Enhanced biosensing strategies using electrogenerated chemiluminescence: recent progress and future prospects

Rashaad A. Husain a, Snigdha Roy Barman a, Subhodeep Chatterjee b, Imran Khan c and Zong-Hong Lin *abd
aInstitute of Biomedical Engineering, National Tsing Hua University, Hsinchu 30013, Taiwan. E-mail: linzh@mx.nthu.edu.tw
bDepartment of Power Mechanical Engineering, National Tsing Hua University, Hsinchu 30013, Taiwan
cInstitute of NanoEngineering and MicroSystems, National Tsing Hua University, Hsinchu 30013, Taiwan
dFrontier Research Center on Fundamental and Applied Sciences of Matters, National Tsing Hua University, Hsinchu, 30013, Taiwan

Received 15th November 2019 , Accepted 3rd February 2020

First published on 4th February 2020

Ultrasensitive and highly accurate bioassays are critically required for the early detection of various biomarkers and diagnosis of cancer. Electrogenerated chemiluminescence (ECL) is one such technique which shows powerful analytical ability by incorporating ECL active species for sensitive detection of targets. In this regard, the development of ECL as an assay technique is constantly being pushed for better performance and lower detection limits. Incorporation of sensitive immunosensing and aptasensing methods with ECL has the ability to multiply the advantages several-fold. The recent progress in and methods utilized for the enhancement and amplification of ECL detection techniques based on highly sensitive immunosensors and aptasensors have been discussed in this review with regard to widely popular techniques.

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Rashaad A. Husain

Rashaad Anwar Husain is currently pursuing his PhD under the supervision of Dr Zong-Hong Lin at National Tsing Hua University. His research interests include the development of thermoelectric and triboelectric nanogenerators for smart sensing, energy harvesting and biomedical applications.

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Snigdha Roy Barman

Snigdha Roy Barman is currently pursuing her PhD under the supervision of Dr Zong-Hong Lin at the Institute of Biomedical Engineering, National Tsing Hua University. Her research interests include development of self-powered and portable triboelectric nanosensors for chemical sensing and environmental applications.

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Subhodeep Chatterjee

Subhodeep Chatterjee received his MS degree in 2016 from Department of Electronic Engineering, Chang Gung University. Currently he is pursuing his PhD under the supervision of Dr Zong-Hong Lin at National Tsing Hua University. His research interests focus on the development of solid–liquid based triboelectric nanosensors for the detection of chemical molecules and bioanalytes.

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Imran Khan

Imran Khan is currently a 2nd Year PhD student at the Institute of NanoEngineering and MicroSystems, National Tsing Hua University, under the supervision of Dr Zong-Hong Lin. His research focuses on the development of highly flexible chemical sensors for in-field environmental monitoring.

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Zong-Hong Lin

Dr Zong-Hong Lin is currently an Associate Professor at the Institute of Biomedical Engineering and Department of Power Mechanical Engineering, National Tsing Hua University. His research interests include the understanding of triboelectrification mechanisms, the development of active portable sensors for biomedical monitoring, the design of highly efficient catalysts for environmental purification and self-powered systems with controllable disinfection performance.

1. Introduction

Electrogenerated chemiluminescence (ECL) incorporates both electrochemistry and chemiluminescence (CL), wherein it generates species at electrode surfaces which then undergo electron-transfer reactions to form excited states that emit light.1–3 The first detailed ECL study by Hercules,4 Visco and Chandross5 and Santhanam and Bard6 was reported in the mid-1960s, and since then ECL has been extensively studied and has emerged as a powerful analytical technique.7–14 ECL possesses several advantages over CL, photoluminescence (PL), and electrochemistry. First, in ECL electrochemical reactions, control of the time and position of the light-emitting reaction is possible through the applied potential. Second, ECL is more selective than CL, since the generation of excited states in ECL can be selectively controlled by changing the electrode potentials. Third, in the majority of cases, ECL emitters can be regenerated after emission, resulting in ECL being a non-destructive technique.2 The ECL method has become popular in analytical chemistry and sensor technology, owing to its wide versatility, simple instrumentation, low background signal, wide linear working range, and high sensitivity.5,15–18

Aptasensors are biosensors that use aptamers as recognition elements. Aptamers are single-stranded DNA or RNA molecules that bind target molecules with high affinity and specificity. They are suitable for fast and easy detection of proteins, and their use allows effective immobilization at high density. Moreover, DNA aptamers are mostly robust and can be synthesized with a high grade of purity and reproducibility, enabling easier fabrication processes of biosensors. Aptasensors can be based on electrochemical, optical, and mass transduction using label-free or label-based techniques. Over the past years, studies have incorporated the use of aptamers for ECL sensing of various biomarkers, enzymes and proteins.19–24

Immunosensors are affinity ligand-based biosensing devices that involve the coupling of immunochemical reactions to appropriate transducers. In recent decades, immunosensors have seen rapid development and wide applications with various detection formats.25–27 The general working principle of immunosensors follows specific immunochemical recognition by antibodies (antigens) immobilized on a transducer of antigens (antibodies) in the sample media, which can produce analytical signals dynamically varying with the concentrations of the analytes of interest. Considerable efforts have been devoted to the development of precise, rapid, sensitive, and selective immunosensors by measurement of the markers or pathogenic microorganisms responsible for diseases, such as proteins, enzymes, viruses, bacteria, and hormones.28–32 Due to the high selectivity and sensitivity of ECL based immunosensors, there has been increased development of such a sensing method for the detection of biomarkers for disease, cancer and other bio agents.33–36

Accuracy and sensitivity for the detection of disease-related biomarkers are critical to many areas of modern biochemical and biomedical research.37 For example, the clinical quantification of cancer biomarkers is crucial for early diagnosis and disease monitoring.38,39 In addition, biomarker detection also enables understanding of the fundamental biological processes responsible for disease development and monitoring patient responses to therapy methods.40,41 To achieve ultrasensitive assays, incorporation of techniques for signal enhancement through amplification has been the most popular strategy. Enhanced signal-based ECL methods for assays have been developed with high sensitivity, selectivity, wide dynamic range, and multiplexing capabilities. Popular enhancement strategies include DNA amplification techniques, incorporation of enzyme-assisted signal enhancement, applying new redox-active probes and luminophores, and nanomaterials for higher loading capabilities and improved ECL properties. In this review, we provide the recent progress on the various strategies incorporated for signal enhancement for the development and application of ECL based immunosensors and aptasensors for detection of various biomarkers and bioanalytes (Fig. 1).

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Fig. 1 Schematic of strategies incorporated in enhanced ECL biosensing assays based on immunosensors and aptasensors: DNA amplification-based enhancement, co-reactant, enzymatic and electrode-based enhancement, and nanomaterials-based enhancement.

2. DNA amplification-based enhancement

2.1 Rolling circle amplification

Rolling circle amplification (RCA) is a powerful, but simple, biochemical method that can be used to generate long, linear, tandem, repetitive, single stranded DNA (ssDNA) with the help of a DNA polymerase in the presence of a growing DNA chain over a short circular ssDNA as the template under isothermal conditions. As the synthesized long DNA molecules contain many repeating sequence motifs, RCA has improved performance, so it is widely used in biosensor fabrication.42

Zhang et al. have reported an ultrasensitive strategy for ECL detection of microRNA.43 First, a Y junction was formed with the primer probe, assistant probe and miRNA, which was then cleaved with the addition of Pb2+ to release the miRNA. Subsequently, the released miRNA could initiate the next recycling process, leading to the generation of numerous intermediate DNA sequences. Next, these intermediate sequences were loaded onto the prepared electrode to displace a dopamine-modified DNA sequence and served as an initiator for RCA. In optimized conditions, numerous repeated DNA sequences coupling with hemin to form hemin/G-quadruplex were generated, which showed strong catalytic activity towards H2O2, thereby resulting in an amplified ECL signal with a low detection limit of 0.3 fM.

Wu et al. reported a paper-based electrochemiluminescence origami device (PECLOD) for the detection of IgG antigen using oligonucleotide functionalized carbon dots (CDs) as the ECL luminophore and coupled with the signal amplification ability of RCA (Fig. 2).44 The sensor was comprised of a gold (Au) paper electrode on which the primary antibodies were bound followed by binding of the secondary antibody immobilized with Au nanoparticles (NPs) and conjugated with RCA primer strands, which were subjected to RCA cycles in the presence of a polymerase enzyme. After the RCA process, longer single strands of DNA were generated. The products of RCA included hundreds of tandem repeat sequences, which served as a template for binding of the complementary DNA sequences (in this case DNA nanotags with CDs) to form a linear assembly and amplify the ECL readout signals. Gel electrophoresis resulted in an anticipated slower mobility of the RCA product due to higher molecular weight, confirming efficient RCA based enhancement for the immunoassay. Moreover, the ECL intensity of the immunosensors increased by 10 times after the RCA reaction compared to the pure CD–DNA probe, proving successful signal amplification.

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Fig. 2 RCA based amplification for enhanced sensing of IgG antigen. Reproduced with permission from ref. 44. Copyright (2015) Elsevier.

A method using hyperbranching RCA (HRCA) and a high selectivity aptamer was developed for an isothermal and highly sensitive and specific assay for the determination of ochratoxin A (OTA).45 This method proceeded by immobilizing the capture probe DNA (CDNA) on the gold electrode surface through Au–S interactions, and thereafter the OTA aptamer was modified on the electrode surface through hybridization with CDNA (Fig. 3). Since OTA can competitively bind with the aptamer due to their high affinity, this induced the release of the aptamer from the electrode surface. Subsequently, the free CDNA on the electrode surface hybridized with the padlock probe and induced the HRCA reaction. Thus, the HRCA products, which contained large amounts of double-stranded DNA (dsDNA) fragments, were accumulated on the electrode surface. Since Ru(phen)32+ can intercalate into the groove of dsDNA and acts as an ECL indicator, high ECL intensity was detected from the electrode surface. The enhanced ECL intensity showed high linearity and a low detection limit of 0.02 pg mL−1. Similarly, an HRCA based amplification technique has also been utilised for the enhanced detection of folate receptors (FRs) and the p53 DNA sequence.46,47

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Fig. 3 Highly sensitive and selective assay based on HRCA for detection of ochratoxin A. Reproduced with permission from ref. 45. Copyright (2015) Elsevier.

2.2 DNA nanoassembly

DNA nanoassembly is a simple, effective approach to signal enhancement via target–probe hybridization. If properly designed, DNA can assemble into different structures in two or three dimensions based on long-range, self-assembled, DNA nanostructures as carriers for ECL signal amplification. Various non-enzymatic signal amplification techniques such as catalytic hairpin assembly (CHA),48,49 and hybridization chain reaction (HCR)50–56 are also regarded as efficient strategies for developing ECL sensors with rapid response time, lower noise, simple design and improved sensitivity.57–61

Recently, a novel ECL immunosensor was developed employing a CHA amplification tool for sensing the target protein cysteine-rich protein 61 (CCN1) with [Ru(dcbpy)32+] as luminophores (Fig. 4).62 The target protein was first converted to a specific oligonucleotide tagged on the detection antibody due to the sandwich immunoassay. In addition, the oligonucleotide activated the CHA amplification in the presence of ferrocene labeled hairpin structures to produce massive hybrid structures consisting of ferrocene labeled sticky ends. The massive hybrid structures were then immobilized on a single strand nucleotide modified electrode by the process of hybridization, which abruptly quenched the ECL signals with an increase in the target analyte concentrations. Owing to the amplified signals, the as-developed ECL immunosensors detected CCN1 with a limit of detection of 3.9 fg mL−1, which is at least 4 times higher than the previously reported analytical strategies, and with a wider linear range of 10 fg mL−1–100 ng mL−1, opening the doors for clinical applications.

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Fig. 4 Catalytic hairpin assembly amplification strategy for enhanced detection of CCN1 protein. Reproduced with permission from ref. 62. Copyright (2019) American Chemical Society.

Furthermore, Yu et al. developed a targeted-catalytic hairpin assembly (t-CHA) strategy for enhanced detection of microRNA-21 (miRNA-21).47 This was achieved by combining the signal-amplification capability of both intramolecular and intermolecular ECL co-reactions. In this strategy, two hairpin DNA probes H1 and H2 were designed as capture probes and detection probes, respectively. The capture probes H1 were immobilized on the multilayer interface of a AuNP and thiosemicarbazide (TSC) assembly on a single-walled carbon nanohorns decorated electrode, while the detection probes H2 were anchored on nanocarriers of gold nanoparticle functionalized reduced graphene oxide (Au-rGO) tagged with a self-enhanced ruthenium complex (PEI-Ru(II)). Based on the t-CHA process, the target miRNA-21 triggered the hybridization of H1 and H2 to further be released for initiating the next hybridization process to capture a large number of H2 bioconjugates on the sensing surface. The designed protocol provided ultrasensitive ECL detection of miRNA-21 down to the sub-femtomolar level with a linear response of about 6 orders of magnitude and a relatively low detection limit of 0.03 fM.

ECL immunoassays through HCR amplification have also been reported for the highly sensitive detection of immunoglobulins (IgG), Cardiac Troponin I (cTnI), antimyeloperoxidase (anti-MPO) etc.58–60 The HCR process consists of two single stranded probes which undergo a hybridization process triggered by ssDNA to form a long strand of nicked dsDNA. Recently, HCR has been combined with catalytic amplification techniques to generate co-reactants in situ to further amplify the ECL readouts. Xiao et al. demonstrated a highly sensitive ECL immunosensor for the detection of IgG using the catalytic effect of glucose oxidase (GOD) and hybridization efficiency of HCR.59 First, L-cysteine/reduced graphene oxide (L-cys/rGO) was employed as the sensor substrate to immobilize anti-IgG, which on reaction with the target IgG due to the biotin–streptavidin interaction formed a sandwich immunocomplex. Following this, the streptavidin present on the surface coupled with the biotin labeled single strand DNA (initiator strand) to initiate the HCR process between the strand tagged with GOD and the complementary strand. Therefore, HCR improved the immobilization of GOD onto the sensor surface by allowing multiple GOD to bind because of the hybridization process, which in turn exhibited outstanding catalytic action towards glucose to generate the co-reactant H2O2 for signal amplification. The ECL signal was increased three fold when the long strand of DNA was formed due to HCR, enabling the presence of multiple GOD for efficient catalysis.

Wu et al. reported a novel ECL closed bipolar electrode (BPE) chip designed based on an HCR-induced ECL amplification strategy for the detection of both DNA and H2O2.63 Here, indium tin oxide (ITO) glass coated with two layers of polydimethylsiloxane (PDMS) slices constituted the chip platform. The ITO cathode was modified with Au NPs for further functionalization of biomolecules, which also amplified the ECL signal at the anode of the BPE. Based on the specific hybridization and HCR process, DNA sequences were greatly extended, leading to a significant increase in the resistance of the cathode. The reduction of H2O2 was inhibited on the cathode of the BPE, resulting in a quenching effect on the ECL intensity on the anode of the BPE. As a result, the biosensor was not only employed for DNA assays but also used in enzyme reactions based on the generation of H2O2. Similarly, a study was reported by Huang et al. where a highly selective and sensitive ECL biosensor for the detection of NF-κB p50 was developed, which combined the high selectivity of the proximity hybridization assay (PHA) with the high efficiency of HCR.57

The rapid advancements in the field of immunosensing have led to the development of novel methods to further increase the sensitivity of the biomarker detection process. One such example is the concept of forming a supersandwich assembly to amplify the ECL signals by utilizing multiple signal probes in a 1[thin space (1/6-em)]:[thin space (1/6-em)]Nn amplification ratio (i.e., a large number of signal probes are introduced for one target) instead of the conventionally used 1[thin space (1/6-em)]:[thin space (1/6-em)]1 ratio of target to signal.64 Relying on their hybridization principle, DNA molecules have been reported for generating supersandwich structures, thereby allowing one to amplify the probe amount and hence enhance the ECL signals. In this system, a porous Au electrode was used as the substrate, which provided a large number of binding sites for graphitic carbon nitride (g-C3N4) nanosheets (NS) modified with Au NPs and ssDNA (Fig. 5). Via the DNA hybridization process, ssDNA formed a double helix due to which a number of g-C3N4 NS were bound to the electrode surface, leading to the formation of a supersandwich assembly, ultimately enhancing the ECL signal. Moreover, the authors have suggested that the ECL intensity increased with the increase in the number of layers of the supersandwich assembly. The highly amplified signal lowered the limit of detection of rabbit IgG to 0.001 fg mL−1, which was three orders lower than the previously reported immunosensors.

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Fig. 5 Supersandwich assembly based highly sensitive immunoassay for rabbit IgG detection. Reproduced with permission from ref. 64. Copyright (2019) Elsevier.

In the ECL system, a ratiometric ECL biosensor is a relatively new ECL detection method and has attracted increasing research interest due to its high detection accuracy.65,66 Based on this strategy, Zhu et al. developed a novel ratiometric ECL–EC hybrid biosensor with high accuracy and reproducibility for the ultrasensitive detection of miRNA-133a.67 In this strategy, three vertices of DNA tetrahedron nanostructures (DTN) with thiol groups were highly ordered and assembled on the surface of an Au electrode through Au–S bonds, while the other vertex had an additional capture sequence. Two Ru(bpy)32+-labeled hairpins (H1 and H2) were used as ECL probes and fuel strands for HCR. With the help of this DTN and HCR-based dual amplification strategy, a detection limit of 12.17 aM for miRNA-133a was obtained.

Strand-displacement amplification (SDA) is another method that can overcome the limitations of other chain reaction-based amplification techniques, where the requirements are high and expensive, while sometimes being prone to mispriming and inadequate template amplification. SDA is carried out under isothermal conditions. It is inspired by normal physiological RNA transcription and DNA replication, which occurs at a constant temperature. Over the past 2 decades, SDA has been widely used as an alternative to polymerase chain reaction (PCR) for the detection of pathogens,68,69 hereditary diseases,70 and cancers.71–73 Moreover, SDA amplified nucleic acids can be multiplexed and readily provide optical and visual readouts.68,74,75 Utilising this strategy, target induced cycling SDA mediated by phi29 DNA polymerase (phi29) was first investigated and applied for miRNA detection (Fig. 6).76 The target miRNA triggered the phi29-mediated SDA, which could produce large amounts of ssDNA (assistant probe) with accurate and comprehensive nucleotide sequences. Furthermore, a self-enhanced Ru(II) ECL system was designed to obtain a stable and strong initial signal to further improve the ECL signal sensitivity. This ECL assay strategy for miRNA-21 detection showed excellent sensitivity of a concentration variation from 10 aM to 1.0 pM and a limit of detection of 3.3 aM. Another study for the detection of miRNA-21 using an ultrasensitive ECL biosensor has been reported.77 Here, a 3,4,9,10-perylenetetracarboxylic acid–luminol composite (PTCA–luminol) was used as a signal tag and applied in a cruciform DNA structure mediated exponential strand displacement reaction (SDR). The target miRNA-21 triggered disaggregation of the cruciform DNA structure was used to mediate exponential SDR for target recycling amplification, achieving excellent sensitivity with a wide linear range from 10 aM to 100 pM and a much lower detection limit of 2 aM. Similarly, more recent methods based on SDA strategies have been reported for ultrasensitive detection of miRNA.78–80

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Fig. 6 Strand displacement amplification strategy for the detection of miRNA. Reproduced with permission from ref. 76. Copyright (2015) American Chemical Society.

2.3 DNA walkers

With the continuous development of DNA nanotechnology, researchers have focused increasing attention on the use of DNA molecules with diverse structural and functional properties to construct nanomachines. Among the various molecular machines, DNA walkers are one of the most sophisticated DNA devices either in design or construction. Due to their excellent abilities, DNA walkers have great potential applications in biosensors,81 molecular computing,82 cargo delivery,83 programmed synthesis,84 logic gate devices,85 and so on. DNA walkers are synthetic molecular machines that move along tracks designed in one dimension,86 two dimensions,87,88 or three dimensions,89,90 and are attracting increasing interest in biosensors for accurate and effective signal amplification.

Recently, an ECL biosensor was constructed on the basis of an amino-modified 3,4,9,10-perylenetetracarboxylic dianhydride/luminol (PTC-NH2/Lu) nanocomposite as an emitter and a bipedal DNA walker signal amplification strategy for ultrasensitive detection of the miRNA-21 biomarker (Fig. 7).91 The target miRNA-21 triggered bipedal DNA walker was powered by a toehold-mediated strand displacement reaction (TSDR) for signal amplification. Consequently, the proposed ECL biosensor achieved ultrasensitive detection of miRNA-21 with a linear range from 100 aM to 100 pM and a limit of detection of 33 aM.

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Fig. 7 DNA walker based amplification process for the detection of miRNA-21. Reproduced with permission from ref. 91. Copyright (2018) American Chemical Society.

Another study reported an ECL aptasensor designed using carbon nitrite with hollow porous structures (HP-C3N4) as the luminophores and trimetallic component AuPtAg as the reaction accelerator for highly sensitive detection of insulin with a DNA walker as the signal-amplifying probe.92 Here, a triple helix molecular switch (THMS) approach was adopted for transforming the aptamer/target protein complex in the presence of insulin into nucleic acids, which were later amplified by the Nb.BbvCI endonuclease powered DNA walkers simply by releasing the quencher molecule from the electrode surface. As a result, the as-developed ECL aptasensor was able to detect insulin concentrations with a detection limit of 17 fg mL−1 and a good linear range of 0.05 pg mL−1 to 100 ng mL−1. Signal enhancement based on DNA walkers has also been employed for the ultrasensitive detection of mycotoxins, namely OTA with CdS QDs as the luminophores.93 For the detection, two different kinds of DNA were used, namely a dsDNA containing aptamer and DNA walker as the first and Cy5-ssDNA consisting of a endonuclease element as the second. In the original state, the signal between the CdS QDs modified on the electrode surface was quenched by Cy5-DNA due to ECL resonance energy transfer (ECL-RET). When OTA was introduced, it cleaved the aptamer away from the dsDNA due to stronger interactions, leaving the free walker to hybridize with the Cys5-ssDNA, followed by cleaving of the DNA by endonucleases, ultimately releasing a large amount of Cys5-DNA molecules from the electrode surface, thereby enhancing the ECL signal. Owing to the significant signal amplification, the proposed ECL aptasensor detected OTA in the range of 0.05 nM to 5 nM with a limit of detection of 0.012 nM.

3. Co-reactant, enzymatic and electrode-based enhancement

3.1 Co-reactant-based enhancement

Generally, nanomaterials like quantum dots, Ru complexes and luminol are used as ECL reagents for construction of immunosensors. Amongst all, Ru tris(2,2′-bipyridyl)ruthenium(II) (Ru(bpy)32+) has gained immense attention as an ECL luminophore owing to its strong luminescence, high quantum yield, longer luminescence lifetime, superior stability in solutions with different pH etc.94–98 To enhance the performance of Ru(bpy)32+ based immunoassays, various co-reactants are introduced into the system as signal amplifiers for detection of ultralow concentrations of biomarkers. Immunosensors functioning on the basis of the mechanism of co-reactant based ECL have displayed numerous advantages such as high ECL efficiency, faster response time, favourable stability and compatibility.99,100

To date, peroxydisulfate, oxalate, and triphenylamine (TPA) have been extensively used as co-reactants to enhance the ECL signals. Specifically, amine group compounds such as TPA are the most widely used co-reactant for Ru(bpy)32+ based ECL systems. Nevertheless, their use is limited due to their inherent toxicity, low biocompatibility and high volatility.101–103 Therefore, researchers have started to direct their attention towards alternative amine based co-reactants to achieve amplified ECL signals. Novel macromolecules such as polyamidoamine (PAMAM) dendrimers with hyper branched structures and a high density of functional groups at the terminal sites are extensively studied for designing biosensors. Moreover, amine terminated PAMAM dendrimers with many secondary and tertiary amine groups have successfully demonstrated the role of co-reactants for significantly amplifying the ECL immunosensing signals. For example, Liu et al. developed an ultrasensitive immunosensor for detection of thyroid stimulating hormone (TSH) integrating a PAMAM dendrimer–norafloxacin complex as co-reactants to enhance the ECL signal of the peroxydisulfate oxygen (S2O82−–O2) system.104 Being a strong oxidizing agent, SO4˙ oxidized the PAMAM–NFLX complex to form PAMAM–NFLX˙+, which was further deprotonated to produce the radical PAMAM–NFLX˙. The formed radical possessed a strong reducing ability due to which it was able to reduce the oxygen dissolved in the solution to form HO2˙. On reaction with another HO2˙ radical, it generated oxygen free radicals (O2), thereby amplifying the ECL emission signals. Owing to the generation of amplified signal, the as-developed immunosensor was able to detect TSH concentrations as low as 0.02 μIU mL−1, which is the concentration of the analyte found in human blood. In addition, PAMAM dendrimers as co-reactant based ECL amplifiers have been studied for precise detection of other biomarkers such as 5-hydroxymethylcytoine (5hmC),105 IgG,106 prostate specific antigen (PSA),99 β-adrenergic agonsits (β-AA)107 and α-fetoprotein (AFP).75

Recently, in situ generation of co-reactants has also been reported as an efficient strategy for improving the sensitivity of ECL immunosensors. In 2016, Jiang et al. reported the fabrication of ECL immunosensors based on the N-(aminobutyl)-N-(ethylisoluminol) (ABEI) compound for detection of laminin.108 The system consisted of PdIr nanocubes on which ABEI and signal-amplifying co-reactant L-cysteine were immobilized, forming a self-enhanced nanocomplex PdIr–L-Cys–ABEI as shown in Fig. 8. The results clearly showed that the ECL intensity was enhanced by 2 times when L-Cys was added. The reason for the amplified signal was that L-Cys loses an electron to form a strong oxidant radical L-Cys˙, which on reaction with oxidative intermediates of ABEI significantly strengthened the ECL signal. Besides the co-reactant, the PdIr nanocubes also behaved as mimic peroxidases catalysing the H2O2 decomposition, further generating free radicals, resulting in synergistically enhanced ECL emission. It is noteworthy that by simply increasing the amount of in situ generated co-reactants such as L-cysteine etc. over the electrode surface, highly sensitive detection of β-AA,109 and Prostagladin E1110 has been reported. Furthermore, other amino acids namely arginine with amine terminals have also been employed as co-reactants to amplify the ECL signals of Ru(bpy)32+ systems for development of an immunosensor for determining levels of apurinic/apyrimidinic endonuclease 1 (APE-1).111 Two molecules of arginine (Bi-Arg) were immobilized on the AuNP/Fe3O4/rGO complex by the self-assembly process. Apart from being a co-reactant, Bi-Arg also acted as a nanocarrier for loading of a secondary antibody. A significant increase of 181% in the ECL intensity of Bi-Arg/AuNP/Fe3O4/rGO was observed compared to the 78% increase in the intensity of Arg/AuNP/Fe3O4/rGO due to the presence of more active groups. The co-reactant based signal amplification relied on the formation of free radical intermediates in the reaction between Ru(bpy)32+ and Bi-Arg. In addition, other in situ generated co-reactants such as hydrogen peroxide (H2O2),112–114N-hydroxysuccinimide (NHS)115 and NADH have also enabled ECL signal amplification for Ru(bpy)32+ systems in order to develop ultrasensitive immunosensors.101,102

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Fig. 8 ABEI compound-based detection of laminin incorporating an L-cysteine co-reactant for signal enhancement. Reproduced with permission from ref. 108. Copyright (2016) Royal Society of Chemistry.

3.2 Enzymatic reaction-based enhancement

Signal amplification by using enzyme labels such as horseradish peroxidase (HRP) and GOD has also proved to be a promising strategy for obtaining higher sensitivity in clinical immunoassays based on ECL. By participating in an enzymatic reaction, these enzymes can generate or consume co-reactants in situ, which contributes towards obtaining lower detection limits of the sensors. For instance, hydrogen peroxide (H2O2) is the most widely used co-reactant, which can either be generated by oxidation of glucose catalyzed by GOD in the presence of oxygen or is consumed by using HRP.

Depending on the type of enzyme used the concentration of co-reactants decreases or increases over the electrode surface in order to achieve intense signal amplification. To date, the HRP enzyme as a signal amplifying agent has been used to enhance ECL immunosensors utilizing quantum dots as luminols.116–121 In 2013, Yao et al. reported an ECL immunosensor fabricated via a layer-by layer approach using CdSe quantum dots (QDs) for the detection of a toxic compound clenbuterol (Fig. 9).118 At a particular potential, the dissolved oxygen was electrochemically reduced to H2O2 due to which strong ECL emission of the QDs was observed. This sandwich type assay comprised a secondary antibody tagged with HRP bound to the electrode surface to form an immunocomplex. The formation of the immunocomplex caused steric hindrance leading to the decrease in the ECL emission of the QDs due to the reduced electron transfer speed of dissolved oxygen. The ECL intensity of the immunosensors decreased upon addition of the enzymatic substrate hydroquinone (HQ) in the solution owing to the consumption of H2O2, which was further amplified by the enzymatic cycle, greatly improving the sensitivity and broadening the detection range of the immunosensors. The sensor response clearly showed that the enzymatic process influenced the ECL quenching ability in the presence of HQ, due to which the decrease in the ECL signal with the decrease in the clenbuterol was more prominent compared to the system without HQ, thus ensuring better performance of the enzyme-cycle amplified sensor. Most importantly, the LOD and detection range of the as-developed sensor for clenbuterol sensing were found to be 0.02 ng mL−1 and 0.05–1000 ng mL−1 respectively, which were much lower compared to previously reported sensors. Recently, attempts have been made to improve the efficiency of the enzymatic amplification process by introducing nanoparticles, which act as a surface matrix for anchoring an increased number of HRP molecules due to their high surface area to volume ratio. Yan et al. have fabricated ECL immunosensors for sensing ractopamine (RAC) utilizing L-cysteine protected CdSe (QDs) and Au NP coupled HRP moieties.116 The sensing mechanism was based on the catalysis of o-phenylenediamine (OPD), instead of HQ mentioned earlier, by the HRP enzyme to consume the co-reactant H2O2. When the concentration of the target analyte RAC increased, there was a decrease in the amount of HRP, ultimately enhancing the ECL intensity. Specifically, the enzyme based amplification increased the sensitivity and detection range of the as-developed sensor and the AuNPs played the role of nanocarriers to increase the loading of HRP, which in-turn sped up the electron transfer for signal amplification. Besides HRP, other enzymes such as GOD are also used as an enzyme label to enhance the ECL intensity of a sandwich immunosensor for determining α-1-fetoprotein levels.122 The immunosensor was constructed employing platinum/gold alloys bound to zinc oxide nanocomposites (Pt–Au@ZnONP), on which the secondary antibody and GOD were immobilized to form the immunocomplex. The loaded GOD catalysed the oxidation of glucose to produce the co-reactant H2O2in situ to enhance the ECL response. Furthermore, the H2O2 was converted to reactive oxygen species (ROSs) catalyzed by the Pt–Au@ZnONP, which dually enhanced the ECL signals for developing ultrasensitive immunosensors.

image file: c9tb02578b-f9.tif
Fig. 9 Detection of the toxic compound clenbuterol from the enhanced ECL signal due to quenching of QD emission by steric hindrance. Reproduced with permission from ref. 118. Copyright (2013) Elsevier.

Additionally, for the detection of miRNA, Sun et al. fabricated an ECL sensing platform for circulating miRNAs utilizing an AuNP@G-quadruplex (G4) spherical nucleic acid enzyme (SNAzyme) as a nanocatalyst, which showed good stability, strong nuclease resistance, and improved catalytic performance toward a luminol–H2O2 ECL system.123 In the presence of target miRNA, the probe hairpin DNA (probe hp) was opened via hybridization with the toehold near the surface of the electrode. Subsequently, the other end of the probe was exposed to initiate cascade amplification, and a long chain with a lot of capture DNA was produced. Finally, the SNAzyme was attached onto the electrode via hybridizing with the capture DNA. Such a strategy not only exerted the strong catalytic ability and stability of the SNAzyme but also combined the nucleic acid amplification technique, resulting in high sensitivity of detecting AMI-related miRNA in real blood samples. In this way, two AMI-related miRNAs were detected accurately with a low detection limit of 0.4 fM.

Ribonuclease A (RNase A) is increasingly considered as a biomarker for tumor diagnosis, and based on the fact that RNase A can specifically hydrolyze RNA at the site of ribonucleotide uracil (rU), Ni et al. designed an ECL based RNase A detector incorporating a rU containing chimeric DNA probe and labelled with Ru(bpy)32+ (acting as an ECL indicator).62 The chimeric DNA probe hardly diffused to the surface of the negatively charged ITO electrode due to the strong electrostatic repulsion between the negatively charged DNA and ITO electrode, resulting in a weak ECL signal. In the presence of RNase A, the chimeric DNA probe was hydrolyzed into small fragments containing less negative charge and hence, due to the decreased electrostatic repulsion, diffused easily to the ITO electrode surface. This strategy for ECL signal enhancement resulted in a linear relationship between the ECL signal and the concentration of RNase A in the range of 0.001–0.10 ng mL−1, with a detection limit of 0.2 pg mL−1.

3.3 Electrode-based enhancement

Recent studies have highlighted that elevating the electrode surface temperature can efficiently increase the ECL intensity, enhancing the performance of immunosensors. Hence, various photothermal agents, which have the ability to convert laser energy and raise the electrode temperature by manipulating the kinetic and thermodynamic aspects of the reactions, have been reported for construction of ECL immunosensors. Lin and co-workers have employed high efficiency near infra-red (NIR) photothermal agent carbon nanohorns (CNHs) modified with polymer dots (Pdots) to enhance the sensitivity of immunosensors for detection of human epididymis protein 4 (HE4) via photothermal amplification.46 When irradiated with an 808 nm laser for 30 s, the ECL intensity of the sensor surface increased by 2.5 times due to the fact that the irradiation process increased the electrode surface temperature, which increased the diffusion and convection of the active materials to the electrode surface, thereby enhancing the sensitivity and detection limit of the immunosensors. Not only a photonic source, but even electrical sources provided temperature control of electrodes. Ni et al. have proposed the use of an electrically heated ITO electrode for temperature treatment in a DNAzyme-based Pb2+ ECL sensor.124 The temperature control during the process of DNA hybridization and DNAzyme reaction was adjusted precisely by a heated ITO electrode instead of the typical bulk solution heating process. Y-shaped double strand DNA (dsDNA) was formed through the hybridization of the DNAzyme, substrate and capture DNA. Subsequently, Ru(phen)32+ was embedded into the grooves of dsDNA and acted as the ECL indicator (Fig. 10). The performance of the biosensor was further improved at an elevated electrode surface temperature because the temperature affected the performance of the ECL of Ru(phen)32+ greatly. The detection limit achieved at 45 °C was 0.2 pM, approximately 5 times lower than that at a 25[thin space (1/6-em)]°C electrode surface temperature.
image file: c9tb02578b-f10.tif
Fig. 10 Electrode heating effect resulting in enhanced ECL signal for ultrasensitive detection of Pb2+. Reproduced with permission from ref. 124. Copyright (2019) Elsevier.

4. Nanomaterial-based enhancement

4.1 Nanoparticles

Nanoparticles exhibit excellent electrochemical, photonic, and magnetic characteristics, which allow them to be superior transducers for ECL sensing designs.

Interestingly, Au NPs are the most widely employed nanomaterials in ECL based biosensors due to their excellent binding affinity towards different biomolecules such as antibodies and due to their large surface-to-volume ratio, low toxicity, good conductivity/catalysis, and biocompatibility.125,126 Liu et al. reported an immune-sensing assay of C-peptide based on label-free ECL signalling in which the basal electrode was fabricated on ITO glass as a conductive substrate and decorated by Au NPs with hydrolyzed (3-aminopropyl)trimethoxy silane as the linker as shown in Fig. 11.127 The sensor could detect C-peptide concentrations between 0.05 ng mL−1 and 100 ng mL−1 with a detection limit of 0.0142 ng mL−1. Enhanced ECL performance was reportedly obtained due to the oxidation of luminol through the increment of d band electrons in the Au NPs. In a related study, Zhai et al. used an Au NP/ITO based electrode to immobilize the I27L gene for realizing a label free aptasensor for diabetes. The single-stranded oligonucleotide as a capture probe (CP) was immobilized via Au–S bonds directly.128 Hybridization between the probe and target strand was achieved by quenching the ECL of the luminol indicator. Without any chemical reaction, the sensor response was triggered by the transfer dynamics of the ECL probe. Hence, a stable, reproducible and sensitive mechanism was obtained. The range of the sensor varied from 100 nM to 10 pM with a LOD of 8.1 pM.

image file: c9tb02578b-f11.tif
Fig. 11 (a) Fabrication and sensing mechanism of an electrochemiluminescent biosensor based on AuNP functionalized ITO for a label-free immunoassay of C-peptide. (b) The effects of the anti-C-peptide concentration on the ECL intensity of AuNP functionalized ITO for a label-free immunoassay. Reproduced with permission from ref. 127. Copyright (2018) Elsevier.

Remarkable enhancement in the optical signals of ECL has been reported due to the phenomenon of localized surface plasmon resonance (LSPR), wherein the free electrons collectively oscillate in metal nanostructures inspired by an electromagnetic (EM) field.129–133 Studies incorporating noble metal nanoparticle-based LSPR enhancement in ECL biosensing assays have been reported recently with good sensing performance.134,135 Li et al. developed an ultra-sensitive biosensor with Au NP dimers, based on the LSPR-enhanced ECL mechanism.96 The Au NP dimers were constructed by hybridization of DNA strands on the surface of an electrode modified with CdS nanocrystals after an enzyme-assisted cyclic amplification process. The plasmon-coupling-induced EM field enhancement effects of the Au NP dimers resulted in an increase in ECL intensity of 6.3-fold, and a detection limit for microRNA-21 as low as 3.6 fM with a wide linear range from 10 fM to 20 pM. Similarly, Feng et al. developed a surface plasmon-enhanced ECL (SPEECL) system for the detection of miRNA-21.97 In this study, DNA templated silver nanoclusters (DNA-AgNCs) acted as ECL emitters and AuNPs as the LSPR source. This system reportedly achieved a lower detection limit of 0.96 aM, and a linear range from 1 aM to 104 fM. Wang et al. reported a new approach for the ultrasensitive assay of carcinoembryonic antigen (CEA) in human serum based on LSPR enhanced ECL of Ru(bpy)32+.98 In this surface-enhanced ECL (SEECL) sensing system, Ru(bpy)32+-doped SiO2 nanoparticles (Ru@SiO2) acted as ECL luminophores, and Au NPs were used as an LSPR source to enhance the ECL signal. Two different kinds of aptamers specific to CEA were modified on the surface of Ru@SiO2 and Au NPs, respectively. One layer of Ru@SiO2–AuNP generated about a 3-fold ECL enhancement compared with the ECL without the presence of Au NPs. Furthermore, a 30-fold ECL enhancement was obtained by a multilayer nanoarchitecture of Ru@SiO2–AuNP. This resulted in an excellent detection limit of 1.52 fg mL−1 of CEA in human serum, one of the lowest limits achieved for CEA assays via ECL sensing.

Highly interesting studies have reported that novel polydopamine (PDA)-based ECL-organic nanoparticles (EONs) have also been utilized for biosensing due to several reasons such as: (i) from the oxidation of catechol, PDA based EONs can be synthesized through dopamine self-polymerization under alkaline conditions by covalent bonding, hydrogen bonding, π–π interactions, and so on, and (ii) the high proportion of immobilized carbonyl and carboxyl groups on the EON surface enhances the ECL reaction with oxygen-containing intermediate radicals and results in high ECL efficiency.136,137 Li et al. utilized such a kind of biosensor for the detection of parathyroid hormone in the range of 0.05–8 ng mL−1 with a detection limit of 17 pg mL−1.138

4.1.1 Nanoclusters. Metallic nanocluster materials have reportedly been employed as luminophores in ECL immunoassays, such as gold nanoclusters (Au NCs), which have become an ideal ECL emitter due to their size-tunable luminescence and high quantum yield.139 In particular, bovine serum albumin (BSA)-templated Au NCs with good biocompatibility can function as signal labels for the indication of the target concentration. Moreover, they also act as a connector for the modification and immobilization of immune molecules via abundant functional groups provided by BSA.140 In a reported study, Jia et al. developed an Au NC based low potential cathodic luminophore for the detection of procalcitonin (PCT) with a detection limit of 2.90 fg mL−1.57 Here, K2S2O8 was catalyzed by highly branched Cu2O, which generated a higher amount of radical anion SO4˙. This oxidized Au NCs˙ to generate an increasing number of high-energy-state Au NCs*, thus doubling the ECL intensity (Fig. 12).
image file: c9tb02578b-f12.tif
Fig. 12 The fabrication and sensing mechanism of an Au nanocluster based low potential cathodic luminophore for the detection of procalcitonin. Reproduced with permission from ref. 57. Copyright (2019) Elsevier.

Apart from Au, other metallic nanocluster-based ECL enhancement systems have also been reported. Huang et al. developed a novel ratiometric ECL sandwich immunosensing assay with a luminol/palladium nanocluster (Pd NC)@graphene oxide probe for the detection of CEA with a linear range of 1.0–100 pg mL−1 and a detection limit of 0.62 pg mL−1.141 The enhanced ratiometric ECL signal was obtained due to the high density and excellent electrocatalytic activity of the loaded Pd NCs. In another study, Liao et al. developed an ECL aptasensor for detecting miRNA based on in situ generation of copper nanoclusters (Cu NCs) as a luminophore and titanium dioxide (TiO2) as a coreaction accelerator.142 The target miRNA (dsDNA) was amplified using the mechanism of HCR, which reduced the self-etching effect of Cu NCs due to aggregation and improved the emission performance of Cu NCs. As an immobilizer, TiO2 was introduced, which enhanced the sensing interface and also acted as the coreactor to accelerate the reduction of the coreaction agent S2O82− for enhancing the ECL efficiency of Cu NCs significantly. The sensor exhibited a detection range of 100 aM to 100 pM with a LOD of 19.05 aM.

4.2 Hybrid nanomaterials

Hybrid nanomaterials or nanocomposite materials are a certain class of materials that have a good capability to improve the sensitivity, selectivity and reliability of ECL based bioassays.

Composites based on Au coupled with 2D materials such as rGO have been widely reported in ECL systems for biosensing. Yang et al. reported a novel ECL signal-amplifying immunosensing strategy using Au NP functionalized rGO capped Fe3O4 (Au-FrGO) hybridized with CeO2@TiO2 for the detection of CEA with a wide linear range of 0.01 pg mL−1 to 10 ng mL−1 and a detection limit of 3.28 fg mL−1.143 In the presence of CeO2, CeO2@TiO2 exhibited better ECL activity than TiO2 with peroxydisulfate as a coreactant. After hybridizing with AuNPs, FrGO exhibited better electrical conductivity and could immobilize a higher amount of CeO2@TiO2 and antibodies, thereby resulting in enhanced ECL detection. In another study, they also proposed an ultrasensitive ECL immunosensor with polyaniline nanorod array grafted reduced graphene oxide (PANI NR/rGO) hybridized with Au NPs (PANI NR/rGO-Au) as a sensing substrate.144 The specificity of the sensor was improved by the HWRGWVC heptapeptide (HWR) as a specific capture-antibody (Ab1) immobilizer, which was introduced to construct a PANI NR/rGO-Au-HWR sensing interface as shown in Fig. 13. The synergistic effect of the proposed interface improved the incubation efficiency of antibodies on the substrate, with faster electron-transfer for ECL enhancement due to the densely oriented 3D PANI NRs. The fabricated sensor was employed to detect PCT with a wide linear range of 100 fg mL−1 to 50 ng mL−1 and a LOD of 50 fg mL−1. In another study, SiO2@PDA was utilized to quench the ECL intensity of luminol. Mesoporous silica (SiO2) nanoparticles have attractive features, such as large area, adjustable pore size, external surface chemistry and inherent biocompatibility.145 Xing et al. utilized this nanocomposite material to develop an ECL immunoassay for the detection of insulin in which SnO2/rGO/Au NP nanocomposites combined with luminol were employed to construct the base of the immunosensor.146 Here, the Au NPs that functionalized SnO2/rGO played an indispensable role as a bridge reagent for the incubation with the primary antibody (Ab1) by Au–N bonds and simultaneously enhanced the sensitivity of the immunosensor. The sensor showed a reliable linear range of 0.0001–50 ng mL−1 with a detection limit of 26 fg mL−1.

image file: c9tb02578b-f13.tif
Fig. 13 Schematic diagrams of the fabrication process of nanorod array grafted reduced graphene oxide (PANI NR/rGO) hybridized with gold nanoparticles (PANI NR/rGO-Au) as a sensing substrate and possible mechanism for ECL emission. Reproduced with permission from ref. 144. Copyright (2018) Elsevier.

In a related study, Liu et al. developed an aptasensor for the detection of miRNA based on nicking enzyme NbBbvcI mediated signal amplification (NESA).147 In this work, GO/Au nanocomposites were used for immobilizing hairpin probe2 (HP2), and nitrogen doped QDs (N-QDs) were linked with hairpin probe1 (HP1) to enhance the selectivity (Fig. 14). HP1-N-QDs were reacted with the assistant probe and target miRNA to form a Y junction structure. The nicking enzyme cleaved the Y junction and released the target miRNA, assistant DNA, and generated intermediate N-CQD-DNA (S1), which provided signal amplification with the coreactant S2O82−. As a result, target miRNAs could be detected quantitatively by adjusting the ECL signal to the concentration. The detection of target miRNA was in the range of 10 aM to 10 fM, with a LOD of 10 aM. As another example, Li et al. used a gold nanoparticle–graphene (GNP–graphene) composite to detect thrombin.148 They reported a sandwich biosensor for detecting thrombin using the ECL method based on a dual amplification strategy. The device detected thrombin concentrations in a range of 0.01–10 nM and the LOD was as low as 6.3 pM.

image file: c9tb02578b-f14.tif
Fig. 14 Schematic of the N-CQD based biosensor with the mechanism and signal enhancement. Reproduced with permission from ref. 147. Copyright (2017) Elsevier.

A polyaniline–gold (PANI–Au) based hybrid nanomaterial which forms nanocomposites with g-C3N4 has been reported for the development of ECL immunosensors. PANI–Au composites exhibit a pair of redox peaks and high catalytic activity. Zhao et al. developed anti-cyclic citrullinated peptide (CCP) antibody sensors based on a dumbbell-like PANI–Au nanocomposite, which exhibited a synergetic enhancement effect on the ECL behaviors of g-C3N4. The proposed immunoassay was utilized for the detection of anti CCP with a broad linear range of 0.001 to 15 ng mL−1 with a lower detection limit of 0.2 pg mL−1.

Incorporation of Ag-based composites in ECL systems has also been recently reported. Wang et al. developed a ratiometric ECL aptasensor for detection of HL-60 cancer cells using chemically and thermally stable g-C3N4 material and Ag–polyamidoamine (PAMAM)–luminol nanocomposites as an ECL sensing system.149 In this work, they functionalized probe DNA (pDNA) and bio-bar-code DNA (bbcDNA) on the Ag–PAMAM–luminol nanocomposite and the resultant composite was hybridized with an aptamer on magnetic beads (Fig. 15). After the addition of HL-60 cells, the process led to the release of pDNA–Ag–PAMAM, and g-C3N4 was immobilized on the electrode surface and functionalized with capture DNA. The complementary DNA on the other hand was hybridized with the released Ag–PAMAM–luminol NCs and immobilized on the electrode. The ECL signal of the g-C3N4 NS decreased while the luminol intensity increased in the presence of coreactant H2O2. The cell concentration directly corresponded to the change in the ratio of the two signals in a range varying from 200 cells per mL to 9000 cells per mL and a detection limit of 150 cells per mL was shown. Khan et al. reported an ECL-RET immunosensor for the detection of insulin with a linear range of 0.0001–80 ng mL−1 and LOD of 0.02 pg mL−1.150 In this immune-sensing assay, silver phosphate (Ag3PO4) was utilized as a novel ECL donor whose emission could be remarkably increased by the synergetic assistance of GO with Ag NPs.

image file: c9tb02578b-f15.tif
Fig. 15 Schematic of HL60 cancer cell detection by a ratiometric electrochemiluminescence sensing system. Reproduced with permission from ref. 149. Copyright (2016) Elsevier.

In an interesting study, Zhang et al. reported for the first time a liquid metal nanoparticle based ECL immunosensor.151 They developed g-C3N4 conjugated PDA coated Galinstan liquid metal shell–core nanohybrids (g-C3N4@Galinstan-PDA) for highly sensitive HeLa cell derived exosome analysis. Galinstan played the role of a nanoprobe and the antibody-modified g-C3N4@Galinstan-PDA was utilized for specific recognition of exosomes, exhibiting stable and strong ECL signals due to the excellent features of the Galinstan NPs, which further facilitated electron transfer and suppressed the g-C3N4 passivation during electrochemical reduction procedures. Thus, the ECL based sensor could analyze HeLa cell derived exosomes with a LOD of 31 particles per μL.

Alternatively, the detection of anti-oncogene has been studied using a nanofiber-based ECL sensing technique.152 Wang et al. fabricated an ECL biosensor for the detection of CdkN2A/p16 antioncogene using functional electrospun nanofibers, which involved multiwalled carbon nanotubes (MWCNTs) doped with polycaprolactam 6 (PA6) forming PA6–MWCNT core–shell luminescent composite nanoparticles. For the luminol, RuAg@AuNP was used. A sandwich structure of ssDNA1-CdkN2A/p16 anti-ssDNA2 (RuAg@Au-ssDNA2) was formed through a hybridization reaction. Adding TPrA to the luminophore amplified the ECL recognition signal and excellent performance was achieved with a detection limit of 0.5 fM, making the sensor a promising biomarker for detecting tumors.

Novel studies involving unique nanostructures have also been reported for ECL based sensing. Jia et al. developed a bioactivity-protected ECL biosensor using Cu2S snowflakes as the co-reaction accelerator for procalcitonin analysis with a detection limit 2.36 fg mL−1.153 With the advantages of good conductivity and high surface area, Cu2S snowflakes served as a satisfying substrate for connecting immune molecules. Moreover, they also acted as a co-reaction accelerator to generate more cationic radicals TEA˙+, which enhanced the ECL intensity for requirements of trace analysis. Fang et al. reported a ECL immunosensing platform for ultrasensitive detection of sialic acid (SA) with Bi nanobelts (Bi NBs) as a coreactant to prepare a self-enhanced Ru(bpy)32+–Bi NB composite, which generated a strong ECL signal by shortening the electronic transmission distance and reducing the energy loss.154 Under optimal conditions, the sandwich-like renewable ECL biosensor enabled sensitive detection of SA in the range of 35 fg mL−1 to 350 ng mL−1 with a low detection limit of 11.7 fg mL−1.

As a separate study, Hu et al. reported an Ru-complex-grafted 2D metal–organic layer (MOL) with excellent ECL efficiency as a sensing platform for simple and ultrasensitive detection of mucin 1 for the first time as shown in Fig. 16.155 They utilized PEI@Ru-Hf-MOL as a highly sensitive detection assay of mucin 1, which showed wide linearity from 1 fg mL−1 to 10 ng mL−1 and a low detection limit of 0.48 fg mL−1.

image file: c9tb02578b-f16.tif
Fig. 16 The (A) construction and (B) sensing mechanism of an Ru-complex-grafted 2D metal–organic layer (MOL) based ECL immunosensor. Reproduced with permission from ref. 155. Copyright (2019) Elsevier.

5. Conclusions

Signal-amplification-based ECL bioassays have become one of the most significant research fields owing to their demand in ultrasensitive biosensing and trend towards early cancer diagnosis. An extensive range of ECL assays have been introduced in the field of biosensing for signal amplification with remarkable sensitivity, high selectivity, and wide dynamic range.

This review discussed the recent progress for the detection of various biomarkers and bio-agents by immunosensors and aptasensors with ultrahigh sensitivity derived from enhanced ECL signalling strategies. The strategies differ based on the kind of reaction constituents and enhancement mechanisms taking part in the ECL process.

Quantitative analysis of genes, cells and proteins, and other bioanalytes giving valuable information for biomedical research and early diagnosis of cancer has been possible and effective due to the strategically enhanced ECL-biosensing methods, with further prospects for improvement. Continuous progress in the development of ECL sensing techniques will include classes of novel and advanced materials such as metal NPs based on Au, Ag and related composites, which have currently shown the highest popularity in ECL-based biosensing studies because of their highly desirable properties and compatibility with diverse materials. Inorganic metal complexes such as those involving Ru-based luminophore species have shown considerable improvement in the past few years. Replacement with luminol and other novel ECL luminophores shows promising ability in ECL enhancement, while advancement in well-studied complexes continues to exhibit desirable and applicable results. Ongoing studies based on 2D ultrathin NS such as rGO and metal organic layers show promising future directions based on material functionalization with highly sensitive species allowing ECL sensitive assays with low detection limits. Organic molecules participating in ECL biosensing assays such as EONs are a new class of materials that show potential in future biocompatible assays of various biomarkers and disease related biomolecules, which could further be extended to cancer cell assays, enabling trace amount detection for early diagnosis. Finally, studies involving sensitive ECL assays continually steered in the direction providing a path for the incorporation of low-toxicity and eco-friendly nanomaterials show immediate concern and priority.

Conflicts of interest

There are no conflicts to declare.


This work is supported by Taiwan Ministry of Science and Technology (108-2628-E-007-004-MY3, 108-2622-M-007-005-CC2 and 108-2218-E-007-057) and Taiwan Ministry of Education (MOE107QR001I5).


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