Photoelectrochemical Immunoassays - Analytical Chemistry (ACS

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Photoelectrochemical Immunoassays Wei-Wei Zhao, Jing-Juan Xu, and Hong-Yuan Chen Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b04672 • Publication Date (Web): 14 Nov 2017 Downloaded from http://pubs.acs.org on November 15, 2017

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Analytical Chemistry

Photoelectrochemical Immunoassays †

†

Wei-Wei Zhao,*, ,‡ Jing-Juan Xu, and Hong-Yuan Chen

†

†

State Key Laboratory of Analytical Chemistry for Life Science and Collaborative Innovation

Center of Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210023, Jiangsu, P.R. China. ‡

Department of Materials Science and Engineering, Stanford University, Stanford, California

94305, United States E-mail: [email protected], [email protected]

ACS Paragon Plus Environment

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CONTENTS PEC Immunoassay Transducers Formats Direct Ag-down Competitive Sandwich Strategies Steric hindrance Biocatalytic precipitation In situ generation of electron donor/acceptor In situ generation of photoactive species In situ generation of light sources Trends Conclusions Author Information Corresponding Author ORCID Notes Biographies Acknowledgments References


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As an essential direction of photoelectrochemical (PEC) bioanalysis,1-30 PEC immunoassays have attracted substantial research scrutiny for its desirable combination of the inherent sensitivity of the PEC bioanalysis and the specific bioaffinity properties of the immunomolecules. The past five years have witnessed substantial advances toward the development of PEC immunoassays, and numerous approaches to advanced PEC immunoassays have been established. Although the summaries on specific topics, such as the PEC DNA biosensors,31 PEC aptasensing,32 PEC enzymatic biosensors33 or the general development of PEC bioanalysis,34 have been presented in previous reviews,31-50 no in-depth survey has yet been made for addressing specifically on the important category of PEC immunoassays. For the first time, this review will depict a general picture of the PEC immunoassays,51-160 with emphasis both on the fundamentals and the-state-of-the-art in this field. Beginning with the brief description from the immunoassays to PEC immunoassays, this work will make a thorough discussion on the photoelectrodes, sensing formats and, especially, the signaling strategies of PEC immunoassays, followed by our perspective on the status of this field and potential future directions. We hope this work could simultaneously provide an accessible introduction to PEC immunoassay and inform the interested audience of the latest developments and applications in this booming field, especially around the past 3 years. Immunoassays are valuable analytical techniques based on the affinity recognitions between the antigen (Ag) and antibody (Ab) that allow for the detection with high sensitivity and specificity. Immunosensors are devices where the immunoreactant associates intimately with a physicochemical transducer.161-165 While the term “immunoassays” is used for the assays based on the immunochemical reactions, the term “immunosensors” is employed specifically to describe the whole instruments, i.e., the immunoreaction-based biosensors. Consistently, the


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cornerstone is the inherent ability of Ab to bind to the specific structure of Ag. The immunoassays could be heterogeneous or homogeneous. In the heterogeneous ones, the Ag/Ab needs to be immobilized on the solid substrates, whereas homogeneous ones usually involve the immobilization of Ag/Ab on the beads and occur in the solution phase. Also, the immunoassays could be either label-free (where the immunorecognition is directly detected by measuring the physical changes caused by the Ag-Ab interaction) or labelled (where a detectable label is combined with the Ag/Ab of interest). However, in most of existing ones, the immunoassay is visualized via an auxiliary reaction, in which Ag/Ab is conjugated with a label for signal generation. Since the debut of radioimmunoassay (RIA) with radioactive label by Yalow and Berson in the 1960s,166-168 a large plethora of different and nonisotopic labels, such as chromophores, fluorophors and enzymes, have been applied in immunoassays. Labels are typically chemically linked or conjugated to the desired Ag/Ab. Correspondingly, several innovative immunologic methods such as chromoimmunoassay (CIA), fluoroimmunoassay (FIA) and enzyme immunoassay (EIA) have been well established in clinical diagnosis and industrial analysis. On the other hand, upon different physical transducers, the immunoassays could be divided into four principal classes: optical (fluorescence, luminescence, absorbance/colorimetry, surface plasmon resonance/local surface plasmon resonance (SPR/LSPR), surface enhanced raman scattering (SERS)), electrochemical (amperometric, potentiometric, capacitative), mechanical

(microcantilever,

acoustic

wave

sensor),

and

thermometric.

Principally,

immunoassays are capable of detections without any analyte enrichment, purification or pretreatment, which is generally needed by standard methods such as high-performance liquid chromatography (HPLC), mass spectrometry or gas chromatography. Especially, for clinical diagnostics where complex samples (e.g., whole blood, serum or urine) contain numerous


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interferents (e.g., DNAs, proteins, amino acids, sugars, hormones etc.), immunoassays have obvious advantages over standard methods in terms of simplicity and sensitivity. Among various immunoassays, enzyme linked immunosorbent assay (ELISA), pioneered by Engvall and Perlman in 1971, has opened a new era in this field and now enjoyed the greatest success. 169,170 It has been the most successful standard EIA and considered as the indispensable “gold standard” of immunoassay for comparison against all newly developed immunoassays. With the great market prospect and rapid development of analytical science in the past years, numerous protocols for new immunoassays have been proposed. While each has distinct advantages, it also suffers from its associated drawbacks. Recent research is focused towards developing miniaturized, portable, reusable, high-throughput, multiplexed and more reliable immunoassays for point of care (PoC) measurements. However, there appears a big gap between the ideality and reality, the situation of which is mirrored by a lack of commercial applications in spite of the increased scholarly articles and well-advanced instrumentations. Photoelectrochemistry is a vibrant scientific domain concerned with the interaction of light with electrochemical systems, and the PEC process refers to the photo-to-electric conversion resulting from the charge excitation and transfer of a material upon absorption of light. The first PEC experiment, performed by Becquerel in 1839,171 was not well understood until Brattain and Garrett launched the modern photoelectrochemistry in 1954.172 Since then, a tremendous surge of interest has emerged in the exploitation of the PEC process for various uses such as photovoltaics, photocatalysis, and photosynthesis.173 Analysts have also been infatuated with the idea of applying such process for the analytical purposes. In 1988, Hafeman et al. fabricated a light addressable potentiometric sensor (LAPS), a kind of semiconductor device based on the surface photovoltage, for biochemical system.174 Willner and coworkers began to construct


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semiconductor/biomolecule nanoarchitectures for bioanalysis via the photocurrent signals in the early 2000s.1 The accelerated fusion of photoelectrochemistry with bioanalysis hence led to the inauguration of more sophisticated methodology of PEC bioanalysis, in which light and electricity are used as separated input and output signals, respectively. On the basis of semiconductive materials, PEC bioanalysis has since stimulated increasing curiosities as such a technique provides great promise for developing versatile miniaturized analyzers compatible with

future

requirements.

Essentially,

its

detection

principle

reverses

that

of

electrochemiluminescence (ECL), i.e., the addressed biological interactions could be transduced by the semiconductive materials as the change of electrical signal.42 As the advanced generation of traditional electrochemical bioanalysis, the PEC bioanalysis naturally inherits its virtues such as low cost, simple instrumentation but with higher sensitivity due to the reduced background that originated from the total separation and the different energy forms of the excitation and detection signals. Because of its attractive merits and huge potential in future biomolecular detection, the popularity of PEC bioanalysis has expanded promptly among the community.1-30 Currently, as evidenced by increased scholarly literatures, there is an increasing impetus for innovative PEC bioanalysis towards various targets of interest, especially DNA and protein targets. PEC immunoassay is the synergy of PEC bioanalysis and immunoassay with complementary advantages. It combines the inherent specificity of immunoreactions and the conveniences of PEC

bioanalysis.

With

semiconductive

materials,

PEC

immunoassay

converts

the

immunobinding into a detectable signal of electricity, and the general detection mechanism is illustrated in Figure 1. Some earlier works have developed the LAPS-based immunoassays, and Cosnier et al. exploited the amperometric PEC immunoassay in 2004.5,127 Although serious


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consideration of PEC systems for amperometric immunoassay is quite recent, research in this area has a rather astonishing momentum. In the past decade, with the rapid progress of PEC bioanalysis technology, PEC immunoassay has experienced an enormous upswing and significant achievements have been recorded in this field. However, while numerous review articles have addressed the optical or electrochemical immunoassays,161-165 to our knowledge, no effort so far has yet been made for the overview of this rapidly developing field. Herein this work has attempted to provide the readers with a clear view of PEC immunoassays, ranging from fundamental principles to the most recent advances with illustrative examples. Specific areas of interest include transducer advances, useful formats and inventive strategies. We apologize for not being able to introduce all of the important works in this field due to the space limitation or our inadvertent omission.

Figure 1. General PEC immunoassay design. The major process involves the probe Ab immobilization on the substrate as the recognition layer for the subsequent immunorecognition, which event is then transduced into an electrical signal.

PEC IMMUNOASSAY PEC immunoassays could be classified into two main categories: potentiometric and amperometric. The potential type basically refers to the early LAPS-based immunoassays, which will not be descripted here due to its stagnation in recent years. However, interested readers are referred to a comprehensive review of this topic and the references therein to pursue their


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interest.175 In contrast, the amperometric type has been developed rapidly in the past decade and still continues on its dynamic path. In a typical amperometric PEC immunoassay, as shown in Figure 1, a solid transducer is initially used for the immobilization of the primary Ab (Ab1), followed by the assay developments with different assay formats and signaling strategies. During this process, the signal evolution before and after the immunorecognition events were tracked to reflect the target concentrations. Following the major development steps, this section will use the typical examples to not only present the basic fundamentals but also highlight the most recent advances in the corresponding domains. Incidentally, the protein immobilization approaches are excluded from this review since they are highly similar to those for DNA or enzyme molecules.31-33 Readers could use the cited reviews to get informed about this topic. Transducers. The principle of the PEC bioanalysis is based on monitoring the biological interactions between various recognition elements and their corresponding targets via current/voltage with an array of electrodes. Specifically, the biological recognition systems transfer the biochemical information (e.g. an analyte concentration) to the variation of specific factor on/surrounding the electrodes, the change of which associates exquisitely with the PEC transducers. When illuminated by suitable light source, the transducer would subsequently convert the physical/chemical interactions between the biological recognition events and PEC active species to electrical signal. Usually, the transducer is also where the immobilization of the recognition layer and the biorecognition of the targets take place. Therefore, the choice of transducer is of vital importance in achieving excellent analytical performance. With the development of PEC immunoassays, many kinds of transducers have been exploited. Previous reports have often classified the PEC transducers in terms of inorganic semiconductors, organic semiconductors, hybrid semiconductors and others.36, 40, 45 Readers could use the cited articles


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and the references therein to pursue their interest in this classification. Herein, given the recent trends in PEC immunoassay, we prefer to summarize the most recent and important developments in the use of traditional and newly emerging materials for novel PEC immunoassay applications. One important direction is to exploit newly emerging materials. Taking the be the most versatile and extensively utilized quantum dots (QDs) for example, since the first CdS QDsbased PEC immunoassay,145 numerous different core (binary, ternary or quaternary) QDs, doped QDs and core-shell QDs have been reported. Specifically, binary CdSe QDs65,94,115,120 and CdTe QDs83,159 have been used by many groups to replace the CdS QDs as light-harvesting species in some PEC immunoassays. Ternary CdAgTe QDs was utilized in an energy-transfer based PEC immunoassay of cardiac troponin I,123 while quaternary ZnCdHgSe QDs has also been used for sensitive PEC Immunoassay of neuron specific enolase.61 Transition metal ion doping has been demonstrated as an effective method for improving the electronic and photophysical properties of QDs, and Mn doped CdS QDs was a typical doped QDs that frequently employed for PEC immunoassays of different targets.59,71,79,108,117 Encapsulating the core QDs by a layer of higher band-gap semiconductor to fabricate the core–shell QDs is an effective method to improve the stability and photophysical properties of the core QDs. For example, CdSe@ZnS core-shell QDs have been reported for PEC immunoassays of carbohydrate antigen 19-9107 and H-IgG,148 respectively, while CdTe@CdS:Mn and CdSeTe@CdS:Mn core−shell QDs have also been developed for PEC immunoassays of polybrominated diphenyl ether93 and carcinoembryonic antigen (CEA),67 respectively. In short, the impetus for advanced QDs-based innovative PEC immunoassays has been growing much faster as evidenced by increasing academic articles. However, as can be seen, Cd-chalcogenide (CdC, C = S, Se, Te) QDs have still been the most


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employed QDs to date. Despite of the availability of precursors and the simplicity of crystallization, Cd-based QDs suffer from high toxicity that comes from the leakage of Cd ions or the decomposition of the QDs. In this regard, some other biocompatible Cd-free candidates are emerged. Carbon dots (C dots) have demonstrated itself as a biocompatible and environment-friendly nanomaterial for PEC immunoassay applications. For example, C dots have been used for PEC immunoassays of aflatoxin B1,73 prostate-specific antigen (PSA),82 and fibroblast-like synoviocyte (FLS) cells,114 respectively. As a type of C dots, graphene QDs (G dots), a class of one- or few-layered graphene sheets with lateral dimensions smaller than 100 nm, have also been used for PEC immunoassay due to their desirable photophysical and physicochemical properties. For example, G dots have recently been used for PEC immunoassay of microcystin-LR.150 Graphene and its derivatives, as interesting two-dimensional (2D) carbon nanomaterials, have also been used in this field. For example, reduced graphene oxide (rGO) has been extensively used for PEC immunoassays of PSA,69,118 indole-3-acetic acid,87 CEA,91,129,134 microcystinLR,146 α-fetoprotein (AFP)155 and cancer antigen 125 (CA 125),154 while graphene oxide (GO) has also been used for PEC immunoassay of carcinoembryonic antigen.80 In addition to graphene, some other carbon materials, e.g. carbon nanotubes,56,

112, 130, 131, 153

mesoporous carbon,148

fullerene C6056,101 and carbon nanohorns59 have also been applied. Similar to graphene, some another 2D nanomaterial with extraordinary physicochemical properties, such as g-C3N4,68, 82, 88, 117, 125, 138, 152, 155, 157

MnO273 and SnS2112 nanosheets have also been used. Besides, various Bi-

related materials, such as BiVO4,69, 74 Bi2S3,92, 106, 121, 157 CuBi2O4,124 and BiOI,126, 156 have also attracted increasing interest for elegant PEC immunoassays.


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Another obvious direction is to utilize specific nanostructures with potential desirable properties. Taking the most used TiO2 for example, its morphologies of nanoparticles (NPs), nanotubes (NTs), nanowires (NWs), nanorods (NRs), nanoneedles (NNs), nanobelts (NBs) and mesocrystals (MCs) have been frequently reported for PEC immunoassay development. For example, using a TiO2 NPs-based electrode, β-galactosidase (β-Gal) catalytic system was integrated with sandwich immunobinding for probing the cardiac marker troponin T (cTnT) at neutral conditions.57 Using a TiO2 NTs-based electrode, the in situ strategy of enzymatically production of electron donor was proposed for general PEC immunoassay.53 More recently, on the basis of TiO2 NWs,58 TiO2 NNs,75 TiO2 NBs134 and TiO2 MCs,78, 98, 142, 149 various TiO2-based electrodes have been fabricated by different groups for PEC immunoassay development. A similar situation also occurs on the ZnO semiconductor.93, 97, 106, 111, 132, 140, 141, 144, 147, 153, 154, 157, 158, 160

Due to the unique LSPR feature, the plasmonic photoelectrochemistry of noble metal nanomaterials deposited on specific semiconductors has been a very hot area that drawing considerable research interest from many fields. For PEC immunoassay, for example, Au NPsdecorated TiO2 NWs has been used for SPR enhanced real-time PEC bioanalysis of cholera toxin subunit B.58 Using Au NPs/TiO2 NTs electrode, a plasmonic strategy was developed for labelfree PEC immunoassay of protein p53.66 Later, many other similar heterostructures, such as Au nanocrystal/BiVO4,74 Au NPs/WO3,118 Au NPs/Bi2S3,121 Au NPs/CuBi2O4124 and Au NPs/gC3N4,152 have been reported for PEC immunoassays. Organic materials are also good candidates as photoactive materials for PEC immunoassays. For example, a photosensitive biotinylated polypyrrole film was generated by electro-oxidation of a biotinylated tris(bipyridyl) ruthenium(II) complex bearing pyrrole groups. In the presence of


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an oxidative quencher (pentaaminechloro cobalt(III) chloride), photoexcitation of this modified electrode will lead to a cathodic photocurrent.5, 127 Recently, Cosnier et al. electrogenerated an organic photoanode of trisbipyridyl Ru(II)-/nitrilotriacetic-polypyrene copolymer film for a label-free PEC immunosensor. In the presence of a sacrificial donor of ascorbate, the targets could be followed by the evolution of the anodic photocurrent.85 Organic complexes combined with inorganic materials have also been exploited since surface sensitization of a wide band gap semiconductor with the chemisorbed or physisorbed dyes can increase the efficiency of the excitation process and expand the excitation wavelength range. For example, cobalt 2,9,16,23tetraami-nophthalocyanine (CoPc)-sensitized TiO2 electrode has been developed for PEC immunoassay of neutrophil gelatinase-associated lipocalin.64 Similarly, polyethyleniminefunctionalized

carbon

nitride,68

5,10,15,20-tetra(4-sulfophenyl)-21H,23H-porphyrin

(TSPP)/TiO2,81 porphyrin/C60,101 naphthalocyanine/ZnO144 and many other poly-dopamine sensitized semiconductors112, 142,149, 160 have also been developed. On basis of the various photoactive materials, either a photoanode or photocathode could be built. Photoanode is an electrode through which the photoexcited electrons flow outwards to the external circuit, generating a photocurrent flows from the photoelectrode to the electrolyte solution. By contrast, photocathode is an electrode where the photoexcited electrons flow outwards to the electrolyte solution, generating a photocurrent flows from the photoelectrode to the external circuit. Depending on how the system operates, both the photoanode and photocathode can have either a negative or a positive voltage, but the chemical oxidation (or reduction) reaction usually dominates at a photoanode (or photocathode) surface. Currently, one significant trend in PEC bioanalysis is the emerging of photocathodic bioanalysis. Since the photocathodes are more prone to interact with electron acceptors (e.g., dissolved oxygen) than


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with electron donors (e.g., ascorbic acid) in the electrolyte, photocathodic bioanalysis generally possesses better capability in anti-interference from reductive substances. In the PEC immunoassays, for example, p-type CuInS2 microflowers has been used as photocathode for PEC immunoassay of PSA.71 p-CuBi2O4 semiconductor has also been used for cathodic PEC immunoassay of AFP.124 Formats. PEC immunoassays appear in many different formats and variations, and mainly could be classified into unlabelled or labelled modes. They may be run in multiple steps with reagents being added and washed away or separated at different points during the assay. The unlabelled PEC immunoassays are able to detect the physical/chemical changes during the immunosensing, and it represents a fascinating direction due to its simplicity. The labelled PEC immunoassays rely on the use of labelled compounds, and could be further carried out in three different ways: the Ag-down assays, competitive assays and the sandwich assays. Various labels have been applied in indirect PEC immunoassays, and these labels are employed to amplify the detection signal and increase sensitivities in the specific protocols. Among the most valuable labels are enzymes such as glucose oxidase (GOx), horseradish peroxidase (HRP), alkaline phosphatase (ALP) and acetylcholine esterase (AChE). For PEC immunoassays, the labelling enzymes should have some properties: (a) high Kcat; (b) stability of enzymes and substrates in buffer; (c) PEC active products; and (d) low side reaction of the enzyme products. It means that stable enzymes with higher turnover number are preferred as labels for the dual benefits of longevity and sensitivity. Although indirect PEC immunoassays are highly sensitive and versatile due to the characteristics of these enzymes, one should also bear in mind that each enzyme has their own deficiencies for specific uses. For instance, ALP necessitates the alkaline working conditions that may cause protein denaturation, and GOx has relatively lower turnover number


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and hence inefficient activity as compared to other enzyme tracers. With recent typical examples, as shown in Figure 2, here we introduce the four commonly used types of PEC immunoassays: direct, Ag-down, competitive and sandwich assays.

Figure 2. (A) Diagram of a direct immunoassay. After addition of analyte, the immunobinding leads to the formation of the AbAg immunocomplex, which would cause the change of specific physicochemical factors that directly lead to signal generation. (B) Diagram of an Ag-down immunoassay. After the immunobinding between the Ag and Ab1, enzyme-conjugated Ab2 binds to Ab1. The addition of the enzyme’s substrate then leads to signal development. (C) Diagram of a competitive binding immunoassay. After addition of both the Ag and the enzyme-conjugated Ag, competition occurs between the two for binding to the Ab. The addition of the enzyme’s substrate leads to signal generation. (D) Diagram of a sandwich immunoassay. The addition of the enzyme’s substrate leads to the signal development.

Direct. As shown in Figure 2A, the direct PEC immunoassay is the simplest type of PEC immunoassay, and its detection mechanism operates directly via the Ab-Ag interaction. In general, one protein probe is firstly anchored onto the surface and an excess of another protein, commonly bovine serum albumin (BAS), is used to block the rest of binding sites on the surface. Then the target is added and links itself to the probe. In such a configuration, measurement of the Ab-Ag interaction doesn’t need any labeling, and the produced signal is directly proportional to the target concentration in the sample. In some cases, a PEC immunoassay may use an Ag to


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detect for Ab that recognizes the Ag. In other words, occasionally, the analyte may be an Ab rather than an Ag. For example, in the pioneering work of Cosnier et al., cholera toxin was used as anchored receptor for the detection of anti-cholera toxin Ab,5,85 whereas cholera toxin (subunit B) was used as analyte in another recent work of Zheng et al.58 The obvious advantages of this methodology are its convenience since only one Ab is used and so the cross-reactivity of secondary Ab2 is eliminated, while its disadvantage is its lack of signal amplification. Using this direct format, increasing PEC immunoassays have been proposed addressing different targets.71,75,79,84 For example, Liu et al. realized the label-free and sensitive PEC immunoassay of neutrophil gelatinase-associated lipocalin (NGAL).64 Zhao et al. developed a plasmonic strategy for label-free PEC immunoassay of protein p53.66 Ag-Down. As shown in Figure 2B, the Ag-down PEC immunoassay is an indirect two-step immunoassay with Ag anchored to the electrode surface rather than Ab. Specifically, the Ag is used to bind Ab found in a sample, which involves two binding processes with the primary Ab1 and labelled secondary Ab2. Experimentally, the PEC electrode is immobilized with Ag, washed and blocked for the following specific binding of Ab1. After washing step, the labelled secondary Ab2 is bound to Ab1. Finally, the substrate is added which produces a detectable signal directly proportional to the Ab concentration. For example, when the sample of human serum is added, human IgE from the sample might bind to the Ag on the electrode, followed by the addition of anti-human IgE labelled with HRP to produce signal. Obviously, the more IgE in the sample, the higher the signal. Generally, Ag-down PEC immunoassay is more sensitive than direct type as more than one Ab is used per Ag. In practice, Ag-down immunoassay could be employed to determine allergy conditions by testing a patient’s blood against different allergens to see if the person has Ab to that allergen. However, compared to the direct mode, the Ag-down


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approach has less been employed in the field of PEC immunoassay. Zhang et al. recently developed Ag-down PEC immunoassay for Ab against tumor-associated carbohydrate antigen.94 Lately, Pavlov et al. reported the use of enzymatically generated CdS QDs to realize a novel Agdown type PEC immunoassay,113 while Zhao et al. also used this format to realize the PEC immunoassay of AFP.115 Competitive. As shown in Figure 2C, competitive PEC immunoassay is based on the competition between the labeled Ag (tracer) and the unlabeled Ag (analyte) for the limited Agbinding sites of the Ab. In a sequential competitive assay, the analyte and tracer are added in steps like other formats, while in a classic competitive assay, these reagents are incubated together at the same time. The key characteristic of this assay is that maximal assay sensitivity is attained using an amount of Ab tending to zero. Typically, a fixed amount of analyte and tracer are incubated, sequentially or simultaneously, with the Ab. According to the law of mass action, the amount of bound tracer is a function of the total concentration of labeled and unlabeled Ag. As the concentration of analyte is increased, less tracer can bind to the Ab and it will decrease the accumulation of labels on the electrode, leading to decline of the measured signals. Thus, the lower the signal, the more analyte in the sample. Therefore, the detectable signal of such assay is inversely related to the analyte concentration in the sample. As to the sensitivity of competitive immunoassay, it is determined by the affinity of the Ab for its Ag. Since the affinity of an Ab can be in the range of 105-1012 M-1, competitive immunoassays using Ab with a Kd = 10-12 M reach their highest sensitivity in the picomolar range. However, the lower detection limit can decrease even further if proper assay conditions are used. This competitive type is often used to measure small analytes since it only involves one Ab rather than two, as in sandwich format. For small analytes, the competitive format is preferable while the sandwich format may not be


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possible. Such competitive assay is also used when a matched pair of Ab to the analytes does not exist. On the other side, its associated disadvantages include: (a) limited sensitivity and working range; (b) slow reaction kinetics; and (c) lower precision. For PEC immunoassay, this format has frequently been utilized.91, 140, 142, 147, 149 For example, Cai et al. developed the sensitive PEC immunoassay of polycyclic aromatic hydrocarbon (PAH) using an anti-PAHs modified nanostructured TiO2 electrode. PAHs will compete with the PAHs/Au NPs/HRP for the binding sites of anti-PAHs, based on which a competitive immunoassay was established for the analyte detection.176 Recently, Du et al. also proposed a competitive PEC immunoassay for estradiol based on in situ generated CdS-enhanced TiO2.104 Very lately, Knopp et al. reported the competitive PEC immunoassay of mycotoxins based on a Ag nanolabels-assisted ion-exchange reaction,65 while Cao et al. realized the competitive PEC immunoassay of PSA on the basis of an energy transfer process.119 Sandwich. As shown in Figure 2D, the sandwich PEC immunoassay configuration, which consists of a capture (primary) Ab1, a target Ag, and a labeled detection Ab2, tends to be more sensitive and robust and therefore is the most popularly approach. In this technique, a significant excess of Ab over the Ag is used, and the measurable Ag is sandwiched between Ab1 and Ab2. Because a single Ag binds to both Ab1 and Ab2, the Ag must have at least two epitopes (the areas on an Ag where the Ab binds), and the Ab1 and Ab2 may be either monoclonal (i.e. recognize a single epitope) or polyclonal (i.e. recognize more than one epitope). Incidentally, polyclonal Ab can identify different epitopes and the same polyclonal Ab can thus be used as the Ab1 and labeled detection Ab2. Typically, the photoelectrode surface is initially bound with Ab1, followed by the addition of Ag for capture by Ab1 and then the addition of labelled Ab2 to bind the Ag, then the addition of substrate and its conversion by enzyme label into a detectable signal.


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Obviously, increased Ag concentration leads to more loading of label and thus a higher signal. Therefore, the detectable signal of such assay is directly proportional to the analyte concentration in the sample. Since a single Ag is captured and detected by two Ab, the key advantage of this immunoassay is its higher specificity than other assays. The disadvantage is that it usually requires long incubation time and more washing steps for non-specific bound reactants, and it requires two Ab bind to one analyte at the same time, which may also cause high probability for steric hindrance. Generally, the sandwich type tends to the most popular mode for PEC immunoassays due to its higher sensitivity.53, 56, 57, 59, 67, 68, 70, 74 Using this format, for example, Tang et al. reported a high-throughput PEC immunoassay of PSA.63 Recently, Zhang et al. proposed a simultaneous PEC immunoassay of dual cardiac markers using specific enzyme tags through the sandwich immunobinding.70 In a word, direct, Ag-down, competitive and sandwich are four frequently used formats for the PEC immunoassays. A common PEC immunoassay will fall into one of these four categories depending on how it is run. Although PEC immunoassays can also be operated in some other formats and variations, the format selected depends essentially on the reagents available and the dynamic range required for the specific purpose. Strategies. Based on those formats discussed above, various signaling strategies have been developed for the PEC transduction of immunobinding events. In fact, the strategy designed hinges closely on the format selected and the way how the format affects the PEC system. Basically, one may consider from three aspects, i.e., using the immunorecognition to influence the solution species (e.g., electron donor), the interfacial electron/mass transfer, or the photoelectrode. According to the specific strategies as will be discussed below, the PEC immunoassays could be operated either in “signal on” or “signal off” types. The former relies on


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the rising signals, while the latter depends on the subsiding signals during the immunorecognition. Both the two types could realize the sensitive immunoassays depending on the specific combination between the sensing formats and signalling strategies. Using the recent illustrative examples, the main established strategies will be reviewed in this section.

Figure 3. Steric hindrance-based cathode PEC Immunoassay of PSA. Reproduced from Fan, G.; Zhu, H.; Du, D.; Zhang, J.; Zhu, J.; Lin, Y. Anal. Chem. 2016, 88, 3392–3399 (ref 67). Copyright 2016 American Chemical Society.

Steric hindrance. Among various strategies, generating steric hindrance is the simplest approach for PEC immunoassays. In such a way, the formation of immunocomplex would cause the steric hindrance that inhibits the diffusion of solution species to the electrode surface for reaction with the photogenerated electrons or holes. Based on this strategy, many works have been reported addressing different targets.5, 25, 85, 93, 145 For example, Cai et. al established several PEC immunoassays for various environmental pollutants, the target concentrations could be measured via the decrease in photocurrent intensity resulting from the increase in steric hindrances due to the formation of the immunocomplex.51, 83, 86, 93, 136 Zhu and Lin et. al also developed many PEC immunoassays towards various tumor markers.59,67 In a recent work, as shown in Figure 3, Zhang et al. reported a novel steric hindrance-based cathode PEC immunoassay, in which PSA was chosen as a model of target while the TiO2/CdS:Mn/ITO


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electrode and the CuInS2 microflowers/ITO electrode served as the photoanode and photocathode, respectively. For immunoassay, the binding of the PSA to the photocathode will cause the decrease of photocurrent intensity due to the obvious steric hindrance of protein molecules.71 Biocatalytic precipitation. Biocatalytic precipitation (BCP) represents another simple method for PEC immunoassays. In this strategy, various enzymes (e.g., HRP, ALP and DNAzymes) and enzyme mimics can be used as biocatalysts to produce an insoluble product that inhibits photocurrent generation. For example, Zhao et. al introduced a QDs-based amplified PEC immunoassay with the integration of HRP-catalyzed BCP. Based on the HRP accelerated oxidation of 4-chloro-1-naphthol (4-CN) by H2O2 to yield the insoluble and insulating product benzo-4-chlorohexadienone on the transducer surface, an isolating barrier would thus be produced against the interfacial electron transfer and thereby altered the light-absorption feature of the electrode obviously. Consequently, the holes of the excited QDs cannot interact with the donor directly due to the blocking by the precipitate, which could decrease the hole trapping capacity of QDs and result in the declined generation of photocurrent.

52,128

Following this

strategy, a lot of BCP-based PEC immunoassays have been reported.128, 143 For example, Tu et. al reported a low potential and competitive PEC immunoassay of CEA,91 Zhang et al. used the HRP-catalyzed BCP to amplified the PEC immunoassay of tumor-associated carbohydrate antigen.94 Since the product of the ALP-catalyzed BCP reaction was of indigo colour, Chen et al. recently realized the simultaneous PEC and visualized immunoassay of β-human chorionic gonadotrophin (β-HCG).109 Using the DNAzyme concatamer, Tang et al. accomplished the PEC immunoassay of PSA on the basis of a DNAzyme-catalyzed BCP process.117 As shown in Figure 4, using silver iodide-chitosan nanoparticle (SICNP) labels, Dai et al. achieved a cathodic PEC


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immunoassay of human interleukin-6 (IL-6), in which the SICNP could act as peroxidase mimetic to induce the BCP for signal amplification.68

Figure 4. Enzyme mimic catalyzed BCP-based PEC immunoassay of IL-6. Reproduced from Gong, L.; Dai, H.; Zhang, S.; Lin, Y. Anal. Chem. 2016, 88, 5775–5782 (ref 68). Copyright 2016 American Chemical Society.

In situ generation of electron donor/acceptor. As known, the presence of electron donor or acceptor is highly essential for the generation of stable photocurrent. This strategy relies on the exquisite catalytic chemistry of various enzymes to in situ generate products for efficient electron donating or sacrificing. Zhao et al. previously have presented a typical example, which used ALP to catalyse the hydrolysis of enzyme substrate (ascorbic acid 2-phosphate, AAP) to in situ produce ascorbic acid (AA) for efficient electron donating. In such a system, increased PSA concentration leads to the improved ALP loading and thus boosts the enhanced AA generation for photocurrent responding. Since the degree of signal increment depends closely upon the target concentration, an elaborate PEC PSA assay can be accomplished by monitoring the final photocurrent intensity.53 Later, they further utilized the β-galactosidase (β-Gal)57 and glucosedehydrogenase (GDH) to in situ generate the electron donors for PEC immunoassays.156 On the basis of this strategy, increasing reports have been published for specific PEC immunoassay purposes.133, 141, 142, 147, 151 For instance, with in situ generated AA for electron donating, an effective PEC immunoassay was developed for detection of subgroup J avian


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leukosis virus by Ai et al.92 Wang et al. then developed another PEC immunoassay using the DNAzyme catalyzed oxidation of hydroquinone to in situ generate the electron acceptor.102 As shown in Figure 5, using specific enzyme tags of ALP and AChE to generate different electron donors, a simultaneous PEC immunoassay of dual cardiac markers was also exploited.70 Incidentally, this strategy has also been frequently applied for probing other biological interactions of interest such as in PEC enzymatic assay or DNA detection.

Figure 5. In situ generation of electron donor-based PEC Immunoassay of Dual Cardiac Markers. Reproduced from Zhang, N.; Ma, Z.; Ruan, Y.; Zhao, W.; Xu, J.; Chen, H. Anal. Chem. 2016, 88, 1990–1994 (ref 70). Copyright 2016 American Chemical Society.

In situ generation of photoactive species. The PEC detection usually needs ready photoactive species as signal sources to transduce the biological events into electrical signals. However, in some cases, one may utilize the biocatalytic processes to in situ produce photoactive species. For example, enzymes could be used as catalysts to produce a product that stimulates the generation of photoactive species. With this strategy, the ALP catalytic process was used to couple with the unique surface chemistry of TiO2 nanocrystalline. The self-coordination of the produced enediolligands onto the under coordinated surface sites of TiO2 would in situ form a ligand-to-metal charge transfer (CT) complex, endowing the inert TiO2 with strong absorption bands in the


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visible region.177 For PEC immunoassay, as shown in Figure 6, Dai et. al proposed an innovative PEC immunoassay based on the in-situ generation of CdS QDs on GO using the HRP catalytic chemistry. In this format, the labelled HRP could catalyse the reduction of Na2S2O3 with H2O2 to generate H2S, which reacted with Cd2+ on the electrode to form CdS QDs. The generated QDs could then be photoexcited to produce a photocurrent as the detection signal.80 Using the same HRP catalytic process, Hong et al. then realized the PEC immunoassay of AFP.98 Du et. al then reported another simple PEC immunoassay protocol based on in situ generated CdS-enhanced TiO2 film.104 Recently, Pavlov proposed the use of ALP catalysed hydrolysis of sodium thiophosphate to produce H2S, which in the presence Cd2+ ions can yield CdS QDs. Detection of CdS QDs “wired” by polyvinylpyridine bearing osmium complex (Os–PVP) was then applied for the PEC immunoassay application.113

Figure 6. In situ generation of photoactive specie-based PEC immunoassay. Reproduced from Zeng, X.; Tu, W.; Li, J.; Bao, J.; Dai, Z. ACS Appl. Mater. Interfaces 2014, 6, 16197–16203 (ref 80). Copyright 2014 American Chemical Society.

In situ generation of light sources. Besides the photoactive species, the common PEC bioanalysis also necessitates the external physical light as irradiation source for the excitation of the photoactive species. Zhang et. al circumvented this necessity creatively by employed the luminescence from the chemiluminescence (CL) reactions of isoluminol-H2O2-Co2+ system or


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bis(2,4,5-trichlro-6-npentoxycarbonylphenyl)oxalate-H2O2-9,1-diphenylanthrancene system to induce the photocurrent production from the photoactive species in the PEC bioanalysis. They first applied this external-light-free PEC system for the PEC bioanalysis of physiological thiols in cancer cells178 and DNA hybridization.179 For PEC immunoassay, Ju et. al then extended such a strategy by integrating the CL system of luminol-H2O2-HRP-p-iodophenol into a sandwich format. The luminol-HRP-Ab2-AuNPs bioconjugate as the detection probe could be confined onto the electrode. Upon addition of H2O2, the CL produced from the oxidation of luminol by H2O2 in the presence of HRP would act as the light source to excite the CdS/RGO composite to trigger the photocurrent generation.129 Yu and Yan et. al then coupled this strategy into the microfluidic lab-on-paper device equipped with a paper supercapacitor for simple and disposable PEC immunoassay applications.131, 140, 151 Recently, as shown in Figure 7, Tang et al. adopted the peroxyoxalate chemiluminescence (PO-CL) self-illuminated system to develop a portable PEC immunoassay with digital multimeter readout, in which the H2O2-induced PO-CL reaction and the H2O2-triggered photoelectron generation of the BiVO4-rGO were combined for the sensitive PSA assay.69

Figure 7. In situ generation of light source-based PEC immunoassay. Reproduced from Shu, J.; Qiu, Z.; Zhou, Q.; Lin, Y.; Lu, M.; Tang, D. Anal. Chem. 2016, 88, 2958–2966 (ref 69). Copyright 2016 American Chemical Society.


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TRENDS Ever since the debut of PEC immunoassays, there have been continuous efforts on developing innovative assay formats and signalling (amplification) strategies. Although desirable progress has been achieved, there is still huge room for developing new detection formats based on novel principles. Currently, some important trends have emerged for future research in PEC immunoassays. New materials. With the rapid development of material science, various traditional and new emerging materials such as graphene and g-C3N4 have been applied for PEC immunoassays. However, more advanced functional materials with unexpected and intriguing properties remain to be sought, on the basis of which new sensing interfaces or novel nanomaterials/biomolecule signal-reporting systems might be developed for PEC immunoassay with innovative principles. One recent example by Wei et al. is the development of TiO2 NNs@MoO3 array for PEC immunoassay of RAW264.7 macrophage cells.75 Currently, compared to the high prosperity of inorganic materials, the exploitation of organic materials for PEC immunoassays lags behind. Various organic small molecules such as porphyrin and its derivatives, phthalocyanine and its derivatives, azo dyes, chlorophyll, bacteriorhodopsin, polymers such as phenylenevinylene (PPV), poly(thiophene), and their derivatives may find their application in future PEC immunoassays. New recognition elements. Recognition elements are important parts of PEC immunoassays since they are responsible for the target recognition. Classical recognition elements for PEC immunoassays are different antibodies. Nowadays, with the advance in biological technique and the need for improved assay characteristics, versatile new recognition elements, e.g. aptamers, phages, whole cells, molecularly-imprinted polymers, and various engineered affinity proteins


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including monobodies/adnectins, anticalins, affibodies, designed ankyrin repeat proteins and nanobodies, have been synthesized in the laboratory or selected in vitro and studied for specific analytical purposes.180 These new recognition elements have aroused the interest of researchers in the field of PEC immunoassays. For example, Liu et al. have used the highly oriented nanobodies as receptor molecules for the successful PEC immunoassay application.64 Besides, the combinations of different recognition elements and their combination with nanomaterials have also been applied for advanced PEC immunoassay.116

Figure 8. Split-type-based PEC immunoassay by the use of liposomes. Reproduced from Mei, L.; Liu, F.; Pan, J.; Zhao, W.; Xu, J.; Chen, H. Anal. Chem. 2017, 89, 6300–6304 (ref 77). Copyright 2017 American Chemical Society.

Split-type immunoassay. Usually the PEC immunoassay necessitates the immunorecognition on the photoelectrode, and the latter will serve as signal source to convert the specific Ag-Ab recognition reaction into electrical signals. However, the immobilization of biomolecules (especially the oriented immobilization) and the subsequent immunoreactions on photoelectrodes are laborious and time-consuming, and may inevitably impair these biomolecules, causing their denaturation and leakage during these steps. Besides, the excited states of the photoactive materials and the light radiation itself may result in biomolecules damage. To break the bottleneck, the facile, simple, and efficient PEC immunoassay protocol, which could eliminate these commonly needed procedures, are extensively pursued. Recently, Xu et al. reported an


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improved PEC immunoassay that separated the immunobinding process from the photoelectrode.60 Such a split-type detection mode could remove the mutual disturbance between the biorecognition and PEC detection system. Using this mode, as shown in Figure 8, Zhao et al. recently proposed the first liposome-based split-type PEC immunoassay by using enediolligands-encapsulated liposomes for directed sensitization of TiO2 NPs.77 Other similar split-type PEC immunoassays were reported soonafter.76, 181 Multiplex immunoassay. Special attention should further be paid to multiplex PEC immunoassay. Multiplexed assays are capable of measuring specific biomolecules that are grouped in panels such as allergens and cardiac markers in biological samples. Advantages of multiplex immunoassay include less sample volume, shorter analysis time and procedure, enhanced detection throughput, as well as lower overall cost as compared to traditional parallel single-analyte assays. However, few reports about the multiplex PEC immunoassays have been reported until recently. Yu et al. then reported the first multiplex PEC immunoassay for determination of three tumor markers by using electrode arrays.132 Hu et al. then established a light-addressable PEC approach for multiple DNA detection182 and immunoassay.121 In addition to these reports based on the spatially separated test zones, Zhao et al. then exploited the simultaneous PEC immunoassay of dual cardiac markers using specific enzyme tags.70 Later, Dai et al. proposed another interesting potentiometric addressable PEC immunoassay by varying the applied bias in the detection process.72 Given the importance of multiplex immunoassays for practical utilization in clinical laboratories, developing specific multiplex PEC immunoassay is highly desired. Future improvement in distinguishability with differences in time, space, labels and so on will certainly lead to advanced multiplex/simultaneous PEC immunoassays.


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Microfluidics and Automation. Tremendous opportunities also exist in taking current PEC immunoassay a further step to combine with microfluidics for automation. Microfluidic networks on a single microchip, integrated with flow-injection systems, will lead to miniaturized and simplified PEC immunoassay systems with obvious advantages, such as simplicity, speediness, automation and high-throughput, the success of which will also have great economic prospects in the diagnostic market for clinical application. In this regard, as shown in Figure 9, Tang’s group has recently exploited a semiautomated PEC immunoassay system for portable and highthroughput analysis. Such a system not only simplified the assay process but also allowed on spot and continuous analysis and thus improved the assay efficiency.74 Using the (Paper-based) microfluidic platform, many other interesting PEC immunoassays have also been reported.93, 111, 130-132, 134, 151

Figure 9. Semi-automated power-free SP-PEC immunoassay coupling with a miniature semiautomatic microfluidic system and digital multimeter readout. Reproduced from Shu, J.; Qiu, Z.; Lin, Z.; Cai, G.; Yang, H.; Tang, D. Anal. Chem. 2016, 88, 12539– 12546 (ref 74). Copyright 2016 American Chemical Society.


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Others. Based on the aforementioned basic formats and strategies, there are many other trends that capable of innovative PEC immunoassays. For example, because of the collective oscillations of CB electrons driven by the applied electromagnetic field of incident light, noble metal NPs, e.g. Ag or Au NPs, exhibit LSPR feature that depends intimately on their sizes, interparticle distances, geometric shapes, dielectric properties of the materials and also the surrounding ambient dielectric permittivity. When deposited on the semiconductive materials and upon light stimulation, LSPR-induced charge separation and transfer could happen at the surface of these noble metal NPs, rendering to a unique plasmonic photoelectrochemistry.183 Significantly, because such a process correlates closely with the LSPR and charge separation and transfer, one may manipulate these related factors and explore its promising applications for plasmonic PEC immunoassay. Zheng et. al recently reported such a novel SPR enhanced realtime PEC assay based on the use of Au NPs-decorated TiO2 nanowires.58 Based on the similar plasmonic photoelectrochemistry, as shown in Figure 10, Zhao et al. reported the use of ALP tagged antibodies on Au NPs/TiO2 NTs electrode for the development of a plasmonic PEC immunoassay. Due to the immunorecognition between the receptor and target, the plasmonic charge separation from Au NPs to the conduction band of TiO2 NTs could be influenced greatly that originated from multiple factors.66 Another recent trend has been the use of exciton–plasmon interactions between QDs and noble metal nanoparticles in the PEC system for biosensor applications.184,185 For immunoassay, Zhu et al. reported the use of DNA sequence functionalized with heterogeneous core-satellite nanoassembly for novel energy-transfer-based sandwich PEC assay of PSA,116 while Cao et al. proposed the competitive PEC immunoassay of PSA on the basis of dual-quenching of photocurrent from CdSe sensitized TiO2 electrode by gold Au NPs


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decorated polydopamine nanospheres.119 Later, Li et al. reported the PEC immunoassay of cardiac troponin I based on energy transfer between CdAgTe QDs and dodecahedral Au NPs.123

Figure 10. Plasmonic PEC immunoassay using an Au NPs-TiO2 NTs electrode. Reproduced from Zhu, Y.; Zhang, N.; Ruan, Y.; Zhao, W.; Xu, J.; Chen, H. Anal. Chem. 2016, 88, 5626–5630 (ref 66). Copyright 2016 American Chemical Society.

CONCLUSIONS PEC immunoassays have made great advances and revolutionary developments with the fusion of PEC bioanalysis and immunoreaction in recent years. It is obvious that evident advantages could be offered in terms of high sensitivity and selectivity by the combination of the inherent properties of PEC bioanalysis and immunorecognition. With recent illustrative examples, this review summarized the latest developments in PEC immunoassay, which could also provide an accessible introduction to this booming field for any interested analyst. As can be seen, although previous efforts have led to many important advances and have raised exciting expectations for real-world applications, scientific challenges have arisen as well, mainly due to the insufficiency of selectivity, sensitivity and/or stability to meet the criteria for practical purposes. The search for advanced PEC immunoassays is hence of constant interest in this field in order to develop more simple, robust, rapid, cost-effective and miniaturized PEC


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immunoassay platforms. Current concerns in this field are essentially improvement of the immunoassay systems in terms of simplicity, sensitivity and reliability for the application in detecting analytes in complex matrices. These issues are expected to be addressed in upcoming years with the advancement of PEC detecting technique, nanofabrication technology, and detection protocols. We anticipate that PEC immunoassays will reach the simple and userfriendly platforms enabling rapid, sensitive, and specific assays for widespread applications such as clinical diagnosis and environmental monitoring.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]; [email protected]. *Phone/fax: +86-25-89684862. ORCID Wei-Wei Zhao: 0000-0002-8179-4775 Notes The authors declare no competing financial interest. Biographies Wei-Wei Zhao obtained his Ph.D. from Nanjing University (NJU) in 2012 and now served as an associate professor at the department of chemistry of NJU. His research is focused on biomolecular detection via various advanced electrochemical techniques. He has published more than 60 international papers, and most of them are related to biomolecular detection. Currently,


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he is doing research work under the supervision of Prof. Yi Cui in the Department of Materials Science and Engineering at Stanford University. Jing-Juan Xu is a full professor in the department of chemistry at NJU and has published more than 300 scientific papers. She gained funding from the National Outstanding Youth Foundation of China (2010) and won the “10th Chinese Young Women Scientists Award” (2013). She was selected as a “RSC Fellow” (2014) and approved as the Chang Jiang Professor (2014). Her research interest focuses on developing various electrochemistry-based biosensors. Hong-Yuan Chen is a professor of chemistry at NJU and also an academician of the Chinese Academy of Science. He is a member of several scientific societies and on the advisory boards of several scientific journals. He has authored and co-authored over 800 papers and several chapters and books. His research interests include electrochemical biosensing, bioelectrochemistry, ultramicroelectrodes, biomolecular-electronic devices, and the micro-total analysis system. ACKNOWLEDGMENTS Financial support from the National Natural Science Foundation of China (Grant nos. 21327902 and 21675080), and the Natural Science Funds of Jiangsu Province (Grant BK20170073) is appreciated. This work was also supported by a Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions. REFERENCES (1) Willner, I.; Patolsky, F.; Wasserman, J. Angew. Chem. Int. Ed. 2001, 40, 1861–1864. (2) Curri, M.L.; Agostiano, A.; Leo, G.; Mallardi, A.; Cosma, P.; Della Monica, M. Mater. Sci. Eng. C 2002, 22, 449–452. (3) Pardo-Yissar, V.; Katz, E.; Wasserman, J.; Willner, I. J. Am. Chem. Soc. 2003, 125, 622–623. (4) Dong, D.; Zheng, D.; Wang, F. Q.; Yang, X. Q.; Wang, N.; Li, Y. G.; Guo, L. H.; Cheng, J. Anal. Chem. 2004, 76, 499–501. (5) Haddour, N.; Chauvin, J.; Gondran, C.; Cosnier, S. J. Am. Chem. Soc. 2006, 128, 9693–9698.


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