (light excitation) and signal detection (current). Consequently, we improved the above signal-amplified system with PEC detection. A tin-oxide nanoparticle electrode instead of ITO electrode was used according to its relatively narrowed band-gap, so that the first case of quantitative PEC detection of a biological affinity interaction was reported [53]. More recently, we reported a PEC DNA sensor based on the photoelectrocatalysis of ruthenium polypyridine complex and guanine [54]. Ruthenium complex was electronically excited by absorption of photo energy, and then the excited electron was injected into the conduction band of tin dioxide, forming Ru3+ (Fig. 3). According to Eq. (2), guanine (G) and/or adenine (A) reduces Ru3+ back to Ru2+. The cycling reaction will not stop until G or A that is reachable to the electrode surface is exhausted, leading to an enhancement of photocurrent. The system was found to be sensitive to DNA structure, so that a metallointercalator-probe PEC DNA sensor for the detection of DNA damage was developed [55–58].

Fig. 3
figure 3

Illustration of the mechanism of photoelectrochemical oxidation of DNA by ruthenium complex. CB: conduction band. VB: valence band. G: guanine. A: adenine

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Apart from PEC detection, another high sensitive detection methodology is electrochemluminescent (ECL) system, in which the signal of Ru(bpy) 2+3 or its analogues attached to DNA can be catalyzed in the presence of tripropylamine (TPA) [43, 59–61]. Electrochemluminescence of the metallointercalator Ru-dppz has been investigated, showing that the signal increased about 1,000 times in the presence of DNA [62]. According to the commonly accepted ECL mechanism, it can be assumed that Ru-dppz2+ is oxidized on ITO to Ru-dppz3+. TPA is oxidized to TPA+, either electrochemically or chemically, which then deprotonates to become TPA•. Light emission is generated after the highly reducing radical species reacts with Ru-dppz3+ to form the excited state of Ru-dppz2+.

In this brief review, we are aiming to introduce our proceeding works in recent years with a focus on the development of electrochemical, PEC and ECL DNA sensors by employing metallointercalator Ru-dppz as probe coupled with electrocatalytic signal-amplified systems. With such DNA sensors we had developed, considerable attentions are given to the investigation of DNA/small molecule interaction (e.g. polycyclic organic compounds) and the detection of DNA damage (by chemically oxidation and formation of DNA adducts). Relevant reports in these fields were also surveyed and discussed.

Early work: signal amplification mechanism

As shown in Eqs. (3) and (4), we have shown in our previous studies that the oxidation current of Ru(bpy) 2+3 was amplified by more than 1,000 fold in an oxalate solution [48]. There are two reasons for us to select Ru-dppz as a DNA intercalating electrochemical indicator. One is that metal complexes with dppz ligand have a high binding constant (106~107 M−1 [33–36, 63, 64]). The other reason is that the formal potential of Ru-dppz is as high as that of Ru(bpy) 2+3 . It has been found that there is a good correlation between amplification efficiency and formal potential [48]. The voltammograms in Fig. 4 demonstrate the power of our signal-amplifying system. Without amplification, anodic peak of 5 μM Ru-dppz at 1.25 V is marginally distinguishable from the background current (Fig. 4-A). In contrast, in the presence of 100 mM oxalate, a substantial rise of current was clearly visible starting from 1.0 V (Fig. 4-B). Measured at 1.25 V, the current was amplified by 60 folds, leading to much higher signal-to-background (S/B) ratio.

Fig. 4
figure 4

Illustration of the signal-amplified system with electrocatalytic mechanism. Cyclic voltammograms of ITO electrodes in phosphate buffer (a) and in oxalate/phosphate buffer (b). Curve (a) buffer alone, and curve (b) with addition of 5 μM Ru-dppz

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Once calf thymus ds-DNA was added into the solution, intercalation of Ru-dppz into DNA duplex led to a reduction in the oxalate-amplified electrochemical current because of slower mass diffusion [50]. The phenomenon is similar to that in early studies conducted by Bard’s group [65, 66]. More importantly, additional signal attenuation mechanism due to the charge–charge repulsion between negative-charged DNA phosphate-backbone and the sacrificial electron donor, i.g. oxalate anions, provided a higher sensitivity than that in those relevant reports. The detection limit of 1 pM DNA is close to some of the reported fluorescence measurements [67, 68]. With the same mechanism, a selective PEC detection of DNA was developed, in which the signal reduction was selective for ds-DNA, as no such effect was observed for single-stranded polynucleotides such as poly-G, poly-C, poly-A, and poly-U [51].

These proof-of-concept results in early works offered a foundation for us to develop an intercalator-probe DNA sensor with high-performance signal-amplifying system. Use of the high-affinity DNA intercalator-label Ru-dppz was expected to permit the electrochemical, PEC or ECL measurements to be carried out in a label-free electrolyte, which is effective to suppress the background signal. The DNA sensor was expected to allow the discrimination of ds-DNA and ss-DNA immobilized on the surface. Subsequently, it would be able to indicate the DNA-binding behaviors of small molecules of interest, and to probe DNA damage by recording the difference in the amount of bound Ru-dppz before and after incubation with damaging agents.

DNA sensor fabrication and development

There are several strategies to immobilizing DNA on solid electrode surface, including physical absorption, covalent attachment, bioaffinity-binding bridge, and film entrapment [2, 11, 13 and cited references]. The layer-by-layer electrostatic self-assembly approach is one of the most convenient methods of constructing multi-layer films. As shown in Fig. 5, DNA film is immobilized to ITO electrode surface through an intermediate film of either avidin protein [52, 55] or cationic polymer poly(diallyldimethylammonium chloride) (PDDA) [56–58, 69, 70]. Avidin protein had been found to adsorb strongly on ITO surfaces by electrostatic interaction due to its high positive charge [71], yielding a surface coverage of 1.6 × 10−11 mol cm−2 estimated by quartz crystal microbalance (QCM) [52]. It can be estimated that 3.2 ng mm−2 nucleic acids were immobilized on the surface of avidin-coated electrode, very closed to that on myoglobin-coated surface in relevant reports [44, 60]. As for PDDA film, the amount of immobilized nucleic acids was estimated to be c.a. 1.2 ng mm−2 [44].

Fig. 5
figure 5

Illustration of the metallointercalator-based DNA sensor with electrocatalytic mechanism. The metallointercalator: Ru-dppz

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Obviously, there are two types of electrocatalytic reaction in the system, as shown in Fig. 5(A) and (B). As discussed in the Introduction section, one happens between the intercalator-label and DNA bases (mostly with G) on the electrode surface, following Eqs. (1) and (2); the other involves a reaction in the double layer of electrolyte between intercalator-label and electron donor species approaching the electrode surface by diffusion, following Eqs. (3) and (4). The latter dominates over the former for the molar concentration of such electron donor species in electrolyte is much higher than that of DNA (bases) on the electrode surface. The amplified signal can be recorded in the manner of electrochemical (EC), ECL, and PEC signals. The as-developed DNA sensor has been mainly used for the determination of surface-immobilized DNA [52], the detection of DNA damage [55–58], and the investigation of DNA/small molecule interaction [69, 70, 72].

DNA binding mode of Ru-dppz

Using 1H and 31P NMR spectroscopies, Barton’s group proved that both of Λ- and Δ-Ru-dppz complex bind to DNA duplex from the major groove side [33–36], whereas Norden’s group believed such intercalation also happens in the minor groove side according to the observation with linear dichroism (LD) spectras [37, 38]. In either case, Ru-dppz complex has unique “light-switch” characteristics, i.g. it shows no luminescence in aqueous solution due to the fact that the excited state of the phenazine nitrogen atoms (*N) can be quenched by the proton of water, while great luminescence is observed after it binds to DNA, in which the *N become protected and this excitation can be sustained. As a result, photoluminescence is preferred as a popular methodology to investigate DNA binding mode of Ru-dppz complexes [31, 32, 67, 68].

With the electrochemical DNA sensor in Fig. 5-B, we revealed the DNA binding mode of Ru-dppz [73]. DNA film voltammetry of Ru-dppz displayed two distinctive oxidation peaks which were, by experimental evidence, related to the oxidation of DNA bases catalyzed by the metal complex at two different rates. At low Ru-dppz concentrations, a single oxidation peak was observed, the potential of which shifted from 1.25 to 1.1 V with increasing Ru-dppz concentration (peak 1). At high metal chelate concentrations, an additional oxidation peak emerged with a potential of 1.25 V which was unaffected by the Ru-dppz concentration (peak 2). A two binding mode hypothesis, intercalative mode for peak 1 and electrostatic mode for peak 2, was evoked to explain the different redox reactivity of the metal complex (Fig. 6). More importantly, it offers valuable information about how to control DNA binding mode of Ru-dppz by varying the molar ratio of the two, which would be helpful for the following studies to probe the interaction between DNA and other small molecules of interest.

Fig. 6
figure 6

DNA binding modes of Ru-dppz revealed by DNA film voltammetry

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This work demonstrates that such DNA film voltammetry is a simple, rapid, but informative method for the study of DNA recognition and redox reactivity of redox molecules, compared with the other approaches mentioned above. It owns many of the advantages as in case of protein film voltammetry developed and popularized by Armstrong and coworkers [74]. An overview on recent reports investigating the binding behaviors of various metallointercalators with DNA by voltammetric titration approach was given in Table 1. In contrast to the solution-phase electrochemical methodology [30, 41, 63–66, 75, 76], the resolution of the voltammetric response is substantially higher because the redox probe is attached to the DNA film on an electrode surface [77–80]. It needs to be pointed out that early in the late 1980s Bard’s group pioneered voltammetric study of the binding interaction between metal chelates and DNA in solution [65, 66].

Table 1 An overview on recent reports investigating the binding behaviors of various metallointercalators with DNA by voltammetric titration approach
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Determination of surface-immobilized DNA

As discussed in the Introduction section, the original objective of a DNA sensor is to determinate the amount of target DNA sequences in sample solution via hybridization. Strategies for electrochemical sensing of DNA can be classified into label-free and label-based approaches. In general, labels include enzymes, nanomaterial and redox small molecules, any of which could be attached to DNA by either covalent or non-convelent means. As far as we known, numerous review articles on this topic have been presented [2, 10–17]. However, few of them have addressed specifically the proceeding works about the determination of surface-immobilized DNA with intercalator-based electrochemical DNA sensors. From our views, it can be cataloged to non-amplifed, pre-amplified and post-amplified detection, as shown in Fig. 7.

Fig. 7
figure 7

Illustration of stratigies for the determination of surface-immobilzied DNA with metallointercalor-based DNA sensors

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In a non-amplified detection format (Fig. 7-a), the hybridized DNA film reacted with intercalator-labels, and the unbound intercalator-labels are washed off from the surface, followed by signal readout in a label-free solution of electrolyte [39, 40, 77, 81–83]. As mentioned before, Barton’s group reported a novel electrochemical DNA sensor based on the “DNA-mediated charge transfer” mechanism that a well-stacked DNA is proved to be an electron transfer intermediate as an “electron wire” connected between bound intercalator-labels and electrode surface [12]. The intercalator cannot be reduced (or oxidized) due to the current flow through the DNA duplex is attenuated by mismatched bases. With this mechanism, a variety of electrochemical DNA sensors have been developed for the sensitive detection of single-based mismatch [12, 84]. There is an interesting report to determinate proteins of interest with aptamer probes [85]. An aptamer was first hybridized with its complementary sequence and the resulting duplex was reacted with intercalator-labels. Next, extra amount of thrombin was added to compete with the complementary DNA sequence to bind to the aptamer, along with a drop of signal due to the bound intercalator-labels released. Therefore, the amount of thrombin is proportional to the reducing signal, thus it can be quantified accordingly.

By specific PCR amplification, the analyte (mutant target sequence) can be hybridized with a special DNA sequence, whereas the hybridization cannot happen as for the control sequence (wild type target) [86–88]. The special sequence can be captured to sensor surface and detected with intercalator-labels, as shown in Fig. 7(b). For instance, a circular target sequence with a point mutation was amplified isothermally by DNA polymerase, yielding an elongation sequence. The elongated tail-like sequence was captured and recognized [86]. Meanwhile, the intercalator-labels can be pretreated with conjugation of nanomaterial (e.g. Ag nanoparticles), which shows a highly characteristic solid-state Ag/AgCl redox process in electrochemical measurement [89]. Another interesting report is that controlling the density of probe sequences on the electrode surface with diluents of alkanethiols was found to enhance the hybridization effect and increase the S/B ratio [90].

Post-amplified detection format can be achieved either by adding electron donor species in the solution of electrolyte to amplify the signal of intercalator-labels [45–47, 52, 69, 70, 72] or by post-labeling procedure [91, 92], as illustrated in (Fig. 7-C). As mentioned in the Introduction section, we used oxalate as a sacrificial electron donor, amplifying the oxidation current of Ru-dppz (Fig. 5-A). As a result, ds-DNA adsorbed from 20 ng mL-1 solution could be detected, which was estimated by QCM to be 160 pg mm−2 on the surface. The catalytic current of ds-DNA was substantially higher than that of ss-DNA and poly-C, indicative of selective binding of the redox indicator to ds-DNA (Fig. 8). Ferricyanide and NADH were employed respectively as electron donor species to amplify the signal of intercalator-labels in the “DNA-mediated charge transport” system developed by Barton’s group (Fig. 2) [45, 46]. Later, Zhou’s and Barton’s groups improved the system by combination with scanning electrochemical microscopy, producing visualized images of DNA arrays [47, 93]. This may indicate an arrival of a new generation of DNA sensor. In addition, Gao’s group reported that the oxidation signal of threading intercalator-labels were catalyzed by electron donor species, such as guanine, ascorbic acid and amine [94–96]. More recently, it has been reported that intercalator-labels were post-labeled with enzymes via avidin/biotin bridge [91, 92]. In these cases, the intercalator-label acts as hybridization indicator, while the enzyme is the signal generator and amplifier.

Fig. 8
figure 8

Illustration of the electrochemical DNA sensor for disguishing defferent types of nucleotides. Voltammograms of ITO/avidin/DNA/Ru-dppz electrodes measured in 30 mM oxalate/oxalic acid buffer, pH 5.8. DNA solutions are 200 μg mL−1 of (a) ds-DNA, (b) ss-DNA, (c) Poly-C, and (d) no DNA. Ru-dppz concentration is 10 μM

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Investigation of DNA/small molecules interaction

Regulation of gene expression by activators and repressors in vivo involves the interaction between DNA and small molecules, which may be the first process of DNA damage and the initiation of genotoxicity. Discovering the mechanism of binding behaviors with DNA will also be helpful for the understanding of the triggering process of cancer. Conventional techniques for the investigation of DNA/small molecules interaction include footprinting, affinity cleavage, NMR, X-ray crystallography, UV–vis spectrophotometry, fluorescence, circular dichroism, and hydrodynamic measurements [97, 98].

With electrochemical DNA sensors, the interaction between DNA and various electrostatic and intercalative binders was investigated [29, 73, 75, 80, 99–101]. Great efforts have been made to investigate DNA/drug [102, 103] and DNA/pollutant [104, 105] interactions by monitoring the electrochemical signal of either the compound itself or the guanine base in DNA. Based on the signal-amplified system in Figs. 5-A and 8, we demonstrated a displacement method for the investigation of the interaction between DNA and five well-known polycyclic organic compounds, such as thiazole orange, 4,6-diamidine-2-phenylindole, H33258, ethidium bromide, and quinacrine [69] (Fig. 9-A). The binding constants (Kb) were calculated to be in the range from 4.3 × 105 to 1.2 × 107 M−1, which are generally consistent with that by some established methods. Later, we utilized such displacement method to measure the binding constants of 26 hydroxylated PAHs (OH-PAHs) with DNA and tried to find the relationship between Kb values and the molecular structure information of OH-PAHs [70]. A plot of the oxidation current measured at 1.25 V as a function of OH-PAH concentration yields a displacement curve for the compound (as illustrated in Fig. 9-B), from which Kb values were calculated to be in the range from 4.5 × 104 to 3.0 × 105 M−1. According to quantitative structure–activity relationship (QSAR) analysis, it was found that q +H , a molecular descriptor associated with hydrogen bonding capability, correlates most strongly with the binding constant among 11 frequently used structural descriptors related to hydrophobicity, electrostatic and hydrogen bond interaction, and steric effect. Specifically, as for the nine hydroxylated benzo[a]pyrenes, strong correlation was evident between the binding constant and the three descriptors related to the molecular size and accessible area. In a more recent report [72], the indicator concentration in the displacement measurement was reduced by 75-fold with ECL detection, which allows the investigation of those OH-PAHs with poor solubility in aqueous solution and week affinity to DNA. It is expected that more details including the binding mode, binding affinity, and sequence selectivity of a series of structurally similar chemicals with DNA can be investigated by this ECL methodology to determine a structure–property relationship.

Fig. 9
figure 9

(A) Illustration of the electrochemical displacement method for the investigation of the binding interaction of polycyclic organic compounds with DNA. (B) Plots of the oxidation current measured at 1.25 V in the voltammogram as a function of OH-PAH concentration in the mixed solution with 30 μM Ru-dppz. Each data point is the average of three electrodes

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Similarly, the observation that the DNA-bound intercalator Co(phen) 2+3 or Cu(phen) 2+3 can be displaced by 2-nitrofluorene and 2,7-dinitrofluorene implicates that under in vitro conditions the nitrofluorenes interact with ds-DNA by intercalation [106].

DNA damage detection

Chronic diseases and medical malfunctions induced by the exposure of toxic chemicals have been serious contemporary public health problems [44]. Cancer can be initiated from DNA damage caused by those chemicals, such as heavy metals or organic compounds. DNA adducts formation and oxidative stresses are the two major routes of DNA damage. Therefore, there is an urgent demand for a rapid and effective detection of DNA damage to accomplish chemical genotoxicity screening. Commonly used genotoxicity tests includes: (a) comet assay, or the single-cell gel electrophoresis; (b) cytokinesis-blocked micronucleus assay; (c) γ-H2AX staining, detected by immunofluorescent microscopy; and (d) DNA adducts detection, relying on modern instrumentations, e.g. HPLC- or GC-MS. Excellent sensitivity and precision can be obtained by these methods. However, they usually require sophisticatedly skilled analysts; moreover, some of these methods are somewhat time-consuming, high-cost, and limited by several factors, including failure to adherent lesions, cross-reactivity of the antibody, and hydrolysis of DNA sample before HPLC analysis. More recently, approaches based on fluorescent [107] and electrochemical [43, 44, 103, 108] detection of DNA damage offers some promising alternatives. For instance, Rusling and co-workers reported a series of electrochemical DNA sensors that the indicator (Os- or Ru-) metellopolymer was immobilized on the electrode surface to detect toxicity of chemicals and oxidative stress [42–44, 60].

The PEC DNA sensor we developed (Fig. 5-A) was employed for the detection of DNA damage induced by Fenton reaction (Fe2+/H2O2) [55]. After the chemical reaction with the damaging agents, a drop in photocurrent was observed (Fig. 10). This can be attributed to that less amount of Ru-dppz binds to the DNA film because of DNA strand breakage induced by hydroxyl radicals [44, 109, 110]. It is also found that the damage of the DNA film was complete in 1 h with Fenton reagents. After optimizing conditions, the sensor permits the detection of DNA damage by as low as 10 μM Fe2+ and 40 μM H2O2 [57], a concentration that is within the physiologically relevant range. For the detection of in situ oxidative DNA damage, glucose oxidase (GOD) was introduced to the system, embedded by double layers of PDDA, forming a PDDA/GOD/PDDA/DNA multilayer [56]. In the presence of glucose, the enzyme catalyzes the reaction that produces H2O2, by which hydroxyl radicals are generated via the Fenton reaction. The incorporation of glucose oxidase into the sensor mimics the metal-mediated reactive oxygen species (ROS) generation pathway in vivo, and eliminates the use of the unstable H2O2 as a reagent. To our knowledge, this is the first report of the detection of metal-induced, enzyme-catalyzed DNA damage. It needs to point out that DNA damage induced by styrene oxide that forms DNA adducts was also conceived by our system [55].

Fig. 10
figure 10

Left: Illustration of the metallointercalator-based photoelectrochemical DNA sensor for the detection of DNA damage. Right: (a) Photocurrent of Ru-dppz intercalated into the SnO2/avidin/ds-DNA film as a function of the reaction time in (a) 1 mM FeSO4/4 mM H2O2, (b) 1 mM FeSO4, and (c) 4 mM H2O2. (b) Photocurrent response of Ru-dppz intercalated into the SnO2/avidin/ds-DNA film incubated for 1 h in (a) 1 mM FeSO4/4 mM H2O2, (b) 1 mM FeSO4, and (c) 4 mM H2O2. The photocurrent measurement was performed in 30 mM oxalate buffer, pH 5.8. The excitation light was switched on and off as indicated

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Another encouraging example is that the PEC DNA sensor was capable to detect DNA damage induced by tetra-halogenated quinines [58], the metabolites of halogenated phenols in vivo. Recent studied show that halogenated quinones are able to produce hydroxyl radicals merely in the presence of H2O2, which is independent of any transition metal ions. It was found that the two benzoquinones exhibited distinctively different reactivity in DNA damage. In the absence of H2O2, more DNA damage is induced by tetra-1,4-chlorobenzoquinone (TCBQ) than that by tetrafluoro-1,4-benzoquinone (TFBQ), presumably because the former could form covalent DNA adducts. Interestingly, in the presence of H2O2, more severe DNA damage was observed with TFBQ rather than TCBQ.

Summary and perspectives

Metallointercalators have been attracted increasing attentions in DNA-associated studies, such as DNA foot-print, quantification, and DNA binding affinity study. This could be attributed to two major advantages of matallointercalator [31, 32]: one is that the binding affinity of such complexes with DNA can be varied by easily altering the molecular structure of the ligands; the other is that the transition metal center offers a rich photo-physical [111] and electrochemical activity. As for the development of DNA sensor, metallointercalator is playing an essential dual-role of structure indicator (for hybridization) and signal generator. As shown in Table 2, the binding affinity of metallointercalators with DNA could reach a relatively high value, i.g. ~107 M−1 or above [33–36, 40, 63, 64, 94–96]. As a label, they are of great conveniences and advantages for the detection: (a) no time-intensive label procedure is needed; (b) the measurement can be carried out in label-free environment. However, lack of catalytic activity on signal generation as in the case of enzyme- or nanomaterial-labels becomes the main weakness of metallointercalator-labels. To overcome this limitation, a number of strategies for signal amplification have been reported (Fig. 7), including selective PCR, label modification, electrocatalysis, and post-labeling. An overview of metallointercalator-based electrochemical DNA biosensors is shown in Table 3, in comparison with sensor parameters (sensitivity, reproducibility, regeneration, etc.). From the table, it is obvious that the PEC and ECL DNA sensors are of higher sensitivity than conventional electrochemical DNA sensors. Signal-amplified strategies (by either pre-amplified or post-amplified mean) can also improve the sensitivity to a certain extend. However, the issue sensor regeneration has not been well-covered in most of reports listed in Table 3. Gold and semiconductor are two major types of materials of working electrode for the listed DNA sensors, whose cost are much higher than carbon electrodes, e.g. screen-printed carbon electrode. Therefore, sensor regeneration should be in consideration in order to open possibility of commercial products in the future.

Table 2 Metallointercalators frequently-used in electrochemical DNA sensors
Full size table
Table 3 Comparison of parameters of various matallointercalator-based electrochemical DNA sensors
Full size table

Thorp’s group discovered the electro-catalytic mechanism of ruthenium complexes and guanine, and developed ultrasensitive electrochemical DNA sensors [30, 41]. The elegant “DNA-mediated charge transfer” model (Fig. 2) has been studied extensively by Barton’s group and other groups [12, 45–47, 84, 93]. This detection format serves as a universal substrate for several types of analysis, including the detection of DNA base mismatch, DNA lesion, the interaction of DNA and proteins/small molecules of interest. In these studies, ferricyanide or NADH was usually employed as electron donor species for signal amplification [45, 46]. By designing threading intercalators with extremely high binding constants with DNA, several DNA sensors were also developed by Gao’s group who employed guanine, ascorbic acid, or amine to electrocatalyze the oxidation signal of the intercalator-labels [94–96]. To this end, we presented a series of EC, PEC and ECL DNA sensors with Ru-dppz probe and electrocatalytic mechanism [49, 55–58, 69, 70, 72]. Catalytic oxidation signals of the metallointercalator amplified by the reduction of oxalate, DNA bases (guanine) or tripropylamine were recorded in order to quantify surface-immobilized DNA [52], to probe the interaction of DNA/OH-PAHs [70, 72], and to detect the DNA damage either by styrene oxide or Fenton reaction [55–57]. Yet the detection of DNA hybridization and defect/mismatch has not been covered. Studies on these topics can be found in some excellent review articles [10–13, 103, 108]. Although encouraging results in our experiments suggest that metallointercalator-based DNA sensors could be a rapid, cost-effective and even high-throughput methodology for the detection of DNA damage/genotoxicity, we need to keep in mind that there would be still a long way to go, for the DNA damage in vivo involves much more complicated cellular processes. Cell-integrated sensor would be the final goal and an ideal tool for genotoxicity detection, yet so far it is the most challenging task in this field.

Coupled with contemporary microfabrication technology, the metallointercalator-based electrochemical DNA sensor could also be a good platform for developing lab-on-a-chip (LOC) analysis [15]. It is exciting that there have already been some reports to combine LOC with DNA sample preparation step (i.g. PCR), which produced intelligent devices with high efficiency and automation [112–115]. For instance, a microfluidic flow-through electrochemical-based qPCR chip was reported by Gong’s group [115], in which both the amplification of the target DNA sequence and subsequent electrochemical detection of the PCR amplicon are realized simultaneously. Commercialization of such device will be available as long as the cost for the microfabrication becomes low enough [2] for marketability in the future.

The other tendency of metallointercalator-based electrochemical DNA sensors is visualized DNA arrays, such as those reported by Zhou’s and Barton’s groups who combined DNA sensor with scanning electrochemical microscopy (SECM) [47, 93]. This principle could be extended by using other imaging technique in the future, such as atomic force microscopy (AFM) [116]. This could be a new generation of DNA sensors that are expected to “look at” not only the image of signals but also the morphology of DNA assembly as well as the binding behavior of target molecules, including the binding mode, binding sites, binding affinity, and sequence selectivity. From this view, multifunctional visualized DNA sensors will be the long-term goal under the help of advanced surface technique (QCM [117], surface plasma resonance, Raman scattering, etc.).