Photoelectrochemical DNA Biosensors - Chemical Reviews (ACS

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Photoelectrochemical DNA Biosensors Wei-Wei Zhao, Jing-Juan Xu,* and Hong-Yuan Chen* State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210093, China DNA base pairing is the basis for the biorecognition events in such DNA biosensors.6,7 To construct DNA biosensors, singlestranded DNA (ssDNA) probe segments are initially immobilized onto the transducer surface as a recognition layer for the subsequent capture of target DNA via the process of hybridization, based on which a detectable signal would be in principle generated, then recognized by the transducer, and finally displayed by a signal processor. In a broad sense, the scope of DNA sensors may also include the following: (a) the CONTENTS detection of DNA association with small molecules of interest, 1. Introduction 7421 such as drugs and chemicals; (b) the investigation of DNA 2. PEC DNA Biosensors 7422 interactions with proteins, including detecting catalytic activities 2.1. Transducers 7422 of DNA-processing enzymes and studying affinity interactions 2.1.1. Inorganic Semiconductors 7423 between DNA and proteins; (c) the construction of aptasensors 2.1.2. Organic Semiconductors 7423 based on aptamer probes for other targets; (d) in vitro DNA 2.1.3. Hybrid Semiconductors 7423 damage detection. According to the mode of signal trans2.1.4. Others 7424 duction, to date, a large spectrum of optical, acoustic, 2.2. Probe Immobilization 7424 gravimetric, electrical, and electrochemical techniques have 2.2.1. Chemisorption 7425 been reported.8−14 2.2.2. Physisorption (Noncovalent Binding) 7425 Ever since the pioneering discovery of the photoelectric 2.2.3. Covalent Binding 7425 effect by Edmond Becquerel in 1839,15 photoelectrochemistry 2.2.4. Affinity Binding 7425 has been actively developing and now attracts substantial 2.3. DNA Interactions 7426 research scrutiny in various fields.16,17 The photoelectrochem2.3.1. DNA Hybridization 7426 ical (PEC) process refers to the photon-to-electricity 2.3.2. DNA Association with Small Molecules 7427 conversion resulting from the charge separation and subsequent 2.3.3. DNA Interactions with Proteins 7429 charge transfer of a photoactive material (usually organic or 2.3.4. DNA Aptamer−Target Interactions 7430 inorganic semiconductors) after absorbing photons upon 2.3.5. DNA Damage 7431 illumination, and the formed electron−hole pairs at the 2.4. PEC Transduction of DNA Interactions 7431 interface would cause the oxidation−reduction reaction of the 2.4.1. Signal On and Signal Off 7431 ground or excited states of molecules or ions. Such a photo2.4.2. DNA Photoelectrochemistry 7431 driven principle has long been applied to photovoltaics, 2.4.3. Label-Free Techniques 7433 photocatalysis, and photosynthesis. Figure 1A shows the basic 2.4.4. Indicator-Based Techniques 7434 concept of a PEC cell. In past years, with the ever-growing 2.4.5. Label-Based Techniques 7435 demand for advanced bioanalysis, enormous efforts have been 3. Conclusions and Perspectives 7437 devoted to the exploitation of new bioanalytical techniques Author Information 7437 compatible with future requirements.18−25 Significantly, the Corresponding Authors 7437 integration of photoelectrochemistry with bioanalysis has Notes 7437 inaugurated an innovative field of PEC bioanalysis.26−35 This Biographies 7437 newly emerged yet promising technique is of special interest for Acknowledgments 7438 its desirable potential in biological analysis. The detection References 7438 principle is that the photocurrent/photopotential change could be brought about by the biological interactions between various recognition elements and their corresponding target analy1. INTRODUCTION tes.34,35 The process of PEC detection is just the reverse of electrochemiluminescence (ECL): the light is used as the Biosensors could register a biological event and convert it into a excitation source and photocurrent is used as the detection measurable signal with specific biosensing elements and signal. Due to this total separation and the different energy transducers.1−5 A DNA biosensor is a device that integrates forms of the excitation source (light) and the detection signal the probe DNA as a biorecognition element and an electrode as a signal transducer. The probes are typically short oligonucleotides that are capable of hybridizing with specific and unique Received: February 19, 2014 regions of the target DNA sequence, and the complementary Published: June 16, 2014 © 2014 American Chemical Society

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dx.doi.org/10.1021/cr500100j | Chem. Rev. 2014, 114, 7421−7441

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articles about the DNA biosensors that are based on different techniques such as electrochemical method have been published,8,9 whereas to date there have been no efforts addressing specifically the survey of this dynamically developing area of PEC DNA biosensors. Given the pace of advances in this area, the PEC routes for monitoring the DNA biorecognition events are the subject of the present review. In more detail, for the first time, this review surveys the methodology related to the PEC DNA biosensor construction, the interactions to be addressed, and the inventive detection principles. The recent progress, current directions, and future prospects in this area are also evaluated and discussed, with the aim to provide an accessible introduction to PEC DNA biosensors for any scientist.

2. PEC DNA BIOSENSORS The PEC experiments need to be performed in a PEC system, including an excitation source (irradiation light such as Xe lamp, a monochromator, and a chopper), a cell, and an electrochemical workstation with a three-electrode system. Under illumination, charge separation and transfer of the photoactive species will occur and hence generate an electric signal between the working electrode and the counter electrode in electrolyte solution consisting of a specific electron donor or acceptor. This electrical output will then be recorded by the electrochemical workstation. Owing to the electronic signal produced directly by the PEC reactions, there is no need for an expensive signal transduction instrument. PEC DNA biosensing commonly involves tracking this transduction signal prior to and after the DNA-based biological event. In a typical configuration, as shown in Figure 1B, anchored probe DNA is initially confined to the electrode substrates for the following DNA interactions, which would be detected via the increased or attenuated electrical signal (that originating from the DNA photoelectrochemistry, or from the covalent or noncovalent labels that were introduced into the system by the end-labeled target DNA or preferentially binding to the duplex DNA instead of the ssDNA probes; see section 2.4). The following sections will focus on the major steps involved in the construction of PEC DNA biosensors and the corresponding applications, i.e., the choice/fabrication of the transducer (section 2.1), the immobilization of the probe DNA (section 2.2), the actual biorecognition interactions to be addressed (section 2.3), and the transduction of the events into the photocurrent signal (section 2.4). As will be demonstrated, the methodology of PEC DNA biosensors is highly interdisciplinary, and the success of such biosensors greatly depends on the integration of DNA−target biorecognition, dynamic processes of semiconductor−liquid interfaces (including photochemistry, electrochemistry, interfacial charge transfer, etc.), surface and material sciences, as well as inventive bioassay formats.

Figure 1. (A) Basic concept of a PEC cell and (B) general PEC DNA biosensor design. The major process involves the probe DNA immobilization on the substrate as the recognition layer for the subsequent hybridization, which event is transduced into an electric signal for analysis.

(electricity), it possesses potentially higher sensitivity than conventional electrochemical methods because of the reduced background signals. Moreover, comparing with optical techniques that necessitate complicated and expensive equipment, the utilization of an electronic readout makes the PEC instrumentation simpler, cheaper, and easier to miniaturize. Due to these obvious advantages and attractive potential in future bioanalysis, its popularity has rapidly expanded within the analytical community and increasing studies have been reported addressing various targets of interest including DNA and proteins.36−112 PEC DNA biosensors, another subclass of DNA biosensors, combine the inherent advantages of PEC bioassay with the high specific bioaffinity properties of the DNA strands. Taking advantage of the photoactive transducers or reporters, such devices convert the DNA recognition events into a useful electrical signal, providing an elegant route for probing various DNA−target interactions at the molecular level. Specifically, the general detection mechanism of the PEC DNA biosensors is based on the fact that the formation of DNA duplex or other DNA−target complexes could be monitored by the changed electrical signal of the employed photoactive species. In the past decade, it has attracted considerable interest, and pioneering work and significant progress have been achieved in its development and bioassay application. On the other hand, as far as we know, an impressive number of comprehensive review

2.1. Transducers

The fabrication way of PEC DNA biosensors influences exquisitely their analytical performance. Although in a few works the PEC DNA detection process could occur in the solution (i.e., the DNA does not be anchored),113−115 the PEC DNA biosensors in principle necessitate the interfacial confinement of the probes onto the surfaces of specific solid transducers. Since this is where the immobilization of the recognition layer and the biorecognition of the targets take place, the choice and the quality of the transducer is of vital 7422


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according to the specific experimental conditions (Figure 3). Unfortunately, so far pure organic materials have been seldom

importance in achieving excellent analytical performance. Generally, the selection of the transducer species is mainly related to the DNA related interactions of interest and the proposed assay strategy. 2.1.1. Inorganic Semiconductors. The great technological advances in nanoscience have made possible the use of manifold nanomaterials as photoanodes in PEC biosensors.116−119 In particular, inorganic semiconductors, such as SnO2,113,120−130 TiO2 nanoparticles (NPs),131−134 and CdS and CdSe quantum dots (QDs),135−142 have been used extensively in these biosensors due to their excellent PEC properties. The photocurrent generation mechanism of the inorganic semiconductor NPs is shown in Figure 2. When the semiconductor

Figure 2. (A) anodic and (B) cathodic photocurrent generation mechanism of semiconductor NPs in connection with electrodes.

NPs absorb photons with energies higher than that of their band gaps, electrons are excited from the occupied valence band (VB) to the empty conduction band (CB), thus forming electron−hole pairs. As soon as the charge separation occurs, the electron−hole (e−h) pairs would be destined for recombination or charge transfer. In the fields of photoluminescence (PL) and ECL, the optical detection signals originate from the spontaneous emission of radiative e−h recombination. In the field of PEC bioassay, however, trapping of the excited electron in the surface states would result in e−h pairs with sufficient long life, permitting the transfer of the CB electrons to the electrode or solution-solubilized electron acceptor to give rise to anodic or cathodic photocurrent, respectively (Figure 2). Simultaneously, the VB holes would transfer to the surface of the semiconductor material and then be neutralized by the electrons supplied by the electron donor in the solution or electrode. The presence of an efficient electron donor (D)/acceptor (A) would inhibit the e−h recombination and hence facilitate the generation of a high and stable photocurrent. 2.1.2. Organic Semiconductors. The PEC active materials also include organic small molecules such as porphyrin and its derivatives, phthalocyanine and its derivatives, azo dyes, chlorophyll, bacteriorhodopsin, and polymers such as phenylenevinylene (PPV), poly(thiophene), and their derivatives.27 For example, tris(bipyridyl) ruthenium complex, [Ru(bpy)3]2+ (bpy = 2,2′-bipyridine), has been used in PEC DNA damage detection and PEC immunoassay.143−145 Upon irradiation, Ru(II) is brought up to the excited state.146 Since [Ru(bpy)3]2+ can react as both electron donor and acceptor, either anodic or cathodic photocurrent could be produced

Figure 3. (A) Chemical structure of the [Ru(bpy)3]2+ and schematic illustrations of the (B) anodic and (C) cathodic photocurrent generation mechanism by ruthenium complex.

exploited as transducers in PEC DNA hybridization biosensors, in spite of their great potential and prospective use as a versatile platform for genetic testing. However, the previously reported PEC determination of nucleotides based on a 5,10,15,20tetra(4-pyridyl)porphyrin (TPyP) modified electrode illustrated the possibilities,147 and the mechanism of that PEC sensor for nucleotides could be applied in the study of the interaction between DNA and organic dyes or other DNArelated interactions. 2.1.3. Hybrid Semiconductors. Another desirable transducer material that needs to be exploited for PEC DNA biosensors is hybrid material, for example, the coupling of two inorganic semiconductors with different band gaps, or organic complexes combined with inorganic materials. The enhancement in the photon-to-current conversion efficiency of the semiconductor materials would be favorable for improving the sensitivity of a PEC sensor. For a single material, the existence of the e−h recombination would decrease the photocurrent. The coupling of small and large band gap semiconductors benefits the charge separation and thus results in improved conversion efficiency. TiO2/CdS is a case in point, and as shown in Figure 4A, excitation of the CdS with a small band gap would lead to charge separation and the subsequent prompt transfer of CB electrons to that of TiO2 with a large 7423


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Figure 4. Schematic diagram of photocurrent generation mechanism for (A) CdS/TiO2 and (B) TCPP/ZnO hybrid materials.

band gap. The spatial separation of the e−h pairs in the different semiconductors retards their recombination. Consequently, scavenging of the VB holes by the electron donor facilitates the electron propagation from the TiO2 to the conductive substrate and the generation of the photocurrent. Alternatively, surface sensitization of a wide band gap semiconductor via chemisorbed or physisorbed dyes can also increase the efficiency of the excitation process and expand the wavelength range of excitation for the use as a PEC transducer. This occurs through excitation of the dye followed by charge transfer to the semiconductor. Charge carriers can form in a semiconductor by exciting a dye attached to its surface. The excited state can inject either a hole or, more commonly, an electron to the semiconductor. Taking the 4,4′,4″,4‴(21H,23H-porphine-5,10,15,20-tetrayl)tetrakis(benzoic acid) (TCPP)−ZnO composite material for example,148 as demonstrated in Figure 4B, the energy of the excited state of TCPP (S*) is higher than that of the CB of ZnO. As a result, S* can inject electrons to the CB of ZnO promptly and then S* turns into its oxidation state S+. The electrons are collected by the conducting substrate to generate photocurrent, which is sustained in the presence of a solubilized electron donor that is acting as a sacrificial agent to reduce S+ to regenerate the ground-state metal complex. Due to the quick transfer of the photogenerated electrons of S* to the CB of ZnO, the enhanced photocurrent can be obtained. In another example of PEC detection of DNA damage, Guo and colleagues immobilized bis(2,2′-bipyridine)(4-methyl-4′-carboxybipyridine)-ruthenium N-succinimidyl ester bis(hexafluorophosphate) (RuNHS) onto a semiconductor SnO2 electrode. Upon illumination, the ruthenium complex absorbs photon energy and becomes electronically excited. Injection of the excited electron into the CB of the SnO2 electrode would produce an anodic photocurrent, with the conversion of Ru2+* to Ru3+, which can thermodynamically oxidize guanine (G) and adenine (A) bases in DNA and get reduced back to Ru2+ to participate in the next cycle of photocurrent generation.121,122 2.1.4. Others. Various bulk electrodes, such as gold electrode,149−157 indium tin oxide (ITO) glass,158−161 and TiO2 nanotubes,162 could also be employed directly in the preparation of PEC DNA biosensors. Recently, on the basis of the conducting property of Au NPs or carbon nanostructures, other composites have further been developed to improve the electron capture and transfer of the illuminated semiconductors, such as fullerene/CdSe, carbon nanotube/CdS, carbon nanotube/CdSe, porphyrin/fullerene/Au NPs, CdS/Au NPs, and graphene/CdSe (Figure 5A).27,138 Besides, as shown in Figure 5B, originating from the unique localized surface plasmon resonance (LSPR) induced charge transfer, the

Figure 5. Proposed mechanism of photocurrent generation for (A) graphene/CdSe and (B) Au NPs/TiO2 under visible light irradiation.

combination of noble metal NPs and semiconductor would also lead to the generation of photocurrent, for instance, from Au NPs to TiO2. The Au NPs/TiO2 hybrid material could be developed as photoelectrodes for biosensing.163,164 In general, any material with good photoresponsibility might find its future application in the fabrication of PEC DNA biosensors. 2.2. Probe Immobilization

Various natural (such as chromosomal DNA and plasmid or viral DNA) and synthetic DNA molecules could be used as capture probes. Synthetically fabricated DNA is mainly serving as a probe in PEC DNA biosensors. The use of these synthetic DNAs is comparatively convenient for its commercial availability, and programmable sequencing/end labeling can be conducted during the chemical DNA synthesis step. The end labels are usually involved with the aim to immobilize the probe onto the transducer surface, and the programmed sequences could be designed for the specific targets. In addition to the DNA, other synthetic nucleic acids, such as ribonucleic 7424


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improve transduction of the hybridization signal, an electrode surface may be modified with a polymer layer that confers desirable properties, such as electrical conductivity, amenability to more stable probe immobilization, or protection of the electrode from nonspecific analyte adsorption.7 Cationic conducting polymers are commonly used for the entrapment of DNA in polymeric films. One example, described by Liang et al., was the use of poly(diallyldimethylammonium chloride) (PDDA, a cationic polymer) deposited on the semiconductor surface for the further electrostatic physisorption of dsDNA.122,123,125,129−131,175 Incidentally, DNA might also be entrapped within hybrids produced by the sol−gel techniques which combined the biomolecule with inorganic material.176,177 2.2.3. Covalent Binding. Covalent binding is another frequently used immobilization technique in PEC DNA biosensors. It relies on the fixation of the chemically modified end of the probe DNA onto the activated surface of the support matrix via the formation of covalent bonds. Schiff base formation has been widely used. For example, an ITO electrode was silanized with 3-aminopropyltriethoxysilane (APTS) and then reacted with the aldehyde-modified capture DNA by Schiff base formation for the capture immobilization.158 In a variation on this approach, an APTS silanized (SnO2 NPs modified) ITO electrode could also be utilized to immobilize amine-capped capture DNA (or aptamers) through the bridging of the cross-linking reagent glutaraldehyde.159,160 Another commonly used method is carbodiimide binding, which is based on the fixation of chemically modified DNA onto the activated transducer surface carrying oxidized groups. For example, amino-linked DNA could be covalently bound to the quantum dots modified electrode using the classic coupling reactions between −COOH groups on the surfaces of quantum dots and −NH2 groups of the DNA sequences.137,139,161 Since the phosphonic acids have a high affinity for the TiO2 surface because the phosphate groups are condensed with OH groups of TiO2 by dehydration,178−180 Tokudome and co-workers revealed the directly efficient adsorption of probe DNA onto the TiO2 surface by covalent chemical bonding between phosphate groups of DNA and the OH groups of TiO2 for the PEC DNA assay.132−134 Click chemistry represents another promising strategy for DNA immobilization.181 As compared to the cases of chemisorption or physisorption, the conditions for covalent binding are much more complicated and less mild that some covalent binding routes may cause damages to the bases and hence injure the original recognition ability of the probe. Therefore, as soon as covalent attachment is selected, the choice is limited by the fact that the binding reaction must be performed under tender conditions that do not affect the subsequent hybridization events. However, one obvious merit of the covalent method is that the binding force between the probe and the support matrix is so strong that not only no leakage of the probe occurs but also weakly bound and nonspecific bound molecules could be easily removed. 2.2.4. Affinity Binding. Affinity binding such as the avidin−biotin system has been frequently used in PEC biosensing. In spite of the different variations, it is generally based on the biorecognition between the biotinylated probe molecules and the avidin anchored to the electrodes. Although the affinity binding presents another effective immobilization strategy, it has been seldom utilized in the field of PEC DNA biosensors. Using supramolecular or coordination complexes is another elegant affinity route for the immobilization of bioreceptors on the transducer. For example, Cosnier and co-

acid (RNA), peptide nucleic acid (PNA), locked nucleic acid (LNA), and aptamers could also be employed for special analytical purposes. After the selection of the transducer, probe immobilization is the initial step that plays a key role in the overall performance of PEC DNA biosensors. Controlling the preparation processes and the surface composition and coverage is essential for assuring high reactivity and stability, orientation, and accessibility of the surface-confined probe, as well as for avoiding nonspecific adsorption or binding events. Therefore, experimental conditions usually need to be optimized for the special application at this step. The exact immobilization schemes in general depend on the utilized electrode material, and various approaches typically used at other biosensors could also be employed in the PEC DNA biosensors. Based on the nature of the anchoring process, these strategies for probe anchoring could be divided mainly into four categories, i.e., chemisorption, physisorption (noncovalent binding), covalent binding, and affinity binding. 2.2.1. Chemisorption. The self-assembled adsorption of probe DNA on gold (or silver, platinum, palladium, etc.) tranducers via the Au−S bonding between the electrode surface and DNA molecules with thiol (or other sulfur-containing derivatives) terminated linkers are commonly used as ways of DNA chemisorption.165,166 In PEC DNA biosensing, many works have used thiolated DNA for its convenient immobilization on Au electrode.149−156,167−170 However, the thiolfunctionalized DNA adsorption suffers from the problem of nonspecific binding of nonderivatized DNA sequences. It may be circumvented by using short-chain alkanethiols (e.g., 6mercapto-1-hexanol) to form mixed monolayers of thiolated probe DNA and alkanethiols, the coassembly of which could minimize the undesired nonspecific adsorption of the thiolated DNA.171,172 Hence, chemisorption makes possible the relatively facile and strong single-point anchoring of the probe DNA to the gold electrode while retaining their conformational capability. This chemisorption process of probe DNA immobilization may be controlled by the electric potential. For example, the application of low positive potential (+0.2 V vs SCE) would result in the faster formation of a more compact thiolated ssDNA layer.173 2.2.2. Physisorption (Noncovalent Binding). Physisorption (noncovalent binding) processes are also typically employed in PEC DNA biosensors. The robustness and stability of physisorbed DNA layer usually rely on the utilization and combination of the following characteristics and factors, including the electrode surface charge, the negatively charged phosphate backbone, the structure of DNA, the electrostatic/hydrophobic interactions of the bases, etc.174 Despite the simplicity and conveniences, one main drawback of the direct noncovalent DNA immobilization avenues is that the accessibility of the anchored probe DNA by the target molecule would be impaired due to the close contact of the DNA backbone with the electrode surface, which might cause poor analytical performance of the sensor. By contrast, layer-by-layer electrostatic physisorption offers a convenient alternative for retaining the accessibility of the anchored probe DNA with the virtue of mild experimental conditions. For example, Guo et al. constructed stable DNA films on a SnO2 electrode by the alternate layer-by-layer electrostatic selfassembly approach; i.e., positively charged avidin-Ru complex and negatively charged dsDNA were sequentially immobilized onto the negatively charged SnO2 surface.121 In an effort to 7425


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workers recently reported the use of supramolecular interactions between nitrilotriacetic acid and histidine to anchor histidine-tagged thrombin aptamer for thrombin determination.182 In addition, DNA assembling via affinity binding for probe immobilization may also be useful for advancements in future PEC biosensor development.183 2.3. DNA Interactions

On basis of the chosen probe DNA layer that was immobilized on a particular transducer surface, numerous DNA-related interactions could occur, be studied, and hence used for specific analytical purposes. In general, these interactions can be divided into the five main categories in sections 2.3.1−2.3.5. 2.3.1. DNA Hybridization. DNA hybridization is the process of establishing a noncovalent, sequence-specific interaction between two complementary ssDNA into a hybrid double-stranded DNA (dsDNA) helix. DNA (or RNA etc.) could bind to its counterpart by hydrogen bonds under normal conditions, so two perfectly complementary strands will bind to each other readily. Generally, the pairs of nucleic bases AT (adenosine/thymine base pairing) and CG (cytosine/ guanine base pairing) are formed, of which the latter is more stable. In a DNA hybridization biosensor, probe DNA with known nucleotide sequence is commonly anchored on the transducer surface to serve as the biorecognition element, followed by the interaction with the target DNA in the sample solution. With a sequence matching that of the anchored probe DNA, the target DNA would combine the probe DNA to form a dsDNA duplex. In laboratory practice, the process of DNA pairing to a complementary sequence is usually called “annealing”. The term is often used to describe the binding of a probe DNA or a primer to a DNA strand during a polymerase chain reaction (PCR). Also, the term is frequently used to describe the re-formation of complementary strands that were separated by heat (thermally denatured). In PEC DNA biosensors, various DNA hybridization sensors have also been reported based on different signaling mechanisms, such as using photoactive reporter molecules labeled target DNA,134,161,184 PEC active intercalators binding preferentially to dsDNA,158,160 the photoelectrochemistry of DNA itself,115,132,133 or resonant energy transfer.135,136,140,185 For example, as shown in Figure 6, Zhao et. al investigated the exciton−plasmon interactions between CdS QDs and Ag NPs in a PEC system for DNA hybridization biosensing application.136 In addition to the target DNA quantization, the DNA hybridization event has also been utilized for the PEC assay with other analytical purposes such as methylated DNA detection167 and physiological thiols in cancer cells.137 In fact, the basic principle of DNA hybridization can further be combined with primer extension-based sensors156 or the introduction of molecular beacons (hairpin DNA)133,168,169,184 and even more complicated DNA structures (e.g., triplex, G-quadruplex, i-motif, Z-DNA, and Amotif).141,186−189 The stability of the formed DNA duplex depends strongly on the compatibility between the two strands. One single inconsistency (sequence mismatching) will make binding between the probe DNA and the target DNA less energetically favorable. Therefore, apart from the testing of the nucleotide sequence for the genetic sequencing information, the DNA biosensor could also be utilized to detect gene mutation (sudden and spontaneous hereditable changes in a genomic sequence). Due to the damaging effects that mutations can have

Figure 6. Schematic mechanism of the energy transfer based PEC DNA sensing system. Major processes involve photon absorption and electron excitation from the VB to the CB to generate e−−h+ pair, hole scavenging by electron donor (d), electron transfer (eT) which is collected by the electrode for electronic readout, e−−h+ recombination consisting of nonradiative decay (nD) and radiative decay (rD) with spontaneous emission, and exciton−plasmon interactions between Ag NP and CdS QD. The whole event is transduced into a photocurrent signal for analysis. (Reprinted from ref 136. Copyright 2012 American Chemical Society.)

on genes, advanced detection of gene mutation is currently and will still be an urgent task in the future. The most common principle for mutation detection is based on the fact that differential hybridization responses between the perfectly matched and mismatched duplexes could be produced due to their different stabilities, which lays the groundwork for measuring the effects of base incompatibility under stringent conditions such as elevated temperature, decreased ionic strength, etc. Gao and Tansil showed for the first time the potential of PEC biosensors for PCR-free ultrasensitive DNA hybridization detection.158 With unique properties, the fabricated sensor was capable of one-base-mismatch discrimination, and it was practically indistinguishable from the background noise when two-base mismatch existed, readily allowing selective detection of genes in a complex DNA mixture and discrimination between the perfectly matched and mismatched genes.158 Likewise, Lu and co-workers described the label-free PEC strategy for the hairpin DNA hybridization on a TiO2 electrode, and the specificity experiment showed that one or more base mismatches of target DNA could be discriminated.133 Using photostimulated-hole transporting DNA immobilized on gold electrode, Okamoto et al. also designed and developed a novel PEC single-nucleotide polymorphism (SNP, point mutation) typing method for use as a versatile platform for gene diagnostics.151 7426


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Figure 7. DNA intercalation mode of [Ru(bpy)2(dppz)]2+ with an intercalating part.

Figure 8. Schematic diagram for the DNA intercalators functionalized as (A) signal reporter and (B) electron mediator, with [Ru(bpy)2dppz]2+ and methylene blue as examples, respectively.

DNA intercalator is defined as “small orgnic molecules that unwind DNA in order to π-stack between two base pairs in DNA structure.”191−193 The intercalation takes place when the intercalator with an appropriate size and chemical nature (mostly compounds of a planar structure with polycyclic aromatic rings) fits itself into dsDNA base pairs. To adapt to the intercalation, the dsDNA must dynamically open a space between its base pairs by slight unwinding, the degree of which depends on the intercalating molecule species. Consequently, the intercalation can cause local structural changes to the dsDNA such as lengthening of the DNA helix or twisting of the base pairs, and even long-range deformation of the dsDNA helix structure. These structural alternations may lead to changes of the original dsDNA functionality, often to the inhibition of transcription and replication and DNA repair processes, making intercalators potent mutagens. As a result, DNA intercalators are often carcinogenic, such as the exo (but not the endo) epoxide of aflatoxin B1, acridines such as proflavine, quinacrine, and ethidium bromide. Nevertheless, DNA intercalators are also often used in chemotherapeutic treatment to inhibit DNA replication in rapidly growing cancer cells. Intensively studied DNA intercalators include berberine, ethidium bromide, proflavine, daunomycin, doxorubicin, thalidomide, and acridone derivatives.194 In the biosensing

In opposite to the DNA hybridization, the process by which dsDNA unwinds and separates into ssDNA through the breaking of hydrogen bonding between the bases is defined as DNA denaturation (also called DNA melting), which can be induced by gentle heating, enzymes, physical force, or some chemicals such as urea. Melting occurs preferentially at certain points in the nucleic acid.190 T and A rich sequences are more easily melted than C and G rich regions. Particular base steps are also susceptible to DNA melting, particularly T A and T G base steps. Incidentally, these mechanical features have been reflected by the use of DNA melting in molecular biology techniques such as the PCR, and in some other research works such as measurement of GC content or detection of sequence differences. 2.3.2. DNA Association with Small Molecules. The investigation of DNA association with small molecules such as drugs and chemicals is usually of importance in disease diagnosis and drug discovery. Noncovalently, these molecules (in this case, also known as ligands) can interact with DNA via three main types, i.e., intercalation (section 2.3.2.1), groove binding (section 2.3.2.2), and electrostatic interaction (section 2.3.2.3). 2.3.2.1. Intercalation. Intercalation represents the insertion of intercalator into the stacked base pair of the dsDNA. The 7427


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Figure 9. (A) Schematic diagram of the PEC biosensor construction process. (B) Photocurrent generation and decrease mechanism of the PEC biosensor. (Reprinted with permission from ref 170. Copyright 2014 Elsevier.)

field, methylene blue (MB) and anthraquinone are two model intercalator labels.7,12 Meanwhile, DNA intercalator labels could also involve transition metal complexes, namely metallointercalators.195,196 For instance, as shown in Figure 7, [Ru(bpy)2(dppz)]2+ (dppz = dipyrido[3,2-a:2′,3′-c]phenazine) binds to DNA avidly in intercalation mode (dppz ligand has the highest binding constant of 106−107 M−1 to date).192,193 In most cases of PEC DNA biosensors, these intercalators are recruited for two purposes, i.e., either use the PEC active species as signal reporter to reveal the biorecognition events, or use the redox active species as mediators to shuttle electron along the DNA double helix (Figure 8). On one hand, after the intercalation of the PEC active indicators binding preferentially to dsDNA, the illumination of the electrodes could generate the corresponding photocurrents to monitor the DNA hybridization or DNA damage. To be used as a PEC signal reporter, besides the effective differentiation between ssDNA and dsDNA, the indicators should possess well-defined, preferably visible-light-activated, higher photon-to-current conversion ability. Such properties are of importance for attaining high sensitivity and selectivity. For example, [Ru(bpy)2dppz]2+ has often been used as an effective photoactive intercalator in DNA hybridization,114,149,160 DNA damage,121,122 and aptasensing159 as well as for metal ion sensing.131 To improve differentiation between ssDNA and dsDNA, bis-intercalators or threading intercalators, which form thermodynamically or kinetically more stable complexes with dsDNA than the simple intercalators, have also been applied for PEC DNA detection. A case in point is that a photoactive threading bis-intercalator consisting of two N,N′-bis(3-propylimidazole)-1,4,5,8-naphthalenediimides (PIND) linked by a Ru(bpy)22+ complex (PIND−

Ru−PIND) was synthesized for ultrasensitive DNA detection.158 In some alternative sensor configurations, photocurrent signals are generated by labeled semiconductor NPs rather than the photoactive intercalators. Regretfully, DNA usually exhibits poor conductivity197 and hence acts as an inefficient charge transporter for the CB electrons. Therefore, as shown in Figure 8B, redox-active intercalators (e.g., doxorubicin and methylene blue) are usually incorporated into the dsDNA to improve the conductivity of DNA, thus facilitating the electron transport through its helix structure.153,154,156 2.3.2.2. Groove Binding. Groove binding means that the binding molecules bind to the grooves of dsDNA. Two sugar− phosphate backbones form the helical structure of the dsDNA, producing another double helix of spaces (grooves) between the backbones. Because the strands are not directly opposite each other, the grooves are unequally sized, and the major and minor grooves are named to reflect their differences in size. These voids are adjacent to the base pairs and may provide a binding site, for proteins (as will be mentioned in following sections) or small binding molecules (such as [Ru(NH3)5Cl]2+),158 which usually hold themselves in the groove through hydrogen-bonding and van der Waals interactions. 2.3.2.3. Electrostatic Interaction. Electrostatic interaction occurs between the negatively charged DNA phosphate backbone and the positively charged guest molecules. For example, the cationic complexes [Co(byp)3]2+, [Ru(byp)3]2+, and [Ru(NH3)6]2+ are considered as electrostatic DNA binders that bind to the phosphate backbone. Just like the function of redox active intercalators to mediate electron transfer, electrostatic binding, for example, [Ru(byp)3]2+ binding to the dsDNA, could also provide the tunneling routes for the CB 7428


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methylation event occurred at the site of 5′-CG-3′ and could be probed by MBD1 protein, which could combine tightly with methylated cytosine, causing a decreased photocurrent due to the hindrance toward the electron donor.170 2.3.3.2. DNA-Modifying Enzymes. The common DNAmodifying enzymes include nucleases, ligases, topoisomerases, helicases, and polymerases. Nucleases cut DNA strands by catalyzing the hydrolysis of the phosphodiester bonds. The nucleases cut from the ends of or within the strands are called exonucleases and endonucleases, respectively. In molecular biology, the most frequently used nucleases are the restriction endonucleases that cut DNA at specific sequences. In DNA biosensors, the use of various sequence-specific nucleases has been well documented.203 Ligases are a specific type of enzymes that can reassemble the broken DNA strands. This means that they repair single-stranded discontinuities in dsDNA molecules. These ligases have applications particularly in DNA replication and DNA repair, and have extensive use in molecular biology laboratories for genetic recombination. Acting on the topology of DNA, the topoisomerases, with both nuclease and ligase activity, regulate the overwinding or underwinding of DNA. For example, in order to overcome the topological problems such as supercoiling in DNA replication caused by the intertwined nature of the double helix, topoisomerases would bind to either ssDNA or dsDNA, cutting the phosphate backbone of the DNA to allow the DNA to be untangled or unwound, and reseal the DNA break again at the end of these processes. Topoisomerases are required for many processes such as DNA replication and transcription. Helicases are a class of molecular motor proteins that are essential for most processes where enzymes need to access the DNA bases. Using the chemical energy derived predominantly from adenosine triphosphate (ATP) hydrolysis, they move directionally along a DNA phosphodiester backbone to break hydrogen bonds between bases and separate the annealed DNA double helix into single strands. Polymerases are enzymes that synthesize polynucleotide chains from nucleoside triphosphates. According to the templates of existing polynucleotide chains and using basepairing interactions, all the polymerases function in a 5′ to 3′ direction by adding nucleotides onto the 3′ hydroxyl group of the previous nucleotide in a DNA strand. They are particularly important in the processes of DNA transcription and replication. Taking advantage of the DNA-modifying enzymes, different laboratories are beginning to exploit the novel strategies for PEC DNA-related sensing. One illustration, described by Willner et al. as in Figure 10, is the construction of a DNA machine and hence self-assembly of semiconductor QDs on electrodes for PEC biosensing. The operating system was composed of a DNA track (a), including a recognition sequence (I), a nicking sequence (II), and a reporter sequence (III). In the presence of polymerase/dNTPs and the nicking enzyme Nb.BbvcI, the DNA machine was activated upon hybridization of (a) and the analyte (b). The subsequent scission of the resulting dsDNA could cause the displacement of ssDNA (c), which would then bridge the CdS QDs to the electrode surface via the hybridization with ssDNA (d) and (e), complementary to the 3′ and 5′ ends of (c), respectively. The self-assembly of the CdS QDs on the electrode would lead to the generation of photocurrent signal.26,156 In yet another innovative application of the DNA-modifying enzymes, Yamada and colleagues used restriction enzymes of HapII and HhaI to develop a PEC approach for the

electrons and thus lead to enhanced photocurrents in PEC DNA sensors.153 Note that the above-mentioned three main modes can be integrated under certain experimental conditions. For example, the dsDNA interaction with a positively charged metal complex with aromatic ligands may involve the intercalation and electrostatic binding simultaneously. Besides these noncovalent ways, some substances may interact with DNA by covalent binding. For instance, some compounds, especially drugs, could combine with DNA bases via covalent bonds to create adducts. Until now, few works have been directed toward the direct PEC detection of small target molecules via the DNA association. Very recently, o-aminophenol (OAP) was detected using a PEC DNA biosensor based on the interaction between DNA and OAP. It was believed that the adsorbed OAP molecules on the photoanode could be quickly oxidized by the photogenerated holes from the CdSe QDs under visible light irradiation.175 In addition, related DNA, such as C−C or T−T mismatches and G quadruplexes that bind metal ions, has also been utilized for PEC metal ion sensing.131,187 2.3.3. DNA Interactions with Proteins. All the functions of DNA rely on its interactions with various proteins. Some proteins can nonspecifically or specifically bind to DNA. Enzymes can also bind to DNA for the fulfillment of their biological functions. Therefore, regarding DNA interactions with proteins, DNA biosensors mainly focus on two aspects. The first concerns the affinity binding of DNA and proteins (section 2.3.3.1), and the second is the employment of the DNA-modifying enzymes (section 2.3.3.2) for the enzymatic DNA conversions in the proposed bioassay formats, or the direct detection of their catalytic activities . 2.3.3.1. DNA-Binding Proteins. Among the numerous DNAbinding proteins, a distinct group is those binding to particular DNA sequences. The most intensively studied of these are the various transcription factors, which are proteins that regulate transcription. Each transcription factor binds to one particular set of DNA sequences and activates or inhibits the transcription of genes that have these sequences close to their promoters.198 Another group of DNA-binding proteins has evolved to bind specifically to ssDNA, which seem to stabilize ssDNA and protect it from forming stem loops or being degraded by nucleases. A case in point of these ssDNA-binding proteins is replication protein A, which takes effect when the double helix is separated, including DNA replication, recombination, and DNA repair.199 In contrast, some proteins bind DNA via nonspecific DNA−protein interactions. For instance, in chromosomes, DNA is held in complexes with structural proteins, which are typically well-understood examples. The nonspecific DNA−protein interactions are usually formed via the ionic bonds between the basic residues in the histones and the acidic sugar−phosphate backbone of the DNA, and hence are largely independent of the base sequence.200 Other nonspecific DNA-binding proteins in chromatin include the high-mobility group of proteins, which are capable of binding to bent or distorted DNA.201 These proteins are essential in the processes of bending arrays of nucleosomes and arranging them into the larger structures that make up chromosomes.202 Figure 9 demonstrates a typical example reported by Ai et. al using methyl binding domain protein (MBD1protein) to develop a novel PEC biosensor for DNA methyltransferase (MTase) activity assay. In this work, M.SssI MTaseas and MBD1 protein were using as the methylation reagent and methylation recognition element, respectively. The cytosine 7429


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Figure 11. (A) PEC protocol for the discrimination of C and mC with the use of restriction enzyme. (B) Photocurrent responses of the Ccontaining duplex (left) and the mC-containing duplex (right). The duplex was enzymatically treated with 1 unit of HapII at 23 °C for 1 h on the electrode. (Reprinted with permission from ref 204. Copyright 2008 Royal Society of Chemistry.)

2.3.4. DNA Aptamer−Target Interactions. DNA (or RNA) aptamers are single-stranded nucleic acid species that are created by repeated rounds of in vitro selection from a large random sequence pool or, equivalently, SELEX (Systematic Evolution of Ligands by EXponential enrichment) to bind specifically to various molecular targets, such as small molecules, proteins, nucleic acids, and even cells, tissues, and organisms. The specific binding affinity of these artificial oligonucleotides makes them desirable in biosensor development because they offer molecular recognition properties that rival that of the commonly used biomolecules, antibodies. For instance, in the case of protein targets, the DNA aptamers could bind their targets with dissociation constants (Kd’s) lower into the picomolar level, discriminating among similar proteins with common sets of structural domains. Besides the high ability and molecular discrimination, aptamers offer a number of advantages over antibodies; for example, they can be engineered completely in a test tube, they can be costeffectively produced by chemical synthesis, they possess desirable storage properties and thermal stability, and they elicit little or no immunogenicity. The design of aptasensors is mainly based on the inherent recognition affinities of the aptamers to specific targets. In a typical assay, Golub et al. presented the first PEC aptasensor for the detection of cocaine using supramolecular aptamer complex and CdS NPs.155 In this study, as shown in Figure 12, one anticocaine aptamer subunit was assembled on a Au transducer, while the second aptamer subunit was labeled with CdS NPs as PEC signal reporter. Then, the addition of cocaine resulted in the formation of supramolecular complexes between the CdS-labeled aptamer subunits and cocaine on the

Figure 10. (A) PEC protocol for the construction of a DNA machine with the use of polymerase and nicking enzyme. (B) Experimental photocurrents observed (a) in the absence and (b) in the presence of the target DNA (1 × 10−6 M). The DNA machine was operated for 90 min. (Reprinted with permission from ref 26. Copyright 2008 WileyVCH Verlag GmbH.)

determination of the methylation status of C bases in DNA (Figure 11). In this example, anthraquinone photosensitizertethered dsDNA bearing 5-methylcytosine (mC) or the normal C at a restriction site of the DNA strands was immobilized on gold electrodes. Under irradiation, the photocurrent responses could be observed due to the photosensitizer-injected hole transfer properties of DNA. Upon enzymatic digestion, the duplexes containing normal C at the restriction site would suffer strand cleavage and hence the elimination of photosensitizer unit from the strand, leading to a significant suppression of the photocurrent response. In contrast, upon similar treatment, the duplex bearing mC at the target site did not experience such an enzymatic digestion and therefore the photocurrent response was preserved. The different photocurrent densities demonstrated that the methylation status of C could be detected by monitoring the photocurrent response in conjunction with restriction enzyme treatment.206 Very recently, using Klenow polymerase, human alkyladenine DNA glycosylase, and apurinic/apyrimidinic endonuclease, two recent strategies have been proposed for PEC DNA hybridization detection184 and PEC DNA methylation detection, respectively.130 7430


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radiation frequencies including X-rays and γ rays. Oxidative DNA damage may lead to aging cancer and mutagenesis.206 The main types of DNA damage caused by endogenous cellular processes include the following: oxidation of bases and interruptions of DNA strand (strand breaks) from reactive oxygen species; alkylation of bases; hydrolysis of bases; formation of various adducts (lesions); mismatch of bases. Damage induced by exogenous agents comes in many forms, such as the direct DNA damage of cross-linking between adjacent cytosine and thymine bases caused by UV-B light, the indirect DNA damage caused by free radicals generated by UVA light, irreparable DNA damage or DNA strand breaks caused by ionizing radiation, and a huge diversity of DNA adducts caused by industrial chemicals (e.g., vinyl chloride and hydrogen peroxide) and environmental chemicals (e.g., polycyclic hydrocarbons). The vast majority of DNA damage affects the primary structure of the double helix, and the unrepaired lesions in critical genes (such as tumor suppressor genes) can impede crucial cellular functions, lead to mutations, and appreciably increase the likelihood of tumor formation. The occurrence of DNA damage (leading to chemical, physicochemical, and structural property changes) could be monitored by its behavior at the transducer surfaces, on the basis of which different techniques have been developed aiming at DNA damage detection. In the direction of the PEC method, through recording the photocurrent alternation, PEC DNA biosensors allow for not only detecting, but also inducing, DNA damage at the electrode surface via PEC generation of the damaging (usually oxidative holes) species (see section 2.4).

Figure 12. (A) PEC analysis of cocaine through the self-assembly of supramolecular complexes of CdS-NPs-functionalized aptamer subunits and Au-surface functionalized with the second aptamer subunit in the presence of cocaine. (B) Photocurrent action spectra generated by various concentrations of cocaine: (a) in the absence of cocaine and in the presence of (b) 1 × 10−6, (c) 1 × 10−5, (d) 1 × 10−4, and (e) 1 × 10−3 M cocaine. (inset) Derived calibration curve with a detection limit of 1 × 10−6 M. (Reprinted from ref 155. Copyright 2009 American Chemical Society.)

2.4. PEC Transduction of DNA Interactions

The PEC transduction of DNA interactions is commonly accomplished via the altered photocurrent signal, which originates from the photoelectrochemistry of the nucleic acid, or from the introduced PEC responsive indicator or a detectable label, or from changes in other parameters that would influence the photocurrent generation resulting from the biorecognition events. 2.4.1. Signal On and Signal Off. The PEC DNA biosensors generate two types of current responses, i.e., “signal on” and “signal off”. While the former is based on the emergence and enhancement of a photocurrent, the latter is based on the decrement of the signal prior to and after the molecular biorecognition. Both the “signal-on” and “signal-off” strategies could be used for the detection of hybridization events or DNA damage. Take two PEC DNA damage sensors, for example; in the “signal-on” sensor configuration, the damaged DNA on the sensor bound less Ru(bpy)2(dppz)2+ than the intact DNA, resulting in a drop in photocurrent. In the “signal-off” configuration, ruthenium tris(bipyridine) was employed as the indicator that immobilized underneath the DNA layer on the electrode. After oxidative damage, the DNA bases became more accessible to PEC oxidation than the intact DNA, producing a rise in photocurrent.122 Generally speaking, the biosensors built on the “signal-on” routes possess better analytical performance than those on the “signal-off” avenues, which is mainly because of the strong background responses with the “signal-off” biosensors. 2.4.2. DNA Photoelectrochemistry. DNA is photoelectrochemically active due to the presence of photoelectrochemically oxidizable components: the four nucleobases (oxidation potential from low to high is G, A, T, and C) and sugar residues. Among these components, oxidation occurs

electrode surface, allowing the quantitative analysis of cocaine. In the presence of triethanolamine as electron donor, the supramolecular CdS NPs aptamer subunits−cocaine complex would allow for PEC detection of cocaine through the photocurrent changes. The major advantage of this aptasensing platform was the lack of background interfering signals, enabling the analysis of cocaine with a detection limit of 1 × 10−6 M.155 The detection methods of PEC aptasensors as well as the application scope can be expanded accordingly. Recently, Zhang and colleagues have also reported PEC aptasensors for adenosine triphosphate,159 thrombin,138 and Ramos cells.139 Zhao and co-workers constructed a visible-light-driven PEC aptasensor for bisphenol A.162 Cosnier reported the label-free PEC aptasensor toward thrombin.182 These aptasensor configurations could be extended to analyze other lowmolecular-weight substrates or proteins. 2.3.5. DNA Damage. DNA damage means any change in the chemical structure of the genetic material caused by interactions with chemical or physical agents that come from the environment or normal metabolic processes inside the cell.205 DNA damage can be subdivided into two main types, i.e., endogenous damage and exogenous damage. Whereas endogenous damages are due to attack by reactive oxygen species produced from normal metabolic byproducts (spontaneous mutation), especially the process of oxidative deamination, exogenous damages are caused by external factors such as viruses, certain plant toxins, human-made mutagenic chemicals (especially aromatic compounds that act as DNA intercalating agents), cancer chemotherapy and radiotherapy, and ultraviolet radiation (200−300 nm) from the sun or other 7431


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Figure 13. (A) Schematic illustration of the label-free PEC DNA hybridization detection processes with hairpin DNA and TiO2 NPs as the probe and signal transducer, respectively. (B) Spectrum dependence of photocurrent for different electrodes in PBS buffer (pH 7.0): (a) TiO2 electrode; (b) 10 μL, 500 nM probe hairpin DNA modified TiO2; and (c) after hybridization with 10 μL, 500 nM complementary target DNA. (C) Dependence plot of photocurrent change vs target DNA concentration, wherein filled squares and solid line are experimental data and fitting line, respectively. (Reprinted with permission from ref 133. Copyright 2006 American Institute of Physics.)

bulges greatly diminish the yields of DNA-mediated charge transport.219 The unnoticeable photocurrents observed with CdS QDs separated by dsDNA from Au electrode might support the insulating features of DNA (the photocurrents were attributed to a small fraction of CdS QDs that were in close contact with the electrode), and suggest the duplex DNA acted as an inefficient charge carrier for the CB electrons and hence behaved as a barrier for the photocurrent generation, which phenomena were consistent with the general conclusion. As mentioned above, yet the ability to intercalate (or electrostatically bind) redoxactive components into duplex DNA could provide tunneling routes for the CB electrons and consequently result in enhanced photocurrents. Based on the insulating nature of DNA, many works have been done correspondingly with the coupling of semiconductor QDs. In contrast, some scientists supported the long-distance charge migration through DNA and explained the process by a multistep hopping mechanism. Experimentally, Giese and colleagues developed a method for site selective oxidation of a single G and for the detection of the charge transfer between the G by incorporating into DNA strands with the 4′pivaloylated nucleotides, the photolysis of which will lead to the formation of carbohydrate radical cations to quantitatively oxidize a G that is situated at its anomeric carbon atom. It was found that the injected positive charge could migrate in a multistep hopping reaction through DNA over long distances and the presence of mismatch could cause a dramatic decrease of the efficiency of the charge transport.220 Alternatively, the DNA sequence could also be labeled with photosensitizer. For example, naphthalimide and phenothiazine (which worked as electron-acceptor and -donor molecules, respectively) were labeled onto DNA to observe the long-distance hole transfer in

most readily at G residues due to its lowest oxidation potential of 1.06 V versus SCE at pH 7 at a mercury electrode, and the major initial product of G oxidation is the highly mutagenic 8oxoguanine, a clinical biomarker for oxidative stress. G is more easily oxidized in ssDNA than in dsDNA because of the better accessibility by oxidizing agents or electrodes.206 As to the charge transferability through DNA, it has been a subject of controversial scientific debates since the discovery of the double helix.207,208 Berg proposed the electron-transfer mechanism of DNA electrochemistry that DNA may have some semiconductor-like characteristics and that the electron migrated by “hopping” from base to base.209,210 On account of this, the photoelectrochemistry of DNA may possibly throw new light on the DNA conducting property.211 Barton et al. later reported their exciting discovery that long distance photoinduced electron transfer (>40 Å) may be mediated by the DNA helix which was described as “molecular wire”;212 however, the photoinduced electron transfer between reactants bound to DNA, or between bases contained within the π stack, has led to different conclusions regarding the nature of DNA as a medium for long-range charge transport.213−215 In addition, based on the DNA photoelectrochemistry itself, PEC detection of DNA damage216 and DNA hybridization115 were then reported. Whereas several studies claimed that DNA acts as a conducting matrix,217 most results suggested that DNA with random, nonspecific base sequences exhibited poor conductivity.197 Consistently, nonetheless, the conductivity of DNA could be improved by appropriate ordering of the base sequences218 or by the incorporation of redox-active species into the helix.7 Also, the integrity of the base stack itself appeared indeed essential for efficient long-range electron transfer, as perturbations caused by intervening mismatches or 7432


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Figure 14. (A) Schematic diagram illustrating the label-free PEC detection of DNA damage caused by direct base oxidation. (B) (top) Photocurrent of the SnO2/avidin-Ru/dsDNA film (corresponding to scheme A) as a function of the reaction time in (a) 2% styrene oxide and (b) 20 mM phosphate buffer. (bottom) Photocurrent response of the electrodes incubated for 3 h in (a) 2% styrene oxide and (b) 20 mM phosphate buffer. (C) (top) Photocurrent of the electrodes (corresponding to scheme A) as a function of the reaction time in (a) 1 mM FeSO4/4 mM H2O2, (b) 1 mM FeSO4, and (c) 4 mM H2O2. (bottom) Photocurrent response of the electrodes incubated for 1 h in (a) 1 mM FeSO4/4 mM H2O2, (b) 1 mM FeSO4, and (c) 4 mM H2O2. (D) Schematic diagram illustrating the label-free PEC detection of DNA damage caused by in situ enzyme-catalyzed, metal-induced reaction. (E) (left) Photocurrent responses of the SnO2/avidin-Ru/dsDNA/PDDA/GOx film (corresponding to scheme D) after reaction for 3 h in (a) 1 mM FeSO4/50 mM glucose, (b) 1 mM FeSO4, and (c) 50 mM glucose. (right) Photocurrent as a function of the reaction time in (a) 1 mM FeSO4/50 mM glucose, (b) 1 mM FeSO4, and (c) 50 mM glucose. (Reprinted from refs 121 and 122. Copyright 2007 and 2008 American Chemical Society.)

dsDNA.221 Two chromophores of SSL16ECz and Rul16SS were labeled to two types of helical peptides for the construction of a molecular photodiode system.222 Naphthalimide-labeled DNA has been used for studying the relationship between charge transfer and charge recombination in DNA film.223,224 Anthraquinone-tethered DNA was also used to initiate the photostimulated hole transport and a sequencedependent cathodic photocurrent was observed, indicating DNA can serve as a good mediator for cathodic photocurrent when an appropriate sequence is selected. Based on this, the PEC SNP typing, the PEC evaluation of alternating duplex− triplex conversion effect, and the PEC investigation of the enzymatic treatment on the dsDNA were reported.150−152,204 In addition to those works relying on the DNA conductibility, many works had been performed based on the PEC oxidation of DNA directly. Although all the components could in principle be oxidized under certain PEC conditions, only the oxidation of the G and A moieties at the semiconductor electrode has been widely selected by the reported label-free PEC DNA biosensors as the source of detection signals for specific analytical purposes. Incidentally, DNA damage has the effect of unwinding the double helix; thus a closer access to these nucleobases is permitted. As we discuss later, the direct oxidation of the G moiety by the TiO2 photoelectrode has been used for the reagentless detection of DNA hybridization.133 Methods to oxidize target DNA indirectly through the use of PEC mediators have also been explored. An especially attractive approach uses ruthenium

polypyridine complex to mediate the PEC oxidation of G. For instance, Liang et al. used ruthenium polypyridine complex not only as a PEC signal reporter but also as redox mediators to shuttle electrons from G to the SnO2 electrode for the detection of DNA damage.143 2.4.3. Label-Free Techniques. Because of the presence of oxidizable component, perhaps the simplest and most straightforward PEC DNA sensing strategy was the direct oxidation of DNA (especially G residues) at a semiconductor electrode as the source of analytical signals. Obviously, the amount of DNA oxidized would reflect the amount of DNA captured or damaged; hence such direct, in situ label-free detection can be achieved by monitoring the intrinsic responses of the target DNA. Such protocols greatly simplify the sensing schemes. Using this method, Lu and co-workers developed the labelfree strategy for PEC detection of DNA hybridization on TiO2 electrode. As shown in Figure 13, hairpin DNA and TiO2 NPs served as the probe and signal transducer, respectively. The detection process can be elucidated as follows: (a) Under illumination, electron and hole pairs were generated in TiO2 and the G of the ssDNA correspondingly served as a hole scavenger. The interfacial electron transfer then enhanced the charge separation and decreased electron−hole recombination, resulting in the significant increase of photocurrent. (b) Once the hybridization between probe DNA and target DNA occurred, the charge transfer rate was inhibited; meanwhile the G cannot interact with the oxidizer directly due to the steric 7433


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complex would transfer an electron to quencher molecules in solution, with the oxidized complex subsequently reduced at the electrode, generating photocurrent. The idea of using Rubpy as a PEC indicator in bioassays was also proposed by the authors, but no experiments were done.225 In a first attempt to detect DNA in solution by the PEC method, Pandey and Weetall used anthraquinone as the indicator. Upon irradiation, photoexcited anthraquinone would be reduced by an electron donor in solution, with the reduced, ground-state indicator then being detected at a modified graphite carbon electrode at an oxidizing potential.226 As mentioned above, such indicators are usually small PEC active DNA intercalators that possess a much higher affinity for the resulting dsDNA than for the ssDNA. Accordingly, the concentrations of the indicators at the electrode surface could be utilized to reflect the recognition events via the altered photocurrent response. Apparently, the simplest application of these indicators is for selective discrimination of the dsDNA on the basis of their effective differentiation between ssDNA and dsDNA. For example, on the basis of the concentration of metallointercalator Ru(bpy)2dppz caused by the formation of dsDNA, Nakamura et al. discriminated between dsDNA and ssDNA from the magnitude of the photocurrent on a gold electrode surface. The authors proposed the photocurrent generation mechanism of an electron-transfer process from the photoexcited Ru(II) to the electrode and subsequent reduction of Ru(III) by a reducing agent in solution.149 Besides the application in the classical biosensor concept (DNA modified electrode), the Ru-dppz complex has also been applied in the solution-based approaches. Liu and co-workers coated SnO2 NPs on ITO electrode for DNA detection in solution along with [Ru(bpy)2(dppz)]2+. When [Ru(bpy)2(dppz)]2+ alone was irradiated, anodic photocurrent was produced on the semiconductor electrode due to electron injection from its excited state into the CB of the electrode. After the addition of calf thymus dsDNA into the solution, the photocurrent dropped substantially. The drop was attributed to the intercalation of [Ru(bpy)2(dppz)]2+ into the helix of the dsDNA and hence the slower mass diffusion of the indicator to the electrode, and electrostatic repulsion between oxalate anion and negative charges on DNA. However, ssDNA had little effect on the photocurrent. The method could be used to selectively detect dsDNA with the detection limit of 1.8 × 10−10 M. Owing to the coupling of semiconductor electrode and the photoresponsive DNA intercalator, this analytical system possesses many attributes of the high quantum yield dyesensitized solar cell, such as high electrode surface area, optimal match of energy level between indicator and SnO2 CB edge, fast charge separation on semiconductor electrode, and recycling of the indicator.114 Advanced detection protocols have also been developed for ultrasensitive detection of DNA hybridization or aptamer− target interaction. Just like the signaling mechanism shown in Figure 8A, Gao and Tansil reported a simple PEC detection of target DNA by forming a DNA/photoreporter adduct layer on an ITO electrode. A monolayer of DNA capture probes was immobilized onto the ITO electrode surface through chemical coupling. The target ssDNA was then hybridized with immobilized capture probes on the ITO electrode, followed by the binding of photoactive threading bis-intercalator PIND− Ru−PIND to allow for the PEC detection of the target DNA. The authors proposed the mechanism of photoinduced charge separation and recombination for the photocurrent generation.

hindrance in the DNA helix, which diminished its hole trapping capacity and thus led to the lower photocurrent. This photocurrent decrease can be utilized to identify the sequence of the target DNA, and the lowest detected target concentration was found to be approximately 2.5 nM.133 When the probe DNA was equipped with Au NPs, the sensitivity and the detection limit for the target DNA could further be improved significantly. This was because the presence of Au NPs could increase the electron−hole separation efficiency and enhance the photocurrent of TiO2/ ssDNA. Hence, the degree of the photocurrent decrease was greater when Au NPs labeled probe DNA was used. The lowest detected target concentration was found at 1 nM in this experiment.132 This label-free methodology has also been applied in the PEC detection of DNA damage. Obviously, the simplest application of this method is for the discrimination between damaged DNA and intact DNA. Based on the indirect oxidization of G (or A) in DNA, Liang et al. first reported the differentiation of chemically damaged dsDNA and normal dsDNA. It was found that different photocurrents could be generated in the presence of normal or damaged dsDNA. Compared with the former, the photocurrent response of damaged DNA increased significantly.143 On the other hand, the change of the photocurrent relative to its intensity yielded by intact DNA could represent the response to damage to DNA. Accordingly, these authors later reported a rapid and in situ method for the PEC detection of DNA damage.121 As shown in Figure 14A, a Ru complex labeled avidin (avidin-Ru) film and a dsDNA film were assembled successively on tin oxide NP film electrodes. Photogenerated Ru(III) oxidized G and A bases in DNA and gave rise to photocurrent. DNA damage was detected after the reaction of DNA film with styrene oxide or Fenton reaction (Fe2+/H2O2), which exposed more DNA bases for photooxidation and resulted in increased photocurrent. Later, as shown in Figure 14D, the glucose oxidase was assembled onto the electrode for the development of the first metal-induced, enzyme-catalyzed DNA damage biosensor.122 In the presence of glucose, the glucose oxidase catalyzed the in situ formation of H2O2, which then reacted with Fe2+ and generated hydroxyl radicals by the Fenton reaction. These radicals would attack DNA in the sensor film, mimicking metal toxicity pathways in vivo. Similarly, after oxidative damage, the DNA bases became more accessible to PEC oxidation than the intact DNA, producing a rise in photocurrent intensity. As mentioned in section 2.3, the PEC aptasensor (which is also label-free in the sense of no chemical modification of targets) has been developed due to the highly specific binding affinity of aptamers for various targets. Besides cocaine and ATP, thrombin and Ramos cells have also been detected using the corresponding PEC aptasensor. The folding of aptamers around the target would lead to enhanced steric hindrances and hence prevent the electron donor from scavenging photogenerated holes on the photoactive film, offering the opportunity for PEC detection.138,139,182 2.4.4. Indicator-Based Techniques. Motivated by the limitations of the DNA’s substantial photoelectroactivity-based routes, many works have relied on the use of PEC active species serving as PEC signal indicators of the DNA-related recognition events at the electrode surface. Weber et al. first reported the PEC detection of ruthenium tris(bipyridine) (Ru-bpy) on a glassy carbon electrode. Under photoexcitation, the ruthenium 7434


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Figure 15. Schematic diagram illustrating the indicator-based PEC detection of DNA damage caused by (A) direct base oxidation and (B) in situ enzyme-catalyzed, metal-induced reaction. (Reprinted from refs 121 and 122. Copyright 2007 and 2008 American Chemical Society.)

measured photocurrent.122 Using the developed PEC DNA damage sensor, the detection of the halogenated benzoquinone125 and polystyrene nanosphere suspension127 caused DNA damage was then reported. Very recently, they further exploited the use of [Ru(bpy)2(dppz)]2+ as indicator for selective PEC detection of chemical DNA methylation damage and Hg2+ in aqueous solutions.130,131 2.4.5. Label-Based Techniques. The signal of a PEC DNA biosensor can be read out in a label-free or indicatorbased manner. As an alternative, covalent labeling represents another useful signaling route. In this approach, the DNA hybridization events are signaled by the introduced129,167 or distanced184 covalent labels (tracer), the presence of which could generate (or influence) the photocurrent signal to get analytically useful responses. By analogy to electrochemical-based techniques, some strategies have been pursued in which target DNA is labeled with photoactive reporter (e.g., dye molecules or CdS NPs). Appearance of the characteristic PEC response of these reporters therefore follows the biorecognition process. In a typical example, as shown in Figure 16, rhodamine B dye labeled target DNA was used to hybridize with the probe DNA immobilized on the nanostructured TiO2 electrode. Light irradiation would generate electrons in the dye molecules, followed by the injection of these electrons into the TiO2 electrode to produce photocurrent, which can be measured corresponding to the concentration of target DNA. This sensor can quantitatively detect target DNA as low as 100 pM.134 As shown in Figure 17, by modifying a dsDNA (of an appropriate sequence) with a photosensitizer (anthraquinone), Okamoto and co-workers observed a sequence-dependent cathodic

The extremely low dissociation rate of the adduct and the highly reversible PEC response under visible light illumination made it possible to perform the ultrasensitive DNA detection with a detection limit of ∼20 fM, a 104-fold sensitivity enhancement over voltammetry. The work demonstrated for the first time the potential of PEC biosensors for PCR-free ultrasensitive DNA detection.158 Similarly, based on the final intercalating of the photosensitizer [Ru(bpy)2(dppz)]2+ into the formed helix of dsDNA on the ITO electrode surface as signal reporter, Zhang and colleagues developed a new PEC DNA biosensor and the target DNA could be detected at 4.5 × 10−9 M without any amplification process.160 These photoactive intercalators have also been applied as indicators of DNA damage (to discriminate intact dsDNA from disrupted DNA that has lost its double-helix structure). For example, Guo et al. used [Ru(bpy)2(dppz)]2+ as a PEC signal reporter to detect the DNA damage. As shown in Figure 15A, the DNA damage was detected by monitoring the change of photocurrent of the indicator. Whereas the DNA damage induced by styrene oxide would lead to the decrease of intercalation sites, the DNA damage induced by the Fenton reaction would cause DNA cleavage at almost every nucleotide site, leading to base loss, chain breakage, and base oxidation. Under both circumstances, the damaged DNA film bound less indicator than the intact DNA, resulting in a drop in photocurrent.121,123 Based on the same signaling mechanism, as shown in Figure 15B, these authors subsequently reported the indicator-based method for the rapid detection of in situ DNA damage induced by the enzyme-catalyzed Fenton reaction. Likewise, the intercalator bound less to the damaged DNA than the native form, leading to a reduction in the 7435


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Figure 16. (A) Scheme of label-based PEC DNA detection for hybridization. (B) Photocurrent versus target DNA concentration. Probe DNA: 5′-AACGTCGTGACTGGG-3′. Filled diamonds, target DNA dye-3′ TTGCAGCACTGACCC-5′; open triangle, target DNA 3′-TTGCAGCACTGACCC-dye-5′; open squares, target DNA 3′-dyeTTTTTTTTTTTTTTT-5′. Insets: (a) time course of current; (b) photocurrent at low DNA concentrations. (Reprinted with permission from ref 134. Copyright 2005 American Institute of Physics.)

photocurrent. The photosensitizer equipped DNA showed an SNP-specific current through photostimulated long-range hole transport and hence could be used for accurate and sensitive PEC SNP typing assay.150,151 In yet another innovative application of this approach, CdS QDs were used as a signal reporter to provide PEC detection of DNA hybridization. Although these QDs lacked contact with the electrode owing to their separation by the DNA tethers, the intercalation of doxorubicin into the dsDNA provided the charge transfer relay units for the CB electrons from the photoexcited CdS particles.154,156 In a DNA-cross-linked CdS NPs array architecture, the association of charge-transfer mediators [Ru(NH3)6]3+ to the DNA array would facilitate the hopping of CB electrons from CdS NPs and enhance the resulting photocurrent.153 Such electrical contact of the photoexcited QDs with the electrode surface via the DNA bridging units is of fundamental interest. Similarly, as has been shown in Figure 12A, these CdS QDs have also been used to signal the aptamer−target interactions.155 A variation on the direct hybridization approach involves a three-component “sandwich” assay, in which the signal indicators are usually labeled to a reporter (signaling) DNA sequence that was specifically designed to bind an overhang portion of the target DNA. Such a dual-hybridization route eliminates the step of target DNA modification, rendering it a potentially promising method for rapid real sample detection. Using hairpin-structure

Figure 17. (A) SNP typing using photostimulated hole transport through a DNA duplex immobilized on a gold electrode. (B) (a) Cluster diagram showing the genotype assignment for an aldehyde dehydrogenase 2 (ALDH2) SNP in 25 individuals. The 91-mer antisense strand of the ALDH2 gene containing a G1459A site was amplified by asymmetric PCR and was hybridized with a probe strand immobilized on a gold electrode. The x-axis shows the photocurrent density of a G probe, 5′-AAQUACACTGAAGTG-(CH2)6-S-Au-3′, and the y-axis shows that of an methoxybenzodeazaadenine (MDA) probe, 5′-AAQUACACTMDAAAGTG-(CH2)6-S-Au-3′. (b) Ratios of the photocurrent density for an MDA probe to that for a G probe are plotted for each sample. Ratios between 1.04 and 1.29 are scored as heterozygous (C/T), while ratios 1.73 are scored as homozygous for the G (C/C) and A (T/T) alleles, respectively. These boundaries are represented on the chart by the yellow areas. (Reprinted from refs 150 and 151. Copyright 2004 and 2006 American Chemical Society.)

DNA as a detecting probe, two “sandwich” protocols have been reported for the novel PEC microRNA detection.168,169 7436


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3. CONCLUSIONS AND PERSPECTIVES Different from traditional DNA assays such as gel electrophoresis and membrane blots, DNA biosensors, with the unique characteristic of monitoring the specific biorecognition events between the probe molecules and the target substances of interest, offer a promising alternative for faster, simple, and cheaper DNA detections. The past decade has witnessed substantial advances toward the development of modern PEC DNA biosensors, which provide excellent sensitivity due to the total separation and the different energy forms of the excitation source and the detection signal. Albeit the enormous opportunities and advantages clearly manifested in this methodology, indeed, the investigation on PEC DNA biosensors is still in its infancy and some important challenges and hurdles remain. For example, the poor stability and the reproducibility of these sensors are problems hindering their competing with the current gold standard for DNA assays (polymerase chain reaction, PCR). There are some aspects for future research in PEC DNA biosensors: 1. Exploiting new photoactive materials: Since inorganic or organic materials are usually essential in PEC DNA biosensors (as transducer, label, or indicator), the resultant analytical performance of the biosensors depends intimately on the properties of the materials utilized. With the development of material science and synthesis techniques, various materials with novel properties might be synthesized to meet the criterion for a particular purpose. For example, the semiconductor transducer with high photon-to-current conversion efficiency such as novel p−n junctions would benefit the generation of improved and stable photocurrents,39 which is obviously desirable in PEC biosensors. Also, the synthesis of new photoactive intercalators, with lower dissociation constants and higher reversible PEC responses, might be useful for ultrasensitive detection. 2. Focusing on the fundamental research: PEC DNA biosensors provide an exciting opportunity for interfacing the DNA and DNA−target recognition at the molecular level. It is anticipated that, with the aid of nanotechnology and biological technology, the innovative assembly and control on the functional interface would be advantageous to the basic understanding of DNA photoelectrochemistry and the DNA− target interactions. 3. Extending the biosensors’ applications: Although much exploitation has been accomplished, much work still needs to be done. For example, PEC screenings of DNA−drug and DNA−protein interactions are of particular interest as they are expected to play a prominent role in the future clinical market for disease diagnosis. In addition, PEC DNA biosensors might also see applications in many other fields, such as the detection of chemical pollutants or other analytes. Especially with the use of DNAzyme or aptazyme, there is great room for developing new detection formats toward numerous targets of interest. 4. Fabricating all-electronic microarrays: The combination of multiple processes, various functional elements, and related microfluidic networks on a single microchip platform will be needed for clinical application. Such miniaturized and simplified devices have also great economic prospects in the diagnostic market. Recently, a PEC lab-on-paper device equipped with a porous Au-paper electrode and fluidic delay switch has already been exploited for sensitive detection of DNA hybridization.227

5. Developing new detection formats based on novel principles: For instance, interparticle energy transfer has recently exploited as a new probing mechanism for PEC DNA hybridization genosensors. Specifically, it has been found that the presence of proximal noble metal NPs could remarkably influence the photoresponse of the QDs, based on which a new kind of energy transfer based PEC DNA bioassay format was proposed.135,136 A chemiluminescence (CL) reaction instead of external light source as a light source has also been reported. This kind of external-light-free PEC system has been demonstrated for the analysis of DNA hybridization or physiological thiols in cancer cells137,160 and then extended to immunoassay applications.51 The coupling of CL and energy transfer has been developed into an interesting strategy, i.e., chemiluminescence resonance energy transfer (CRET) for new PEC bioassay without external irradiation.141 On the basis of the great progress that has made in the existing work and the prospective technological breakthroughs, it is expected that advanced PEC DNA biosensing strategies will be proposed in future research. With the advancement of the PEC detecting technology and DNA-based assay, we envision that PEC DNA biosensors will continue to evolve and advanced PEC DNA biosensors will certainly offer simple, powerful, and user-friendly tools enabling rapid, sensitive, and specific detection for disease diagnosis, genetic testing, environmental monitoring, food safety and security, drug discovery, medicine research, and so on.

AUTHOR INFORMATION Corresponding Authors

*Tel.: +86-25-83597294. Fax: +86-25-83597294. E-mail: xujj@ nju.edu.cn. *Tel.: +86-25-83594862. Fax: +86-25-83594862. E-mail: [email protected]. Notes

The authors declare no competing financial interest. Biographies

Wei-Wei Zhao was educated at the Nanjing University of Aeronautics and Astronautics (NUAA) and received his B.S. and M.E. degrees in 2005 and 2008, respectively. Then he moved to Nanjing University, he received his Ph.D. from Nanjing University in 2012 under the supervision of Prof. Hong-Yuan Chen and Prof. Jing-Juan Xu. Currently, he works at Nanjing University and his research involves photoelectrochemical DNA detection, immunoassay, and biocatalytic sensing. He has published over 20 scientific articles. 7437


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Jing-Juan Xu, born in 1968, graduated from Wuhan University in 1990 and earned her M.Sc. and Ph.D. degrees from Nanjing University in 1997 and 2000. Currently she is a full professor in the Department of Chemistry at Nanjing University and has published more than 150 scientific papers. She is the recipient of the National Outstanding Youth Foundation of China (2010) and the National Nature Science Award of China (second class). Her research interest focuses on the development of various electrochemical, electrochemiluminescent, and photoelectrochemical sensors based on nanostructured materials with enhanced sensitivity and selectivity, and the fabrication of lab-on-chip detectors for biological analysis.

Hong-Yuan Chen was born in 1937 in Sanmen of Zhejiang Province in China. After graduation from Nanjing University in 1961, he has worked in the Department of Chemistry, Nanjing University. From 1981 to 1984, he worked at Mainz University as a visiting scholar. During 1986−1999, he was a guest professor or visiting professor in Germany four times. In 2001, he was elected as an academician of the Chinese Academy of Science. He is a member of several scientific societies and several advisory boards of scientific journals. He has authored and coauthored over 650 papers and several chapters and books. Research interests include electrochemical biosensing, bioelectrochemistry, ultramicroelectrodes, biomolecular-electronic devices, and micro total analysis systems.

ACKNOWLEDGMENTS We thank the 973 Program (2012CB932600), the National Natural Science Foundation of China (Nos. 21327902, 21025522, 21135003, 21121091, and 21305063), the Natural Science Funds of Jiangsu Province (BK20130553), the open funds of SKLACLS (1314), and the Fundamental Research Funds for the Central Universities (20620140158) for support. The authors are also thankful to all the referees for their useful suggestions and constructive comments. 7438


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