Graphene and graphene oxides: recent advances in chemiluminescence and electrochemiluminescence

Abstract

Due to the high surface area, excellent conductivity, high mechanical strength, and good biocompatibility, graphene has become a growing area of interest since it was first discovered in 2004. Despite these important achievements in the design of fluorescent, colorimetric and electrochemical sensors, the merging of graphene and chemiluminescence (CL) or electrochemiluminescence (ECL), especially CL is still in its infancy. In this review, according to the roles that graphene and its derivatives play in various CL or ECL systems, we discuss the new CL and ECL sensors in particular, in the last two years. Furthermore, we discuss some future prospects and critical challenges in this field. Additionally, possible solutions to overcome these challenges are presented.

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Yingying Su

Yingying Su received her B.Eng. in Applied Chemistry from the College of Chemistry & Chemical Engineering, Central South University in 2000. Then she obtained her M.S. degree in Analytical Chemistry from Fuzhou University in 2004. After three years of research experience with Professor Xiandeng Hou at Sichuan University for her Ph.D degree, she joined the faculty of Analytical & Testing Center, Sichuan University as a lecturer, then as an associate professor. Her current research interests mainly focus on the development of chemo/bio-sensors with novel nanomaterials. She is the author or co-author of about 30 publications in peer-reviewed scientific journals.

Yi Lv

Yi Lv is a professor in college of chemistry at Sichuan University, China. He received his bachelor's degree, master's degree and Ph.D. degree from Southwest China Normal University (currently Southwest University) in 1997, 2000 and 2003, respectively. After a two-year stay at Tsinghua University as a postdoctoral researcher, he joined the faculty of the College of Chemistry at Sichuan University in 2005. His research interests are mainly in the areas of analytical spectrometry and nano-materials for analytical chemistry. He has published over 80 papers on the journals of analytical chemistry and one book chapter on the topics.

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Introduction

Graphene is a two-dimensional (2D) crystal, composed of monolayers of sp² carbon atoms arranged in a honeycombed network with six-membered rings.^1–3 It has attracted tremendous research interests in the fields of physics, chemistry and material science since it was discovered in 2004.⁴ Due to its many unique properties, such as the quantum Hall effect at room temperature,⁵ the high surface-to-volume ratio (2600 m² g⁻¹),⁶ the high electron transfer rate (up to 200 000 cm² V⁻¹ s⁻¹) and the outstanding thermal stability,⁷ graphene has been demonstrated its great potential applications in various fields such as novel superconductive materials, nanoelectronics, supercapacitors, energy storage devices, catalysis and imaging and therapy. And in the recent three years, an explosive growth in studies relating to the use of graphene and its derivatives as enhanced materials or carriers for probes and recognition elements in the development of new analytical devices has occurred. In these analytical devices, fluorescent, colorimetric and electrochemical sensors are the most widely studied and so are predominant.

Chemiluminescence (CL) is the production of electromagnetic radiation (usually in the visible or near-infrared region) by a chemical reaction between at least two reagents in which an electronically excited intermediate or product is obtained and subsequently relaxes to the ground state with emission of light.⁸ Due to the advantages of simple instrumentation, high sensitivity, wide linear range, no interference from background scattering light, and versatility for the determination of a wide variety of species, CL is of extensive interest and has been developed to be a powerful tool in analytical fields over the past several decades. With the vigorous development of nanomaterials, the analytical application of nanomaterials-assisted CL-reaction strategies and ECL sensors based on the enhanced and amplified CL or ECL signal has been a new trend. Through 177 references mainly from 2008 to 2012, the recent analytical applications of metal nanoparticles, magnetic nanoparticles, quantum dots (QDs), and carbon-based nanomaterials (carbon nanotubes and graphene) in liquid-phase CL systems were summarized by us.⁹ QDs with excellent optical and electronic properties have been the potential alternatives of CL emitters. The group of Lin described the mechanism of QDs in CL reaction, and summarized QDs-enhanced ultraweak CL systems, QDs-based CRET and their applications.¹⁰ This review demonstrates that the combination of QDs with CL is a good platform to develop analytical methods and design new optical sensors. In addition, a review on ECL of nanomaterials for bioanalysis was offered by group of Ju.¹¹ They believed that nanomaterials biofunctioned with multitudinous biomolecules can provide excellent ECL signal-transduction platforms for fabricating novel biosensing devices.

Several reviews gathering the latest advances in graphene related to the analytical areas of application have appeared.^12–17 Guo and Dong described the recent research efforts on the design of new electrochemical sensors, including amperometry, ECL, field-effect transistor (FET), electrochemical impedance, photoelectrochemical and surface plasmon resonance (SPR) electrochemical sensors and optical sensors, such as fluorescent, colorimetric and surface enhanced Raman spectroscopy (SERS) sensors¹² Liu et al. comprehensively review the emerging graphene-based electrochemical sensors, electronic sensors, optical sensors, and nanopore sensors for biological or chemical detection.¹³ Liu and Wu not only review the recent progress of graphene-based nanomaterials in optical and optoelectronic applications but also summarized their synthesis techniques and main electronic and optical properties.¹⁴ In the review reported by Kochmann et al.,¹⁵ electrochemical and optical chemical sensors and biosensors using graphenes was discussed in detail. Graphene quantum dots (GQDs) with high fluorescent property were systematically discussed ranging from the mechanism, the influencing factors to the optical tenability by group of Zhu.¹⁶ In addition; the synthesis and application of this material were also reviewed by them. Meanwhile, a comprehensive discussion on the development of the optical sensors, mainly fluorescence sensor using graphene materials as the energy acceptors was provided by group of Liu.¹⁷ In this review, the detection mechanisms of the sensors were emphasized.

As far, there is no a review only on the latest advances in graphene related to CL. The reason is that graphene related CL is still in its infancy when compared with the other analytical devices, and many graphene-based CL systems and sensors were reported in the recent one to two years.

This current review provides recent research advances using graphene in chemiluminescent technique. Graphene and its derivatives not only act a catalyst in liquid-phase CL but also in cataluminescence. Moreover, it also acts as a platform for multipurpose sensing and bioassays, or a super-quencher of a CL donor or fluorophore in chemiluminescence resonance energy transfer (CRET). The dynamic combination of unique structure and electronic/optical properties of graphene also makes it a wonderful platform for ECL, a kind of CL. Various ECL systems are involved, such as Ru(II) complex-based ECL systems, luminol-based ECL systems, QD-based ECL systems, peroxydisulfate systems, and some new ECL systems. Finally, we also provide some perspectives on the future trends of graphene used in CL or ECL. We hope this review can offer valuable insight for the investigation of graphene participated in CL or ECL systems and stimulate more exciting developments in this still young yet very promising field of research.

Chemiluminescence

As catalysts in liquid phase chemiluminescence

Similar to metal nanoparticles, graphene oxide (GO), one of water-soluble derivative of graphene has also been found to act as liquid phase CL catalyst and enhance CL intensity due to its high efficiency, large surface area, and good water solubility.

GO was at first found to catalyze the CL of luminol–H₂O₂ system in a weakly alkaline medium mainly through singlet oxygen, which was greatly different from the traditional catalyst in such CL system that occurred in a strongly basic medium.¹⁸ Six-fold enhancement CL intensity can be obtained compared with that in the absence of GO. Afterwards, GO was also found to catalyze the luminol-O-2 reaction.¹⁹ In this CL system, the intrinsic catalytic effect of GO is from it acting as the radical generation proliferators and electron transfer accelerators. Based on the GO catalyzed luminol-O-2 system, a new CL sensor to detect glucose was successfully developed. Under the optimized conditions, glucose could be assayed in the range of 0.05 mM to 5 mM with a detection limit of 0.044 mM. For the detection of clinical serum samples, it is well consistent with the data determined by commercially available method in hospital, indicating that the new CL method provides a possible application in clinical diagnostics. In addition, many prominent works about chemiluminescent functionalized GO composites have been reported by group of Cui including GO/silver nanoparticle nano-composites,²⁰ dendritic platinum nanoparticles/lucigenin/reduced GO hybrid²¹ and luminol and lucigenin bifunctionalized gold nanoparticles/graphene oxide nanocomposites with dual-wavelength chemiluminescence.²² In the last work, AuNPs/GO nanocomposites is proposed that lucigenin molecules and AuNPs were located at the surface of GO by pi–pi stacking and electrostatic force respectively, and luminol existed on the surface of AuNPs by virtue of Au–N covalent interaction in the as-prepared nanocomposites. Because luminol and lucigenin were attached to the surface of the nanocomposites, the obtained nanocomposites could react with hydrogen peroxide resulting in a good dual-wavelength chemiluminescence activity. Based on the work, label-free logic gates (AND, OR, and INHIBIT) based on chemiluminescent outputs by taking advantage of the unique CL activity of luminol–lucigenin/AuNPs/GO nanocomposites was developed by them.²³ The proposed logic gates were successfully used to detect Fe²⁺, Ag⁺, and L-cysteine, respectively.

Different from GO, graphene always needs to be decorated metal nanoparticles or hybridized with luminescent reagent or other catalyst and then acts as liquid phase CL catalyst. Group of Cui reported the synthesis of chemiluminescent functionalized graphene composites.²⁴ The as-prepared nanocomposites, chemically converted graphene/N-(aminobutyl)-N-(ethylisoluminol) (ABEI), luminol and isoluminol, exhibit good CL activity. By decorated PtCo bimetallic alloy nanoparticles on graphene, Yang et al. designed and developed a kind of highly catalytic nanocomposites for detection of H₂O₂ and glucose with good sensitivity and low detection limit.²⁵ Compared with some catalysts consists of enzymes, the catalyst are very stable and can be easily stored at room temperature for long time without any deactivation. In order to further improve CL performance of graphene hybrids,²⁴ group of Cui accumulated a CL reagent (ABEI) and a catalyst (hemin) simultaneously on a graphene nanosheet (GN) substrate by non-covalent functionalization.²⁶ They found multilayers of ABEI and hemin accumulated on the graphene surface result in high chemiluminescence performance, good solubility and stability in aqueous solution. The synthesis of dual functionalized graphene hybrids (A–H–GNs) was shown in Fig. 1. The forming of dual-functionalized GNs not only provides great potential for investigating novel CL reagent functionalized nanoparticles and allows further exploration of graphene based CL sensors in the biological field.

Fig. 1 Illustration of preparation of chemiluminescence reagent and catalyst dual-functionalized graphene hybrids. Adapted from Liu et al.²⁶ with permission from RSC Publications.

As sensing materials in cataluminescence (CTL)

Since CTL was first observed by Breysse et al.²⁷ during the catalytic oxidation of carbon monoxide on the surface of thoria surface, CTL based gas sensors have attracted great interests with high sensitivity, short response time, simple implementation, low cost, and good suitability for the design of portable instruments. During the development of CTL, Zhang and co-workers did a lot of prominent work, especially nanomaterial-based CL sensor array.^28–33 For a specific analyte, such as alcohols and flavors,^28,29 CO,^30,31 proteins and cell,³² natural sugars and artificial sweeteners,³³ the nanosized catalysts show different catalytic activities and generate unique CL response “fingerprints” for discrimination.

Although new techniques, for example, Plasma assisted catalysis has been introduced CTL to improve the sensitivity for the detection of benzene, toluene, ethylbenzene, and xylenes on the surface of nanosized ZrO₂.³⁴ High sensitivity and good selectivity is still one of the most difficult challenges to develop cataluminescence (CTL) sensor. With the development of nanomaterial, specific material was more and more concerned to resolve the problem. The graphene sheets decorated with nanoparticles or metal oxide composites provide new opportunities to develop new CTL sensing materials, owing to its greater versatility in carrying out adsorption, selective catalytic and sensing processes, more porosity and large specific surface area. Platinum nanoparticles supported by graphene were firstly used in CTL for CO.³⁵ In this work, a faster catalytic reaction rate and a higher CTL intensity were obtained when Pt nanoparticles are well distributed on graphene. Recently, graphene sheets decorated with SnO₂ nanoparticles via the hydrothermal-assisted in situ oxidation reduction reaction between graphene oxide and SnCl₂ was prepared by our group.³⁶ Owing to the catalyzing oxidization of propanal on the surface of SnO₂/graphene composite, a new CTL sensor for propanal was designed. The method with a detection limit of 0.3 mg mL⁻¹ (S/N = 3) and linear ranges magnitude of two orders shows perfect analytical performances. Moreover, the CTL-based propanal sensor showed good stability and relatively long-time durability.

Although few work were reported, with the development of the combination of graphene or its derivatives and the new CTL techniques, such as sensor array and plasma assisted catalysis, graphene will exhibit its importance both in analytical application and catalysis industry.

As a platform for multipurpose sensing and bioassays

By taking advantage of the concept of the preferential binding of GO to single-stranded DNA (ssDNA) over rigid double-stranded DNA (dsDNA) or aptamer to target complexes over GO, GO was widely used as a platform for multipurpose sensing and bioassays. Recently, the concept was introduced to CL aptasensor and CL immunoassay. For example, a new homogeneous GO-horseradish peroxidase (HRP)-mimicking DNAzyme (GO-HMDNAzyme) based CL biosensor for sequence-specific DNA detection was developed based on that GO greatly inhibits the peroxidatic activity of a horseradish peroxidase-mimicking DNAzyme, combining the unique DNA/GO interactions.³⁷ Song et al. found that GO-aptamer complex showed strong CL intensity instead of CL quenching based on the instantaneous derivative reaction between phenylglyoxal (PGO), a special CL reagent as the signaling molecule, and guanine nucleobases of aptamer strands adsorbed on the surface of GO.³⁸ A rapid and simple CL aptasensor for adenosine triphosphate (ATP) based on GO nano-platform and a structure-switching aptamer without any label was established (Fig. 2). In the absence of ATP, the aptamers adsorbed onto the surface of GO leading to a strong background CL signal. Conversely, in the presence of ATP, the aptamers formed the aptamer–ATP complexes which had weak binding ability to GO resulting in a significant CL signal decrease. The CL intensity was adversely related to the ATP concentration and CL aptasensor for ATP was established.

Fig. 2 Schematic illustration of the label-free CL aptasensor for ATP based on GO and an instantaneous derivatization of guanine bases. Adapted from Song et al.³⁸ with permission from Elsevier.

Additionally, using pi–pi stacking interaction between magnetic Fe₃O₄ graphene oxide nanoparticles and impurities (e.g., biomolecules, free prostate specific antigen (PSA) aptamer-conjugated TEX615 that does not bind with PSA) in human serum, a rapid aptasensor with 1,1′-oxalyldiimidazole chemiluminescence (ODI-CL) detection was developed for the quantification of a tumor marker, PSA.³⁹ Most recently, biocompatible GO sheets as a sensing platform combined with CL immunoassay labeled with enzyme, has been extensively exploited for miniaturization.⁴⁰ GO sheets not only provide an abundant domain for biomolecules but also play a role of signal amplification detection. Combine the advantages of GO with the good water permeability of chitosan, a highly efficient CL immunosensor for ultrasensitive and rapid flow-through CL immunoassay of ChIFN-γ only in 25 min was constructed. The detection limit of the proposed method is 0.36 pg ml⁻¹ (S/N = 3), which is 138-fold lower than the current lowest value of 50 pg ml⁻¹ for ChIFN-γ. The GO-chitosan film provided a large surface area for high-capacity loading of proteins and displayed a biocompatible microenvironment for long activity retention of the biomolecules. The flow-through CL enzyme immunoassay system for ChIFN-γ was illustrated in Fig. 3.

Fig. 3 Schematic illustration of the flow-through chemiluminescent immunosensing system for ChIFN-γ. (S) Sample, (WB) wash buffer, (HS) HRP substrate, (PMT) photomultiplier. Adapted from Zhu et al.⁴⁰ with permission from Elsevier.

Graphene or GO used as a platform for multipurpose sensing and bioassays often involve chemiluminescence resonance energy transfer (CRET). So it will be discussed in the next section in detail.

Chemiluminescence resonance energy transfer (CRET)

Graphene, along with its water-soluble derivative, GO, is a single-atom thick and two-dimensional carbon material that recently acted as a super-quencher of a CL donor or fluorophore in CRET, due to the non-radiative transfer of electrons from CL-donor excited states to the π system of GO or graphene.^41,42 Moreover, graphene oxide (GO) can differentiate single- and double-stranded DNA structures.⁴³ As a result, many novel CRET methods for DNA or proteins by virtue of the characters of GO were developed. Bi et al. reported a sensitive and selective detection of DNA (H1V1) and protein (thrombin) by using GO–luminol–H₂O₂-based CRET system, which could highly adsorb single-stranded DNA (ssDNA) and effectively quench the emission of a fluorescein-based dye (FAM).⁴¹ In the same year, Lee et al. demonstrated CRET between graphene nanosheets as energy acceptors and luminol as energy donors.⁴² They designed a graphene-based CRET system for homogeneous immunoassay of C-reactive protein (CRP) using a luminol–H₂O₂ CL reaction catalyzed by HRP. Based on the super quenching efficiency of graphene oxide (GO) and exonuclease III-assisted target recycling amplification, group of Li reported an amplified CL biosensing platform for ultrasensitive DNA detection.⁴⁴ In the presence of target DNA, the target-probe hybrid forms a double-stranded structure, and exonuclease III catalyzes the stepwise removal of mononucleotides from the fluoresce in labeled probe DNA, resulting in the recycling of the target DNA and CL signal amplification of the luminol–H₂O₂–HRP-fluorescein CRET system. Then, using exonuclease III-assisted target recycling amplification and the same CRET system, they also developed a simple and highly sensitive CL biosensing platform for site-specific determination of DNA methylation.⁴⁵ The analytical procedure was shown in Fig. 4. The biosensing platform exhibits excellent high sensitivity, and it can ever distinguish as low as 0.002% methylation level from the mixture, which makes the CRET system promising application in cancer risk assessment. More new CRET systems for the detection of DNA were still constantly established. For example, trace levels of mutated ssDNAs was detected by a rapid hybridization method using graphene oxide and highly sensitive 1,1′-oxalyldiimidazole chemiluminescence (ODI-CL) and the detection can be completed in a sample containing mismatched ssDNAs within 15 minutes without any interference.⁴⁶ Group of Cui also investigated a new CRET system for DNA.⁴⁷ GO can directly quench the CL emission of an acridinium ester (AE)–H₂O₂ system effectively. By taking advantage of the different ionic attraction of double stranded DNA and GO toward AE, a novel label-free and homogeneous DNA sensor for M. tuberculosis was designed. In this CL system, electron transfer or resonance energy transfer would occur between GO and the excited state N-methylacridone (NMA*, the chemiluminescent luminophor of the AE–H₂O₂ system).

Fig. 4 Schematic illustration of the procedures of the gene-specific DNA methylation determination. (A) Assembly of FAM-labeled probe DNA on GO surface; (B) treatment of unmethylated DNA with bisulfite, hybridization with FAM-labeled probe DNA/GO, and indication with CRET system; (C) treatment of methylated DNA with bisulfite, hybridization with FAM-labeled probe DNA/GO, ExoIII-assisted target recycling amplification, and indication with CRET system. Adapted from Chen et al.⁴⁵ with permission from Elsevier.

Graphene quantum dots were also used as energy acceptors in CRET system. An immunoassay has been developed for the detection of the ovarian cancer biomarker CA-125 by utilizing the CRET to graphene quantum dots.⁴⁸ This biosensor shows a wide linear range from 0.1 U mL⁻¹ to 600 U mL⁻¹ with a limit of detection of 0.05 U mL⁻¹ for CA-125. In a more recent study, Lei et al. reported a strategy of CRET using graphene as an efficient long-range energy acceptor.⁴⁹ Magnetic nanoparticles were used for simple magnetic separation and immobilization of horseradish peroxidase (HRP)-labeled anti-HCG antibody. In the CRET system, the sandwich-type immunocomplex was formed between human chorionic gonadotropin (HCG, antigen) and two different antibodies bridged the magnetic nanoparticles and graphene (acceptors), which led to the occurrence of CRET from chemiluminescence light source to graphene. After optimizing the experimental conditions, the quenching of chemiluminescence signal depended linearly on the concentration of HCG in the range of 0.1 mIU mL⁻¹ to 10 mIU mL⁻¹ and the detection limit was 0.06 mIU mL⁻¹. The proposed method was successfully applied for the determination of HCG levels in saliva and serum samples, and the results were in good agreement with the plate ELISA with colorimetric detection.

Luminol-functionalized chemically reduced graphene oxide (RGO) was successfully formulated through simple noncovalent interaction between luminol and RGO.⁵⁰ The CL of luminol was completely quenched in RGO/luminol assembly, confirming that RGO platform is acceptable for not only fluorescence resonance energy transfer (FRET) but also CRET in this study.

Others

Molecularly imprinted polymer (MIP) with GO has been introduced to chemiluminescence sensor by Qiu et al. in the recent years. Based on graphene oxide–magnetite-molecularly imprinted polymer (GM-MIP), the flow injection chemiluminescence (FI-CL) system of KMnO₄–SnCl₂–CHOH for (L)-tryptophan ((L)-try)⁵¹ and the FI-CL system of luminol–NaOH–H₂O₂ for epinephrine⁵² was described, respectively. In the same year, they also reported a CL array sensor for determination of benzenediol isomers simultaneously using the system of luminol–NaOH–H₂O₂ based on a graphene–magnetite-molecularly imprinted polymer (GM-MIP).⁵³ The array sensor was finally used for the determination of hydroquinone, resorcinol and catechol in waste water samples simultaneously. Afterwards, chitosan/graphene oxide-molecularly imprinted polymers (CG-MIP) was used as recognition element for sulfamethoxazole (SMZ).⁵⁴ The SMZ–CG-MIP was synthesized in acetone as solvent and chitosan/GO for support, using acrylamide as functional monomer, ethylene glycol dimethacrylate as crosslinker and 2,2-azobisisobutyronitrile as initiator. The CG-MIP showed satisfactory recognition capacity for the SMZ. Then the synthesized CG-MIP was employed as recognition by packing into a lab-made tube connected in FI-CL analyzer to establish a novel CL sensor respectively. The CL intensity responded linearly to the concentration of SMZ in the range 1.0 × 10⁻⁷ mol L⁻¹ to 2.3 × 10⁻³ mol L⁻¹ with a detection limit of 2.9 × 10⁻⁸ mol L⁻¹ (S/N = 3).

Electrogenerated chemiluminescence

Electrochemiluminescence or electrogenerated chemiluminescence (ECL) is a form of CL in which the light-emitting reaction is caused by an electrochemical reaction. At the electrode surface, excited states that emit light are upon an energy relaxation process. Due to its fascinating two-dimensional structure, unusual electrochemical properties, large accessible surface area, as well as good biocompatibility, graphene or its derivatives is an attractive candidate in ECL. Graphene or its derivatives presents excellent electron transfer ability for some enzymes, excellent catalytic behavior toward small molecules, and an excellent character as a carrier to load more active probes and active domains for biomolecules binding which offering a significant amplification on the ECL sensing signals. As a result, the indirect ECL of graphene or its derivatives has been investigated in detail, and combining it with various ECL systems, especially Ru(II) complex-based ECL systems, luminol-based ECL systems, and QD-based ECL systems, more and more novel and sensitive analytical methods have been developed.

Direct ECL of graphene and GO

Since semiconductor nanocrystals are well known to exhibit ECL, it comes as no surprise that graphene and GO have aroused interest for ECL studies. ECL of GO nanoparticles with TPrA as coreactant was first found by group of Bard.⁵⁵ They believed when the electrode potential is scanned to a value positive of ∼1.15 V, further oxidation of GO NPs takes place either directly on the Pt electrode during collision or via TPrA radical cations. The deprotonation reaction of TPrA generates a highly reductive radical intermediate and the radiative recombination of this radical and the grapheme oxide NP leads to the formation of the excited-state of grapheme oxide for the generation of ECL. Four years later, strong electrochemiluminescent emission was observed from the GO colloid in the presence of K₂S₂O₈.⁵⁶ The electrochemiluminescent signal of this system was quite stable with time, and its mechanism was ascribed to the electron-transfer of GO colloid and SO₄⁻ radicals produced by electroreduction of S₂O₈²⁻ ions. It's interesting that ECL intensity of GO can be amplified through combining GO and Se nanoparticles.⁵⁷ In this system, the chitosan (CHIT) was used as improver. Under optimal conditions, the GO/Se nanocomposite produced an intense ECL emission and maintained long-term ECL stability. Significantly, it was used to detect the biomolecule dopamine (DA) with a detection limit of 0.025 μM.

With H₂O₂ as coreactant, a newly anodic ECL was observed from the water-soluble GQDs for the first time.⁵⁸ The ECL induced a strong light emission at a low potential (ca. 0.4 V vs. Ag/AgCl). Employing SiO₂ nanospheres as signal carrier, a novel SiO₂/GQDs ECL signal amplification labels were synthesized based on which an ultrasensitive ECL aptamer sensor was proposed. Under the optimized experimental conditions, the proposed ECL aptamer sensor exhibited excellent analytical performance for ATP determination, ranging from 5.0 × 10⁻¹² to 5.0 × 10⁻⁹ mol L⁻¹ with the detection limit of 1.5 × 10⁻¹² mol L⁻¹.

L-Cysteine (L-Cys) as a new coreactant of GQD-based ECL system was found by group of Chen and Chi.⁵⁹ The study shows that the ECL signal attributes to the oxidation of L-Cys, the presence of dissolved oxygen and the reduction of GQDs and the possible ECL mechanism was proposed as Fig. 5. Pb²⁺ can inhibit the ECL intensity through prohibiting the formation of the three kinds of free radicals (RSO˙, RSO₂˙, and RSO₃˙) from the oxidation of L-Cys. Based on this, a sensor for the detection of Pb²⁺ has been developed.

Fig. 5 Diagram for the ECL reaction mechanism of the GQD/LCys system. Adapted from Dong et al.⁵⁹ with permission from ACS Publications.

The above foundation studies about the ECL properties of graphene and GO would promote, without doubt, the development of new ECL systems and the related analytical application.

Ru(II) complex-based ECL systems

N,N,N-Tripropylamine (TPrA) as coreactant

Various requirements, such as a good solubility and high chemical stability, matching redox potentials and fast charge transfer kinetics and, finally, a rapid degradation route to produce a high-energy radical capable to initiate the ECL process need to be met by a chemical species in order to be a good coreactant for Ru(II) complex-based ECL systems.

Currently, however, alkyl amines has become the most representative class of oxidative-reduction coreactants and, among them, TPrA is the most widespread one. For example, it is used in combination with the luminophore Ru(II) complex and graphene or GO in all the commercial systems for biosensor, immunoassay and DNA analysis based on ECL detection.

Based on Ru(phen)₃²⁺–graphene–Nafion and bis(2,2′-bipyridine)-5-amino-1,10-phenanthroline ruthenium(II) (Ru(II)–NH₂)–graphite oxide composite films, ECL sensors for oxalate in urine samples,⁶⁰ propranolol hydrochloride in pharmaceutical samples,⁶¹ and 2-(dibutylamino) ethanol (DBAE)⁶² was designed, respectively. Without any auxiliary medium, such as Nafion, Gao et al. also developed a facile ECL sensor for oxalate in urine samples based on Ru(phen)₃²⁺–graphene.⁶³ Then they developed a novel ECL ethanol biosensor based on Ru(phen)₃²⁺ and alcohol dehydrogenase (ADH) immobilized by graphene/bovine serum albumin composite film.⁶⁴ Such a design of Ru(phen)₃²⁺–graphene/BSA film to modify electrode holds a great promise as a new biocompatible platform for the development of enzyme-based ECL biosensors. Another universal biosensing platform, AuNP dotted reduced graphene oxide composite was used as simple and sensitive sandwich-type electrochemiluminescence biosensor for alpha-fetoprotein (AFP).⁶⁵

Utilizing a simple p–p stacking self-assembly noncovalent strategy, bi(2,2′-bipyridyl)-pyrene derivative functionalized ruthenium(II) complexes anchored on graphene sheets (B–Ru–P/GS nanohybrid) were reported.⁶⁶ With TPrA as a coreactant, the ECL behavior of the B–Ru–P/GS nanohybrid modified electrode has been studied in detail. Graphene platelet (GP)–Ru(phen)₃²⁺ assembles have been prepared through self-assembly of poly sodium styrenesulfonate (PSS) functionalized GPs and Ru(phen)₃²⁺ driven by electrostatic attraction interactions in aqueous solution.⁶⁷ The resultant assembled Ru(phen)₃²⁺ hybrid structure modified electrode exhibits excellent ECL behaviors because of the ECL active species Ru(phen)₃²⁺. Via the aromatic–aromatic π-stacking interaction and simultaneously the reduction of graphene oxide to graphene, another non-covalent modification method to obtained ruthenium(II) complex/3,4,9,10-perylenetetracarboxylic acid (PTCA)/graphene nanocomposites (Ru–PTCA/G) was introduced by Xia and his coworkers.⁶⁸ In this approach, graphene facilitates interfacial electron transfer and offers large surface area for loading functional unites. PTCA protects the surface of graphene sheets from aggregation, and simultaneously provides abundantly carboxylic acid groups for covalent attachment of Ru(II) complex. The functional Ru–PTCA/G nanocomposites show good electrochemical activity and ca. 21 times higher luminescence quantum efficiency than the adsorbed derivative ruthenium(II) complex. In addition, a new ECL immunosensor based on steric hindrance effect is fabricated for detection of AFP.

To sum up, in these Ru(II) complex-based ECL systems, graphene or GO could be non-covalently functionalized by π-stacking interactions, hydrogen bonding interaction or van der Waals forces. Moreover, these non-covalent modification methods have been proven as an effective strategy for enhancing the chemical properties of graphene while the structure and electronic properties of graphene can be retained. However, to some extent, the introduction of many functional groups by covalent bonds may be a better method.

Amidation reactions are one of the most common approaches used for linking molecular moieties onto oxygenated groups of GO. In work of Yu et al., a Ru(II) complex designed with a long alkyl amino functional group on one of its diimine ligands, bis(2,2′-bipyridine) (N-(2-aminoethyl)-4-(4′-methyl-2,2′-bipyridine-4-yl) butanamide) ruthenium(II) (denoted as [Ru–NH₂]²⁺[Ru–NH₂]²⁺), was used to covalently graft onto GO by the amide reaction between the amino group of [Ru–NH₂]²⁺[Ru–NH₂]²⁺ and the carboxyl group of GO.⁶⁹ Using this hybrid material, a reagent-free ECL method was developed for the detection of tripropylamine. Afterwards, based on the Ru–GO composite, a novel homogeneous label-free aptasensor for 2,4,6-trinitrotoluene (TNT) detection was designed by them.⁷⁰

Using amidation reactions, an ECL aptasensor for thrombin based on GO was also developed.⁷¹ In this case, GO is attached on glass carbon or gold electrodes through physical adsorption, and the amino-tagged aptamer is immobilized on the electrode surface via an amide linkage between the amino group at the end of aptamer and the carboxyl groups on GO. The oligonucleotide (FO) is designed to contain two parts: the complementary strand and an intermolecular duplex for the intercalation of Ru(phen)₃²⁺ as ECL probe. The hybridization between aptamer and its complementary part at FO achieves the introduction of Ru(phen)₃²⁺ probe onto the electrode surface for high ECL emission. The hybrid between aptamer and thrombin leads to the release of FO containing the intercalated Ru(phen)₃²⁺ probe. The decreased ECL emission is used to quantify thrombin.

In the recent one year, based on the grephene composites for immobilization of Ru(bpy)₃²⁺, paper-based chips were investigated by group of Wang.^72,73 Using a simple solvothermal method, they prepared graphene-nanosheet-based highly porous magnetite nanocomposites (GN–HPMNs) and tune the structures and sizes by varying the proportions of the solvents ethylene glycol and diethylene glycol.⁷² Then, the relatively highly porous ones with an average diameter of about 65 nm were combined with Nafion to form composite films on an electrode surface for immobilization of Ru(bpy)₃²⁺. It was found to be much more favorable for detecting compounds containing tertiary amino groups and DNAs with guanine and adenine. A detection limit (S/N = 3) of 5.0 nM was obtained for tripropylamine. Based on the composite film of poly(sodium 4-styrenesulfonate) functionalized graphene (PSSG) and Nafion, they also prepared another paper-based chips.⁷³ And a same low detection limit (S/N = 3) of 5.0 nM for tripropylamine was obtained.

S₂O₈²⁻ as coreactant

Regarding the so-called reductive-oxidation kind of coreactants, the most important ones are peroxides, and the first example reported in literature of this class was the peroxydisulfate (S₂O₈²⁻). It shows a low solubility in acetonitrile, and therefore it is largely used in water and the reduction of S₂O₈²⁻ generates a strong oxidant intermediate such as SO₄²⁻.

Ru-derivatized graphene have been applied as labels in most common bioassays protocols to obtain amplified ECL responses. In particular, immunosensors for rapid, selective, sensitive, and low-cost sensing of antigen–antibody interactions attract increasing attention. A very interesting example is the detection of CA 125 on a nanoporous gold (NPG) modified glassy carbon electrode (GCE).⁷⁴ The Ru–AuNPs composite assembled with poly(diallyldimethylammonium chloride) functionalized graphene nanosheets (GR) (Ru–AuNPs/GR) was used as ECL labels, which has large surface area, good biocompatibility and electronic conductivity. The ECL intensity observed by the application of as-prepared Ru–AuNPs/GR composites was enhanced 6-fold compared to those of Ru–AuNPs. The primary antibody of CA 125 (Ab₁) was first immobilized on the NPG modified electrode, and the antigen and the secondary antibody (Ab₂) were conjugated to form a sandwich-type immunocomplex through the specific interaction. The proposed ECL immunosensor provided a wide linear response range over 0.01–100 U mL⁻¹ with a detection limit of 0.005 U mL⁻¹. The sandwich immunoassay procedure was also used by Hao et al. to obtain carcinoembryonic antigen (CEA) ECL detection.⁷⁵ They coated Ru(bpy)₃²⁺–graphene–Nafion composite on the glassy carbon electrode surface. The sandwich-type immunoreactions between the first antibody and the second antibody bridged the donors Ru(bpy)₃²⁺ and acceptors QDs, which led to the occurrence of ECL quenching of Ru(bpy)₃²⁺ by QDs. Under optimal conditions, the ECL signal depended linearly on the logarithm of the CEA concentration within a range from 0.005 to 0.5 pg mL⁻¹, and the detection limit of CEA at very low levels was 0.002 pg mL⁻¹. A novel ECL ethanol biosensor was designed base on Ru(bpy)₃²⁺ and alcohol dehydrogenase (ADH) immobilized by graphene/bovine serum albumin composite film.⁷⁶ The graphene film was directly formed on a glassy carbon electrode surface via an in situ reduction of GO and Ru(bpy)₃²⁺ was immobilized during its formation. The graphene film acted as both a decorating agent for immobilization of Ru(bpy)₃²⁺ and a matrix to immobilize BSA, while BSA not only reduced GO, but also provided a friendly environment for ADH immobilization. Such a design of Ru(bpy)₃²⁺–graphene/BSA film to modify electrode holds a great promise as a new biocompatible platform for the development of enzyme-based ECL biosensors.

Based on electrochemiluminescence resonance energy transfer (ERET), a novel sensing strategy for sensitive detection of mucin 1 protein (MUC1) and MCF-7 cells was documented by Wei et al.⁷⁷ Due to the strong noncovalent interaction between the Ru1-aptamer and GO, the ECL of Ru1 was efficiently quenched because of the ERET. As shown in Fig. 6, in the presence of a target MUC1 protein, the binding between the Ru1-aptamer and MUC1 disturbed the interaction between the Ru1-aptamer and GO. These interactions led to the release of the Ru1-aptamer from GO, and resulted in the restoration of Ru1 ECL. This was shown to detect MUC1 protein sensitively in a linear range from 64.9 to 1036.8 nM with a detection limit of 40 nM. With further application in the detection of MCF-7 cells, the presented method could respond at concentrations as low as 30 cancer cells per mL. On the basis of the similar concept, an ECL biosensor for thrombin was reported.⁷⁸ In this case, a new ECL reagent, iridium(III) complex instead of Ru(II) complex was conjugated to aptamers and adsorbed on magneto-controlled magnetic graphene oxide. In the presence of thrombin, the aptamers will release into solution and result in increased ECL intensity, and the enhanced ECL intensity has a relationship with the logarithm of thrombin concentration in the range of 2.0 × 10⁻⁹ to 5.0 × 10⁻⁸ mol L⁻¹. The detection limit was 1.3 × 10⁻⁹ mol L⁻¹ (S/N = 3).

Fig. 6 Schematic representation of GO-induced electrochemiluminescence quenching of Ru1-aptamer and biosensing mechanism. Adapted from Wei et al.⁷⁷ with permission from RSC Publications.

Other coreactant

It is well known that with strong water-solubility, it is difficult to immobilize the TPrA into the electrode. Thus, the TPrA was usually added into the electrolyte as additives, which might lead to a more complex assay system and increase the analytical steps and expense. Very recently, a new coreactant, poly-L-lysine, has been reported for Ru(bpy)₃²⁺ ECL system by Yuan and his coworkers.⁷⁹ The poly-L-lysine film containing many primary amino and secondary amino groups not only can modify the electrode but also act as coreactant to significantly enhance the ECL of Ru(bpy)₃²⁺. In this ECL system, they assembled T3 capture antibody (anti-T3) on AuNPs loaded electro-deposited L-lysine film modified GCE as sensing interface and used magnetic Fe₃O₄ loaded graphene nanosheet as nanoprobes, which can achieve an impressive detection limit of 0.03 pg mL⁻¹ human total 3,3′,5-triiodothyronine (T3), a kind of diagnostic markers of thyroid disease. This strategy avoids the addition of the co-reactant to the electrolyte and significantly simplifies the immunoassay procedure, shortens the analytical time, and thus provides a new promising platform for clinical immunoassay.

Luminol-based ECL systems

Recently, in luminol-based ECL systems, graphene or its derivatives or their composites modified electrodes was widely investigated. In 2011, the cathodic ECL behavior of luminol at a positive potential on a graphene-modified electrode is first reported.⁸⁰ On the basis of the strong and stable cathodic ECL signal, an ECL sandwich immunosensor for sensitive detection of cancer biomarkers, PSA, was proposed with a multiple signal amplification strategy from functionalized graphene and enzyme antibody-conjugated gold nanorods as the sensor platform. Afterwards, the cathodic ECL of luminol at the electrochemically reduced graphene (ERG) modified electrode was also investigated.⁸¹ The ECL intensity increased 123 times compared with that of the GO modified electrode or the bare electrode, which is due to the strong adsorption of dissolved oxygen by ERG film. Recently, a graphene modified glassy carbon electrode (GR–GCE), a carbon nanotube modified glassy carbon electrode (CNT–GCE), a carbon nanotube–graphene modified glassy carbon electrode (CNT–GR–GCE), and a gold nanoparticle–carbon nanotube–graphene modified glassy carbon electrode (GNP–CNT–GR–GCE) were prepared. ECL of luminol was comparatively studied at these modified electrodes in neutral solution.⁸² The results revealed that carbon nanotube could intercalate into the graphene film and improve the conductivity of the CNT–GR composite significantly. The CNT–GR composite is an ideal matrix for the deposition of gold nanoparticles. At the GNP–CNT–GR–GCE, the effect of GR on luminol ECL is more predominant than that of CNT while the electrocatalytic effect of GNP on luminol ECL is well preserved. These nanomaterials exhibit a good synergic effect towards luminol ECL, which is more efficient than that of the sole material. At the polyaniline (PANI)–GCE, the GR–GCE, and the PANI–GR–GCE in neutral phosphate buffer solution (PBS), ECL of luminol was also comparatively investigated by Dong et al.⁸³ The PANI–GR composite exhibited apparent electrocatalytic effect on luminol oxidation, and as a result, the intensity of anodic ECL located at 0.50 V was enhanced two magnitudes compared with other two modified electrodes.

Graphene or its derivatives or their composites as a biosensing platform for labeling CEA has been widely investigated in luminol-based ECL system. So far, many ECL immunosensors for CEA have been fabricated. A sandwiched ECL immunosensor for CEA using ZnO nanoparticles (ZnONPs) and glucose oxidase (GOD) decorated graphene as labels and in situ generated hydrogen peroxide as coreactant was reported by Cheng et al.⁸⁴ The as-prepared ECL immunosensor exhibited excellent analytical property for CEA with a detection limit of 3.3 pg mL⁻¹ (S/N = 3). Cao et al. constructed an ECL immunosensor CEA based on an amplified cathodic ECL of luminol at low potential.⁸⁵ Firstly, AuNPs were electrodeposited onto single walled carbon nanotube–graphene composites (CNTs–Gra) coated GCE with enhanced surface area and good biocompatibility to capture primary antibody (Ab(1)) and then bind the antigen analytes. Secondly, Pd and Pt nanoparticles (Pd&PtNPs) decorated reduced graphene oxide (Pd&PtNPs@rGO) and glucose oxidase (GOD) labeled secondary antibody. (Pd&PtNPs@ rGO–GOD-Ab(2)) could be captured onto the electrode surface by a sandwich immunoassay protocol to generate amplified cathodic ECL signals of luminol in the presence of glucose. The Pd&PtNPs@rGO composites and loaded GOD promoted luminol cathodic ECL response by efficiently catalyzing glucose to in situ produce amount of H₂O₂ working as a coreactant of luminol. The as-proposed ECL immunosensor exhibited sensitive response on the detection of CEA ranging from 0.0001 ng mL⁻¹ to 160 ng mL⁻¹ with a detection limit of 0.03 pg mL1 (S/N = 3). Using Au nanoparticles and Pt nanoparticles (nano-AuPt) electrodeposited on graphene–carbon nanotubes nanocomposite as platform, another ECL immunosensor for CEA based on luminol cathodic ECL was fabricated.⁸⁶ For this introduced immunosensor, graphene (GR) and single wall carbon nanotubes (CNTs) dispersed in chitosan (Chi–GR–CNTs) were firstly decorated on the bare gold electrode (GE) surface. Then nano-AuPt were electrodeposited (DpAu-Pt) on the Chi–GR–CNTs modified electrode. When glucose was present in the working buffer solution, GOD immediately catalyzed the oxidation of glucose to in situ generate H₂O₂ then to amplify cathodic ECL of luminol. The same limit of detection, 0.03 pg mL⁻¹ (S/N = 3), for CEA can be obtained. Besides, with the aid of the catalytic properties from graphene nanosheets attached to spiky MnO₂ nanospheres GS/MnO₂, an advanced ECL immunoassay for the ultrasensitive detection of CEA was also proposed through signal amplification of nanoporous gold (NPG)–GS platform.⁸⁷ Very recently, a novel and ultrasensitive ECL immunosensor, which was based on the amplifying ECL of luminol by hemin-reduced graphene oxide (hemin-rGO) and AgNPs decorated reduced graphene oxide (Ag-rGO), was constructed for the detection of CEA.⁸⁸

Except for CEA, other bimolecular, such as AFP⁸⁹ and immunoglobulin G (IgG)⁹⁰ were also labeled and detected by functionalized graphene or rGO or their composites as a biosensing platform. Combining a functionalized graphene nanosheets and gold-coated magnetic Fe₃O₄ nanoparticles (GMPs) labeled AFP antibody (GMP similar to Ab1). The poly(diallyldimethylammonium chloride) functionalized graphene nanosheets (PDDA–G) were used to combine with luminol-capped gold nanoparticles (Lu–Au NPs). The resulting PDDA–G@Lu–Au composite displayed an enhanced capability for the immobilization of horseradish peroxidase (HRP) and signal antibody (Ab2). Under the optimized conditions, the ECL method shows a linear range of AFP from 0.002 to 20 ng mL⁻¹ and an extremely low detection limit of 0.2 pg mL⁻¹ (S/N = 3).⁸⁹ Recently, L-cysteine functionalized reduced graphene oxide composite (L-cys–rGO) was used to capture anti-IgG through the interaction between the carboxylic groups of the L-cys–rGO and the amine groups in anti-IgG. And then biotinylated anti-IgG (bio-anti-IgG) was assembled onto the electrode surface based on the sandwich-type immunoreactions. By the conjunction of biotin and streptavidin (SA), SA was immobilized, which in turn, combined with the biotin labeled initiator strand (S1). In the presence of two single DNA strands of glucose oxidase labeled S2 (GOD-S2) and complementary strand (S3), S1 could trigger the hybridization chain reaction (HCR) among Sl, GOD-S2 and S3. Herein, due to HCR, numerous GOD was efficiently immobilizated on the sensing surface and exhibited excellent catalysis towards glucose to in situ generate amounts of hydrogen peroxide, which acted as co-reactant of luminol to significantly enhance the ECL signal. The proposed ECL immunosensor presented predominate stability and high sensibility for determination of IgG with a detection limit of 33 fg mL⁻¹ (S/N = 3).⁹⁰

Since cholesterol can be oxidized to H₂O₂ which an ECL coreactant of luminol, many ECL sensors for it were developed. For example, Zhang et al. successfully developed a simple cholesterol ECL biosensor by synthesizing hemin–graphene nanosheets (H–GNs).⁹¹ As for the principle of the biosensor: cholesterol is oxidized by ChOx to form cholest-4-en-3-one and H₂O₂. Luminol reacted with H₂O₂ to produce the signal of ECL, and hemin could catalyze the cathodic ECL of luminol. Using the similar principle, they prepared another cholesterol ECL biosensor.⁹² In this case; cerium oxide–graphene (CeO₂–graphene) composites could catalyze the ECL of a luminol–H₂O₂ (hydrogen peroxide) system.

QDs-based ECL systems

Semiconductor nanocrystals (NCs) or QDs have received considerable interest due to their excellent luminescent properties and applications in many areas of fundamental importance. High fluorescence quantum yields, size-controlled luminescence properties, and stability against photobleaching are the most remarkable properties of QDs that make them particularly attractive for development of novel ECL biosensors. In these biosensors, owing to its large surface area and excellent electroconductivity, graphene or its derivatives or other nanomaterials functionalized with them was usually used as a sensing platform to capture a large number of analysts or a conducting bridge to promote the electron transfer between QDs and the electrode.

Using gold–silver nanocomposite-functionalized graphene as a sensing platform, Zhang et al. reported a novel ECL immunosensor for the sensitive detection of cancer antigen 125, a tumor marker in human serum.⁹³ In their work, the as-prepared CdTe QD coated carbon microspheres (QD@CMs) acted as bionanolabels, and a good ECL performance was exhibited through a sandwich-type immunoassay. The proposed method provides a new promising platform for clinical immunoassays of other biomolecules. Very recently, the CdTe QD coated PtFe nanoparticles was used to label the signal DNA, while magnetic graphene nanosheets were selected as carriers for the capture DNA due to their excellent biomagnetic separation capability and electrical properties. Based on this, another disposable microdevice suitable for sandwich-type ECL detection of DNA was reported.⁹⁴ Under optimal conditions, the biosensor responds linearly to DNA in the 0.02 fM to 5000 fM concentration range, with a detection limit as low as 15 aM. Similarly, CdTe QD coated hollow ZnO nanoparticles was used to label the signal DNA, while GS, CNT and AuNPs to form AuNPs dotted CNT–GS composites (Au@CNT–GS) was acted as platform for the capture DNA. In the presence with S₂O₈²⁻ coreactant, a new sandwich-type DNA ECL biosensor was designed.⁹⁵

The GO with unique electrical properties was used as nano-amplified platform to immobilize a large number of capture DNA (c-DNA1) was described.⁹⁶ In this work, the endonuclease-assisted amplification technique was applied to amplify the ECL signal change induced by target cells. Specifically, the bifunctional composite QDs with excellent magnetic property can be conveniently labeled, separated, and developed the ECL signal probe, thus an ECL method for rapid and sensitive detection of cancer cells was developed.

Graphene or GO conjugated with gold nanoparticles was widely used to capture great deal of antibody. Gold nanoparticle–graphene nanosheet (Au–GN) hybrids were prepared and served as an effective matrix for initial antibodies (Ab1) attachment.⁹⁷ The NIR ECL nanoprobe (SiO₂–QD-Ab₂) was designed by covalent assembly of goat antihuman IgG antibody (Ab2) on CdTe/CdS QDs tagged silica nanospheres. After a sandwich immunoreaction, the functionalized silica nanosphere labels were captured onto the glass carbon electrode surface. Integrating the dual amplification from the promoting electron transfer rate of Au–GN hybrids and the increasing QD loading of SiO₂–QD-Ab2 labels, the NIR ECL response from CdTe/CdS QDs enhanced 16.8-fold compared to the unamplified protocol and successfully fulfilled the ultrasensitive detection of human IgG (HIgG) with a detection limit of 87 fg mL⁻¹. Compared with the conventional two-dimensional (2D) structure, 3D graphene with larger surface area conjugated with gold nanoparticles (AuNPs) (3D-GR@AuNPs) was used to provide an effective matrix for antibody immobilization.⁹⁸ Base on the new sensing platform, a sensitive ECL immunosensor for detection of PSA was developed. Nanoporous sliver (NPS) @ CDs composites was as good ECL label. The good electrical conductivity of 3D-GR@AuNPs and high loading of CDs of NPS@CDs composites provided the significant advantage of dual-signal amplification technique. The new biosensor displayed good analytical performance for the detection of PSA in the range from 1 pg mL⁻¹ to 50 ng mL⁻¹ with a low detection limit of 0.5 pg mL⁻¹. A sandwich-type ECL aptasensor for sensitive determination of thrombin is designed employing CdSe/ZnS quantum dots served as an ECL label and Au–GN hybrids used as capturer of thiol-terminated aptamer.⁹⁹

With SO₄²⁻ as coreactant, ECL of the graphene/AuNCs hybrid in aqueous solution was studied and an ECL biosensor for H₂O₂ in the practical clinical analysis was developed.¹⁰⁰ The cathodic ECL was originated from the formation of excited-state AuNCs* via electron-transfer annihilation of negatively charged AuNCs and the strongly oxidizing SO₄⁻˙ radicals produced by electro-reduction of S₂O₈²⁻. After H₂O₂ was injected into the ECL testing system, the cathodic ECL intensity of the Indium Tin Oxides (ITO) electrode modified with the graphene/AuNCs hybrid displayed an obvious decrease. The decrease was attributed to the quench of the excited state of AuNCs*.

Using GO, 5-fold ECL amplification of CdTe QDs platform was reported. This platform showed a detection limit of 8.3 μM (S/N = 3) for glutathione and a selective detection linear dependence from 24 to 214 μM in the presence of 120 μM cysteine and glutathione disulfide.¹⁰¹ Combining CdSe quantum dots (QDs) with GO–chitosan (GO–CHIT), the enhancement of ECL was also found by Wang et al.¹⁰² The sensitive ECL sensor was used to detect of cytochrome C (Cyt C) with the detection limit of 1.5 μM. One year later, via layer-by-layer assembly, graphene oxide sheets/polyaniline/CdSe quantum dots (GO/PANi/CdSe) nanocomposites were used as a sensing platform for Cyt C was also proposed.¹⁰³ Under the optimized conditions, the ECL intensity decreased linearly with the Cyt C concentrations with detection limit of 2.0 × 10⁻⁸ M.

Graphene nanosheets also were selected as signal amplification agents for ECL of CdS NCs,¹⁰⁴ C-dots¹⁰⁵ and CdTe QDs.¹⁰⁶ In the latter, on the basis of the effect of organophosphate pesticides (OPs) on the ECL signal of acetylcholinesterase (AChE)–QDs–GNs modified glassy carbon electrode (GCE), a highly sensitive GNs-anchored-QDs-based signal-on ECL biosensor was developed for sensing Ops. Using multicolor quantum dots as labels and graphene as conducting bridge, an ECL immunoassay for simultaneous determination of two different tumor markers, AFP and CEA was developed.¹⁰⁷ 30-fold enhancement of the ECL intensity can be obtained. As a result, the detection limit (LOD) for both analytes at 0.4 fg mL⁻¹ was very low. This novel multiplex ECL immunoassay provided a simple, sensitive, specific and reliable alternative for the simultaneous detection of tumor markers in clinical laboratory. In another work, a nearly 48-fold ECL amplification can be obtained by immobilizing carbon quantum dots (CQDs) on graphene.¹⁰⁸ Base on this phenomenon; an unprecedented sensitive ECL sensor has been first developed for the determination of chlorinated phenols, a typical group of persistent organic pollutants in the environment. Graphene doped with K was also found to amplify the ECL signal of SiO₂@CdS nanocomposites and a new ECL biosensor was developed for the successful detection of transcription factor TATA-binding protein (TBP).¹⁰⁹ It's ingenious that a dual amplification strategy for ultrasensitive ECL immunoassay based on a Pt nanoparticles dotted graphene–carbon nanotubes composite (Pt/Gr–CNTs) and carbon dots functionalized mesoporous Pt/Fe (Pt/Fe@CDs) was established by Deng et al.¹¹⁰ They fist synthesized Pt/Gr–CNTs via facile ultrasonic method to modify the working electrode and then capture a large amount of primary anti-CEA antibodies. The other amplification is from the Pt/Fe@CDs nanocomposites as signal tags can increase CDs loading per immunoreaction in comparison with single CDs. As a result, a very sensitive method for CEA with a low detection limit of 0.8 pg mL⁻¹ was designed. The fabrication of the ECL immunosensor was shown in Fig. 7.

Fig. 7 Schematic representation of the fabrication of the ECL immunosensor. Adapted from Deng et al.¹¹⁰ with permission from RSC Publications.

Recently, our group found ECL of thiol-capped CdTe QDs in aqueous solution was greatly enhanced by PDDA-protected graphene (P-GR) film.¹¹¹ Two ECL peaks at −1.1 (ECL-1) and −1.4 V (ECL-2) in pH 11.0 were observed and ECL-1 showed higher sensitivity for the detection of H₂O₂ concentrations than that of ECL-2. Thus, a novel CdTe QDs ECL sensor with detection limit of 9.8 × 10⁻⁸ mol L⁻¹ for H₂O₂ was designed. ECL of PDDA-protected graphene–CdSe (P-GR–CdSe) composites was also reported for the sensitive detection of human IgG (HIgG).¹¹² Graphene oxide nanosheets/polyaniline nanowires (GO/PANi) nanocomposites was found to enhance ECL of CdSe QDs. Base on this, Liu et al. reported an ECL immunosensor for detection of human interleukin-6 (IL-6).¹¹³ Electrochemically reduced graphene oxide (ERGO) was also found to amplify ECL of QDs to construct a nanobiosensing platform. In the presence of dissolved O₂ as coreactant, the QDs/ERGO modified electrode showed ECL intensity increase by 4.2 and 178.9 times as compared with intrinsic QDs and QDs/GO modified electrodes due to the adsorption of dissolved O₂ on ERGO and the facilitated electron transfer. After choline oxidase (ChO) or ChO–acetylcholinesterase was further covalently cross-linked on the QDs/ERGO modified electrode, two ECL biosensors for choline and acetylcholine were fabricated, respectively.¹¹⁴

Interesting, Guo et al. found that AFP could effectively scavenge the ECL of graphene–CdS quantum dots–alginate (G–CdS QDs–AL) composite¹¹⁵ or G–CdS QDs–agarose composite,¹¹⁶ and the quenched ECL intensity depended linearly on the logarithm for AFP concentration. As a result, two ECL immunosensors for ultrasensitive detection of AFP was fabricated, respectively.

ECL of peroxydisulfate systems

Different nanocomposites conjucted by RGO or graphene were used to modify electrodes and indirect ECL of S₂O₈²⁻ was investigated at these modified electrodes in detail.

In a recent literature, RGO/CdCO₃ nanocomposites can not only enhance the ECL intensity of S₂O₈²⁻ but can also decrease its onset potential.¹¹⁷ ECL of S₂O₈²⁻ also was found to be efficiently enhanced by nanocomposite of electrochemically RGO and gold nanoparticles.¹¹⁸ 15 times enhanced ECL was observed with the formation of nanocomposite at GCE and immunoreaction occurred at electrode surface can suppress corresponding ECL emissions. A new immunosensor for HIgG with a detection limit of 1.3 pg mL⁻¹ (S/N = 3) was fabricated. A dopamine (DA) ECL sensor also was prepared by the similar composite and ECL system. The as-prepared solid-state ECL DA sensor showed a wide linear response of 0.02–40 μM with a detection limit of 6.7 nM (S/N = 3).¹¹⁹ Using RGO/multiwall carbon nanotubes (MWCNTs)/AuNPs composite modified GCE, another ECL sensor for DA based on ECL of S₂O₈²⁻ was proposed.¹²⁰ DA was found to be able to enhance the ECL. The applicability of the proposed sensor was also evaluated by detecting DA in dopamine hydrochloride injection, human urine and serum.

Similar to DA, phenolic compounds was found can enhance the ECL of S₂O₈²⁻ at the graphene (GP)/MWCNTs/AuNCs modified glassy carbon electrode. Based on this, an ECL sensor was proposed for the determination of phenolic compounds with high sensitivity, good repeatability and stability.¹²¹

Apart from enhancing the ECL intensity of S₂O₈²⁻, graphene or GO or RGO functionalized with nanomaterials are particularly valuable in the framework of the ECL immusensor for some of their unique characteristics as mentioned above and here more detailed: (i) their high surface area/volume ratio that allows a great increase of the electrode surface, and their loading to form multiple ECL labels; (ii) their conductivity to facilitate the transfer of the electrons; (iii) their strong interaction with amines and sulphur containing moieties, that can be very advantageous in the preparation of ECL biosensors since it allows an easy immobilization of biomolecules.

A novel tracer, 3,4,9,10-perylenetetracarboxylic dianhydride (PTCDA) functionalized GS composite (GS–TCDA) is employed to label the secondary anti-thrombin aptamer (TBA) to construct an ultrasensitive electrochemiluminescent sandwich-type aptasensor.¹²² The GS provided large surface area for loading abundant PTCDA and TBA with good stability and biocompatibility. Because of the excellent electroconductivity of GS and the desirable optical properties of PTCDA, the as-formed Apt II bioconjugate considerably not only amplified the ECL signal of S₂O₈²⁻ but also worked as the desirable label for Apt II. On the basis of this, an ultralow detection limit of 0.33 fM for thrombin was obtained.

Three-dimensional (3D) nanoflower-like MnO₂ functionalized graphene (GN/MnO₂) as bionanolabels was prepared and provided an effective matrix for antibody immobilization with good stability and bioactivity.¹²³ With a sandwich-type immunoassay format, the amount of functionalized GN/MnO₂ labeled antibodies increased with the increment of antigens in the samples. And the ECL signals of S₂O₈²⁻ enhanced with the increase of in situ generated O-2 due to the catalysis of MnO₂ (a substitute for horseradish peroxidase) to the H₂O₂. The immunosensor displayed excellent analytical performance for the detection of PSA in the range of 0.005–10 ng mL⁻¹ with a detection limit of 2.5 pg mL⁻¹ (3 sigma).

A novel electrochemiluminescent immunosensor was designed for cancer biomarker detection based on peroxydisulfate cathodic ECL using a new enhancer, one kind of perylene derivatives, synthesized by integrating the arginine covalently onto the 3,4,9,10-perylenetetracarboxylic acid (PTCA) (abbreviation as PTC-Arg).¹²⁴ Combining the supramolecular assembly method, the aromatic compound of PTC-Arg was effectively immobilized on the gold nanoparticles functionalized graphene (Au@Gra) by pi–pi stacking (PTC-Arg/Au@Gra). And then PTC-Arg/Au@Gra complexes were further used as multifunctional nanocarriers for the absorption of the second antibody of alpha-fetoprotein (Ab(2)), which formed sandwich-type immunoassay format through associating with AFP and the first antibody (Ab(1)). With PTC-Arg/Au@Gra as enhancer of peroxydisulfate system, the immunosensor exhibited a wide dynamic range of 0.001–10 ng mL⁻¹ and detection of limits (LODs) of 0.3 pg mL⁻¹, respectively. It also suggested a great potential development and applications for sensitive bioassays in clinical detection.

Some new ECL systems

Apart from QDs, other nanomaterials, such as upconversion nanomaterial (NaYF 4/Yb,Er)¹²⁵ and metal–organic complex nanowires¹²⁶ combined with graphene have been introduced to ECL systems. And the ECL intensity of these nanomaterials can be significantly amplified by graphene due to its wonderful conductivity, extraordinary electron transport properties and large specific surface area.

Graphitic carbon nitride (g-C₃N₄) is regarded as a valuable extension of carbon in material applications and has experienced a renaissance of activity in recent years. In particular, its ECL behavior first reported in 2012 demonstrated that semiconductor g-C₃N₄ may be a new class of efficient and promising luminophore for ECL sensing.¹²⁷ Two years later, the ECL intensity of carboxylated g-C₃N₄ was found to be much enhanced after being combined with graphene.¹²⁸ The g-C₃N₄–graphene was deposited on electrodes and antibodies were immobilized on the surface of carboxylated g-C₃N₄ through amidation. Based on this sensing platform, a novel label-free ECL immunosensor was developed for the detection of squamous cell carcinoma antigen (SCCA).

A new ECL system, a phenyleneethynylene derivative-based ECL and its ultrasensitive immunosensing application has also been reported by Yan et al.¹²⁹ Combined gold/graphene modified screen-printed working electrode (SPWE) with their previous reported ECL system,¹²⁹ an ECL microfluidic origami immunodevice was developed by the same group.¹³⁰ Initially, a graphene layer was immobilized on the surface of carbon working electrode, and then gold was assembled on the graphene surface to capture the antibody. Then, phenyleneethynylene derivative modified nanotubular mesoporous Pt–Ag alloy nanoparticles were used as signal amplifier for the highly sensitive determination of CEA with a low detection limit of 0.3 pg mL⁻¹.

On the basis of these studies, more sensitive and special ECL sensors may be fabricated with the development of more novel ECL systems combined with graphene and its derivatives.

Conclusions and perspectives

The interest in chemiluminescence and electrochemiluminescence of graphene and its derivatives has evolved along with interest in their electronic properties. The chemiluminecsence, especially electrochemiluminescence of graphene and its derivatives has encouraged numerous explorations of them in chemsensors and biosensors. In chemiluminsencence system, graphene and its derivatives usually act as catalysts, a platform for multipurpose sensing and bioassays, or a super-quencher of a CL donor or fluorophore in CRET. In ECL systems, such as Ru(II) complex-based ECL systems, luminol-based ECL systems, QD-based ECL systems, and some new ECL systems, graphene and its derivatives present excellent electron transfer ability for some enzymes, excellent catalytic behavior toward small molecules, and an excellent character as a carrier to load more active probes and active domains for biomolecules binding which offering a significant amplification on the ECL sensing signals. Moreover, graphene and its derivatives often indirectly enhanced ECL intensities of different ECL systems.

Up to now, it is still a developing field and less literatures of graphene or its derivatives participated in ECL and CL, particularly, CL was reported. However, by suitable doping, chemical manipulation, or as essential components of nanocomposites, graphene and its derivatives may open the door to a host of unforeseen applications in CL and ECL for bioanalysis.

Recently, optical imaging and therapy using nanosized graphene and graphene oxide was reviewed by Wang et al.¹³¹ and the challenges of graphene in biomedical applications were summarized by Bitounis et al.¹³² Just as they said, although graphene has great potential in bioimaging cells or drug delivery, toxicity of graphene to cells, organisms and the environment has not yet been completely assessed. To solve this problem, one way is the biocompatibility of graphene needs to be better understood and to be controlled. Another way is the exploration of other two-dimensional atomic crystals such as graphitic carbon nitride (g-C₃N₄). Very recently, ECL,^{127,128,133,134} persistent luminescence¹³⁵ and CL¹³⁶ of g-C₃N₄ have been reported, respectively. Among them, the persistent luminescence of g-C₃N₄ has been successfully applied for imaging detection of biothiols in human urine, plasma, and cell lysates with excellent biocompatibility and nontoxicity. So, we believe there are still many two-dimensional graphene-like atomic crystal to be discovered and more sensitive and selective analytical methods can be developed based on these new discoveries.

In addition, there are still two obstacles for developing graphene sensors. One is the lack of methods for controllable, reproducible, scalable, and facile preparation of graphene materials with defined structures and properties. The other is a better understanding of graphene properties, the interactions between graphene and molecules/cells, and the detection mechanisms. To move forward, the collaborations between different disciplines and technologies are necessary. With the development of material science and chemometrics, we envision that the emerging graphene sensors would soon bring significant impacts on environmental and safety monitoring, diagnosis, bioimaging, and drug screening.

Acknowledgements

Authors gratefully acknowledge financial support for this project from the National Natural Science Foundation of China [no. 21105067 & 21375089].

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