Anal Bioanal Chem DOI 10.1007/s00216-014-7946-x

REVIEW

Analytical applications of nanomaterials in electrogenerated chemiluminescence Paolo Bertoncello & Alasdair J. Stewart & Lynn Dennany

Received: 19 March 2014 / Revised: 25 May 2014 / Accepted: 4 June 2014 # Springer-Verlag Berlin Heidelberg 2014

Abstract This critical review covers the use of carbon nanomaterials (single-wall carbon nanotubes, multi-wall carbon nanotubes, graphene, and carbon quantum dots), semiconductor quantum dots, and composite materials based on the combination of the aforementioned materials, for analytical applications using electrogenerated chemiluminescence. The recent discovery of graphene and related materials, with their optical and electrochemical properties, has made possible new uses of such materials in electrogenerated chemiluminescence for biomedical diagnostic applications. In electrogenerated chemiluminescence, also known as electrochemiluminescence (ECL), electrochemically generated intermediates undergo highly exergonic reactions, producing electronically excited states that emit light. These electron-transfer reactions are sufficiently exergonic to enable the excited states of luminophores, including metal complexes, quantum dots and carbon nanocrystals, to be generated without photoexcitation. In particular, this review focuses on some of the most advanced and recent developments (especially during the last five years, 2010–2014) related to the use of these novel materials and their composites, with particular emphasis on their use in medical diagnostics as ECL immunosensors. Keywords Electrochemiluminescence (ECL) . Immunosensors . Quantum dots . Carbon nanomaterials Published in the topical collection Analytical Bioluminescence and Chemiluminescence with guest editors Elisa Michelini and Mara Mirasoli. P. Bertoncello (*) Centre for NanoHealth, College of Engineering, Swansea University, Singleton Park, Swansea SA2 8PP, UK e-mail: [email protected] A. J. Stewart : L. Dennany WestCHEM, Department of Pure and Applied Chemistry, University of Strathclyde, Glasgow G1 1XL, UK

Introduction Electrochemiluminescence (ECL) is defined as a process in which electrochemically generated species combine to undergo highly energetic electron-transfer reactions (usually of redox or enzymatic origin) that lead to the emission of light from excited states. ECL was first reported in the early 1960s in seminal works from Hercules [1], followed by Visco [2] and Bard [3]. ECL has been used in many analytical applications and these have been covered in several reviews [4–14]. Notably, ECL is used not only as a “stand-alone” technique, but also in combination with other analytical techniques, in primis high-performance liquid chromatography (HPLC) [15], electrophoresis [16], and flow-injection analysis (FIA) [17]. ECL is initiated and controlled by applying a suitable potential at the electrode surface. In contrast with other spectroscopic techniques, ECL, as well as providing accurate control of the time and position of the light-emitting reactions, does not require the use of an external light source, and therefore problems related to light scattering are avoided. Sensitive measurement of proteins or other biomarkers is critical to many aspects of biochemical and biomedical assays. A biomarker is defined as “a laboratory measurement that reflects the activity of a disease process” [18]. An important class of biomarkers are specific proteins that, when present in elevated or depressed concentrations in serum, tissue, or saliva, may be indicative of disease. The fabrication and development of reliable, cost-effective, and powerful analytical detection methods for early-stage detection and ongoing monitoring of disease are of crucial relevance, in particular for cancers and cardiovascular diseases because of their prevalence, high rates of recurrence, and potential lethality. ECL immunosensors combine the high sensitivity of ECL detection, the specificity of immunoreaction, simple instrumentation, and easy signal quantification and are therefore ideal for use in this field.

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ECL can be produced by two dominant pathways: annihilation and coreactant pathways. Most ECL applications are based on the coreactant pathway and this review tends to focus on these. These two mechanisms of ECL reaction have been described in previous reviews [12, 13].

Semiconductor quantum dots Miniaturization of biosensors is an attractive topic for investigation in the development of sensor arrays, and this is also true in the field of ECL sensors. The optical and luminescent properties of semiconductor nanocrystals, or quantum dots (QDs), make them highly attractive for a large variety of applications in nanotechnology [19]. In particular, high-fluorescence quantum yields, size-dependent luminescence, and stability against photobleaching make them a very attractive material for biosensing applications [20]. However, QDs are synthesized in organic solvents and capped with hydrophobic ligands to prevent aggregation. For full exploitation in bioanalytical applications, it is necessary to functionalize the QDs’ surface with hydrophilic ligands to make them water soluble and biocompatible. For this purpose different approaches have been used, including ligand exchange with hydrophilic ligands (dendrons, peptides, and thiol derivatives) or encapsulation in amphiphilic copolymers, silica coating, and phospholipids [21]. Since Bard’s first reports on the electrochemiluminescence (ECL) properties of CdSe [22, 23] and CdTe nanocrystals [24], analytical applications of ECL from QDs have greatly increased. Most ECL QD biosensors are based on the quenching or enhancement of ECL intensities via these well-established co-reactant ECL systems [1, 8, 9], with the ECL response related to the presence and concentration of the QDs. The common ECL processes corresponding to the production of ECL through the interaction with the coreactant H2O2 are [19]: QDs þ 1e− →QDsðe− 1Se Þ

ð1Þ

QDsðe− 1Se Þ þ H2 O2 →QDs þ OH− þ OH•

ð2Þ

OH• þ QDs→OH− þ QDsðhþ 1Sh Þ

ð3aÞ

QDsðe− 1Se Þ þ OH• →OH− þ QDs

ð3bÞ

QDsðe− 1Se Þ þ QDsðhþ 1Sh Þ→QDs

ð4Þ

QDs →QDs þ hν ð640 nmÞ

ð5Þ

During the cathodic scan, electrons are injected into the QDs (Eq. 1), after which the electron-injected QDs (QDs(e− 1Se)) reduce H2O2 to produce OH− and OH• (Eq. 2). OH• is the crucial species that can easily inject a hole into the 1Sh quantum-confined orbital of QDs (Eq. 3a), resulting in the formation of QDs(h+ 1Sh). This process is possible because of the high standard redox potential of the OH−–OH• pair [12, 13]. At the same time, the excited states, QDs*, are formed by the reaction of the reduced QDs with OH• or by the recombination of the injected electrons (e−) with the injected holes (h+) of QDs (Eq. 4). Both these processes (Eq. 3b and 4) lead to the formation of the luminophore, QDs*, even though the two processes are mechanistically different, the former being a coreactant ECL process and the latter an annihilation process. QDs* will emit light at a wavelength that depends on the size of the quantum dots [19, 25–27]. Numerous systems using QDs have been used as labels within ECL detection systems [19, 28–30] as a result of their above-outlined advantages over more common emitters. Liu et al. [30] used CdSe QDs to detect α-fetoprotein (AFP), a specific biomarker for carcinoma of the liver. Anti-AFP was covalently linked to a gold electrode, with secondary AFP antibodies labelled with the CdSe QDs. The electrode-bound antibodies were allowed to capture AFP and then the QDlabelled antibodies were allowed to bind to the captured AFP (see Fig. 1). Attaching the QDs on to an antibody for subsequent analysis, through a “sandwich” immunoreaction, is a common approach to these types of ECL systems. In this system, in the presence of H2O2, the ECL intensity increased with the increase in analyte concentration. Detection limits for AFP were improved further in [31], where a CdS-QD–graphene–agarose composite coated on to a glassy carbon electrode was used and no label was required. Anti-AFP was linked to the modified electrode via glutaric dialdehyde. ECL was produced as a result of the interaction of the reduced forms of CdS and the coreactant, K2S2O8 (CdS•− and SO4•−, respectively), to produce the excited state (CdS*) via electron transfer [32]. The ECL intensity of the QD– graphene–agarose composite was seven times greater than that of the pure CdS QD film, because of the improved porosity and conductivity of the composite. Complexation of anti-AFP with AFP resulted in a logarithmic reduction in the ECL intensity, enabling the concentration of AFP to be determined with a detection limit of 0.2 fg mL−1 [32]. A similar system was developed that used a CdS-QD– graphene–alginate composite coated on to a glassy carbon electrode; however, in this case, CdSe–ZnS core-shell QDs were used [33]. Anti-AFP was attached to the QD– graphene–alginate system, with secondary AFP antibodies bound to the CdSe–ZnS QDs. AFP was allowed to complex with the composite-bound anti-AFP, and then

Analytical applications of nanomaterials in electrogenerated chemiluminescence Fig. 1 Biosensor fabrication for the detection of AFP using CdSe QDs. Reprinted from Ref. [30], with permission of Royal Society of Chemistry (2011)

caused by the scavenging of ECL energy by the CdSe–ZnS QD label, preventing the formation of excited states. CdSe– ZnS QDs have an absorption range of 240–620 nm, and the emission of the CdS QDs is in the range 500–700 nm: this

the secondary antibodies were allowed to bind to the formed immunocomplex (see Fig. 2). The formation of this complex resulted in a reduction in the ECL intensity of the CdS-QD–graphene–alginate composite

BSA

anti-AFP-biotin

a

streptavidin-CdSe/ZnS QDs

Ab2-CdSe/ZnS QDs

b

G-CdS QDs-AL G CdS QDs AL

OH OH OH OH OH OH OH OH OH

O O O O O O O O O

APS

Si(CH2)2NH2

GLD

Ab1

BSA

Si(CH2)2NH2

Si(CH2)2NH2

GCE c O O O O O O O O O

Si(CH2)2NH=CH(CH2)3CH=

AFP Si(CH2)2NH=CH(CH2)3CH= Si(CH2)2NH=CH(CH2)3CH=

O O O O O O O O O

Si(CH2)2NH=CH(CH2)3CH= Si(CH2)2NH=CH(CH2)3CH= Si(CH2)2NH=CH(CH2)3CH=

ECL energy scavenging

Ab2-CdSe/ZnS QDs

O O O O O O O O O

Si(CH2)2NH=CH(CH2)3CH= Si(CH2)2NH=CH(CH2)3CH= Si(CH2)2NH=CH(CH2)3CH=

Fig. 2 Fabrication of a dual QD biosensor with detection based on the quenching of CdS-QD ECL by the CdSe–ZnS-QD label. Reprinted from Ref. [33] with permission of Elsevier (2012)

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spectral overlap enables the ECL resonance transfer process to occur, resulting in extremely efficient quenching of the ECL signal after binding of the anti-AFP CdSe–ZnS QD label [33]. This enables very low limits of detection, down to 20 pg mL−1 in this case, with a working range of 0.05 to 500 fg mL−1. Each of these AFP detection systems was successfully used in the determination of AFP from clinical samples (serum and saliva) and none were affected by the presence of other bioma rk ers (c ar bo hy dr ate an tig en 1 9– 9 ( CA 1 9– 9 ) , carcinoembryonic antigen (CEA), human chorionic gonadotropin (HCG), and hepatitis B surface antigen (HBsAg)), revealing the specificity of this technique. This most sensitive system is five million times more sensitive for AFP detection than an equivalent system that does not use nanomaterials in its set-up [34]. Therefore, these systems reveal that the benefits of incorporating QDs into ECL immunosensors include not only vast improvements in sensitivity, as a result of fast electron-transfer kinetics and high quantum yields, but also the wide variety of systems in which they can be used. Their high surface-to-volume ratio and biocompatibility make them ideal for immobilization of biomolecules, and their sizetunable emission enables simple alterations in wavelength to meet specified requirements. Biosensors have been developed for several other cancer biomarkers, including carcinoembryonic antigen (CEA) and human chorionic gonadotropin (HCG). A further development described a system for the detection of CEA using a composite nanostructure comprising gold–silica–CdSe–CdS core-shell QDs [35]. CdSe–CdS QDs were surface passivated with silica-coated gold nanoparticles and immobilized on an electrode. Antibody-bound gold nanoparticles were then attached. The ECL intensity of this nanostructure was 17 times greater than that of pure CdSe–CdS QDs. This amplification was caused by an increase in the rate of electron transfer in the ECL reaction, resulting from the high concentration of gold nanoparticles coated on to the surface of the QDs and an improvement in structure porosity, enabling improved diffusion of the coreactant, K2S2O8, towards the electrode [35]. Formation of the immunocomplex in the presence of CEA resulted in a reduction in the ECL intensity caused by an increase in steric hindrance, impeding electron transfer and diffusion of K2S2O8 in the ECL reaction. A linear working range for CEA of 0.32 pg mL−1 to 10 ng mL−1 was achieved, with a detection limit of 0.064 pg mL−1. Following on from this, there has also been much progress in recent years in combining QDs with other metals for enhanced ECL responses. One such example is a biosensor using CdTe-QD-functionalized Pt–Ru alloys for the detection of α-HCG [36]. CdTe QDs were immobilized within the nanoporous structure of the Pt–Ru alloy, followed by attachment of anti-HCG. Chitosan-coated Fe 3 O 4 magnetic

nanoparticles were coated with primary anti-HCG, and in the presence of HCG these formed an immunocomplex with the QD-modified alloy (Fig. 3). The presence of HCG resulted in an increase in the ECL intensity because the formation of the immunocomplex enabled QD-driven signal amplification. ECL detection using a single QD label was ca. four times less intense because of reduced signal enhancement associated with the decrease in QD loading. A working linear range of 0.005–50 ng mL−1 and detection limit of 0.8 pg mL−1 were obtained [36]. This system was tested using human clinical samples (serum) and had better sensitivity than a standard ELISA assay, which has a detection limit of approximately 70 ng mL−1 (Pishtaz Teb Diagnostics, Inc.; Rapid hCG ELISA kit). These systems provide evidence of the versatile nature of these QDs, which are revealed to be useful ECL luminophores both by themselves and when combined with other species to form nanocomposites. These systems also help to reveal the commercial opportunities associated with QD ECL systems, which are now being realized as a result of excellent research into this exciting field of biosensor analytical applications. Aptamers are single-stranded DNA or RNA molecules that can form tertiary structures after binding to a specific target (proteins, small molecules, or metal ions) [29]. They are prepared by an in-vitro method known as systematic evolution of ligands by exponential enrichment (SELEX), and have several advantages over natural receptors. These include simple synthesis and labelling, good chemical stability, and excellent flexibility for biosensor design [37]. As a result, aptamers lend themselves to incorporation into novel biosensors and, in combination with QDs, have been the subject of much research and development in recent years. For example, Jie et al. [38] have developed an ECL sensor for cancer cells using novel dendrimer–CdSe–ZnS QD nanoclusters. Gold nanoparticles were absorbed on to an electrode surface and then functionalized with the specifically designed aptamer. Dendrimer nanoclusters were loaded with CdSe–ZnS QDs, on to which a probe DNA strand was attached; the loaded nanoclusters hybridized with the electrodebound aptamers, resulting in an ECL signal that was 13 times more intense than that of a film of pure QDs (Fig. 4). This was probably caused by the increase in the number of QDs that could be loaded on to the dendrimer nanoclusters, increasing the surface area and improving electron transfer rates. Aptamer recognition by target cells led to dehybridization of the nanocluster–QD-DNA biocomplex, resulting in a reduction in ECL intensity that was proportional to the concentration of target cells. A working linear range of 400–10,000 cells mL−1 and a detection limit of 210 cells mL−1 were achieved. Shan et al. [39] developed a thrombin sensor that uses the quenching of CdS–Mn-nanocrystal ECL by CdTe-QD-doped

Analytical applications of nanomaterials in electrogenerated chemiluminescence Fig. 3 Fabrication of the immunosensor based on QDmodified Pt–Ru alloys and magnetic beads. Reprinted from Ref. [36] with permission of Royal Society of Chemistry (2012)

silica nanoparticles. CdS–Mn nanoparticles were immobilized on an electrode and then modified with the specific aptamer for thrombin. Silica nanoparticles were doped with CdTe QDs and then functionalized with a probe DNA strand that complemented the aptamer. Hybridization of the QD-doped silica nanoparticles with the aptamer brought them within the effective distance of energy scavenging, resulting in ECL signal reduction caused by quenching of CdS–Mn ECL (585–660 nm) by the QD-doped silica nanoparticles, which revealed a wide absorption in the visible region (400– Fig. 4 Fabrication of a QD-based aptasensor for detecting cancerous cells. Reprinted from Ref. [38] with permission of American Chemical Society (2011)

700 nm). Non-doped silica nanoparticles showed no signs of ECL quenching, and quenching was therefore attributed to the presence of the CdTe QDs. ECL-quenching efficiency was dependent on the number of doped CdTe QDs. ECL quenching was attributed to long-distance (>10 nm) energy scavenging, because interactions with the co-reactant and charge transfer between excited CdS–Mn states and CdTe QDs were ruled out. Thrombin selectively interacts with the electrode-bound aptamers, displacing the QD-doped silica nanoparticles and thus inhibiting ECL quenching. Therefore,

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the ECL signal is related to the concentration of thrombin, and a working linear concentration range of 5 pmol L−1 to 5 fmol L−1 was reported for this system, with a detection limit of 1 pmol L−1. This compares to a detection limit of approximately 0.3 ng mL −1 for a standard ELISA kit for thrombin (AssayMax Human Thrombin ELISA kit). QDs are often incorporated into biosensor techniques to amplify the ECL signal; however, in this case the QDs efficiently amplify ECL quenching, indicating the versatility and flexibility of these nanomaterials when used in ECL systems. An adenosine 5 -triphosphate (ATP) sensor was developed by Huang et al [40]. Anti-ATP aptamers were attached to a gold electrode surface and biotinylated complementary DNA strands were allowed to hybridize with the aptamers. Avidinmodified CdSe–ZnS QDs could then bind via the biotin– avidin system to these complementary DNA strands, resulting in ECL emission after the reaction of the reduced forms of the QDs and the co-reactant (K2S2O8) to produce excited QD states. ATP preferentially binds with the aptamers on the electrode surface, reducing the number of QDs that can interact with the electrode and resulting in a reduction in ECL intensity that is related to ATP concentration. A working linear range of 0.018 to 90.72 μmol L−1 was achieved, with a detection limit of 6 nmol L−1. This assay was not as sensitive as a standard ELISA kit for ATP, which has a detection limit of approximately 1.5 nmol L−1 (Antibodies Online ATP ELISA kit). A very similar system was developed by Yao et al. [41]; however, their system used a ruthenium complex as the lightemitting species. This system had a detection limit of 0.02 nmol L−1 and is clearly superior to both the QD-based and ELISA systems. However, neither the QD nor rutheniumbased systems were tested using human samples, whereas the ELISA kit was. The attractiveness of the QD system is their ease of modification, resulting from the flexibility and versatility of QDs, and this is one of the reasons QD-based biosensors have received so much attention in recent years. An excellent example of such a system modification was the introduction of ECL detection via resonance energy transfer (RET), first used for immunosensors in 2012 by Wu et al. [42]. To optimize the ECL-RET efficiency greater energy overlap between donor and acceptor pairs is essential [43], and therefore energy-tunable materials, including QDs, are appealing. The feasibility of ECL sensing in biological matrices also makes QDs attractive materials, because they can be tuned to emit in the near-infrared (NIR) [44]. Using ECL systems based on NIR-ECL materials enables lower background interference [44]; however, use of such systems revealed one of the main challenges of this field, namely producing stable and strong ECL emitters. To this end, electron transfer (ET) ECL was investigated and revealed improved sensitivity in this region

[45]. These systems established the effectiveness of ECL-ET resulting from the excellent overlap between donor emission and acceptor absorption. This enables efficient ECL-ET quenching that, in turn, improves the sensitivity of these types of system.

Carbon nanomaterials Since their discovery carbon nanotubes (CNTs), with their excellent mechanical and electrical-conductivity properties, have been widely used in electroanalytical sensing applications [46–56]. Single-wall carbon nanotubes (SWCNTs) are small graphene sheets rolled up to form single-wall carbon nanocylinders. Graphene sheets can be rolled up in different ways, and this is represented by the variation of a pair of integers (n, m). The values of these integers relate to the structure of SWNTs, in terms of both diameter and chirality. Depending on the value of these integers, SWNTs are classified as having metallic or semiconductor character. Multi-wall carbon nanotubes (MWCNTs) are co-axial assemblies of SWCNT carbon cylinders placed within each other. Clearly, for electroanalytical use, metallic CNTs are preferable. CNTs are entirely composed of sp2-hybridized carbon and this confers unique properties, including tensile strength, chemical stability, and electrical conductivity in metallic SWNTs [56, 57]. A critical aspect of the use of CNTs in electroanalysis is functionalization with metal nanoparticles, biomolecules, redox mediators, and other molecules, which confers high selectivity and sensitivity properties to the composite material. For sandwich-type immunoassays, signal amplification and noise reduction are crucial to achieve detection limits at ultra-trace level and high sensitivity. Carboxylated and aminefunctionalized carbon nanotubes are useful in this respect, because they are suitable for conjugation with a variety of biomolecules. In recent years, several works on CNT-based ECL immunoassays have been reported. For example, Sun et al. developed an ECL immunosensor for the detection of a protein marker (carcinoembryonic antigen (CEA)) using Lcysteine and in-situ generation of the coreactant for signal amplification [58]. A primary antibody anti-CEA (Ab1) was immobilized on to Au nanoparticles (AuNPs) produced by electrodeposition on glassy carbon electrode. Then, L-cysteine and AuNPfunctionalized MWCNTs were used as a base for the enzyme immobilization (glucose oxidase (GOD), horseradish peroxidase (HRP), and secondary antibody (Ab2)). The CEA antigen and MWCNT–AuNP–enzyme-labelled Ab2 were conjugated to form a sandwich-type immunocomplex through the specific interaction between the antigen and the antibody. The whole procedure is summarized in Fig. 5. The developed ECL

Analytical applications of nanomaterials in electrogenerated chemiluminescence

immunosensor had high sensitivity and specificity for CEA detection, with a linear concentration response in the range 0.02–80 ng mL−1, and a detection limit of 0.67 pg mL−1. Using a similar approach, and using MWNT-functionalized carbon-coated magnetic nanoparticles (MWNT–Fe3O4–C), Chu et al. were able to achieve a very similar detection limit (0.7 pg mL −1 ) [59]. However, in this study the ECL immunosensor was tested in the presence of other biomarkers as potential interferences, including cancer antigen 125, human serum albumin, and prostate-specific antigen. The asprepared ECL immunosensor had excellent specificity and the measurements were not appreciably affected by the presence of such interferences. These detection limits were even further decreased by Cao et al. using a combination of metal nanoparticles (Au, Pt, and Pd) with a SWNT–graphene composite [60]. However, these authors used a different strategy based on amplified cathodic ECL of luminol at low potential, as summarized in Fig. 6. The SWNT–graphene–metal-NPs composite material loaded with GOD promoted the cathodic ECL of luminol response by catalyzing the enzymatic reaction of GOD, with the generation in situ of hydrogen peroxide as a coreactant of the luminol reaction. The as-proposed ECL immunosensor had a highly sensitive response towards CEA detection in the concentration range 0.1 pg mL−1 to 160 ng mL−1, with a detection limit of 0.03 pg mL−1 [60]. Using a very similar approach, in another paper the same authors developed an ECL immunosensor using a base made of carboxyl-functionalized MWNTs as a label, on which Au NPs were deposited for further functionalization with glucose oxidase [61]. Then a secondary antibody (Ab2) and glucose oxidase were bound to the Au-NP-functionalized MWNTs. The enzymatic reaction produced hydrogen peroxide as a coreactant, causing a large increase of the luminol ECL signal. Fig. 5 Schematic diagram of the preparation of the ECL immunosensor for CEA detection. Reprinted from Ref. [58] with permission of Elsevier (2014)

The as-prepared immunosensor had a linear range of concentration for the detection of α-1-fetoprotein (AFP) from 0.1 pg mL−1 to 80 ng mL−1, with a detection limit of 0.3 pg mL−1 [61]. Similar results, but with a slightly higher detection limits, were obtained by the same authors using a MWNT–Nafion and [Ru(bpy)3]2+ composite [62]. The use of a cation-exchange polymer (Nafion) to incorporate [Ru(bpy)3]2+ is attractive, because it enables preconcentration of the luminescent positively charged specie at a molar concentration range within the polymer matrix [63]. Yan et al. obtained very similar results in terms of sensitivity (linear range of concentration from 1 pg mL−1 to 100 ng mL−1, and a detection limit of 0.3 pg mL−1); however, in their work they used an ECL immunosensor based on a paper-based microfluidic origami and Au NPs grown on graphene layers [64]. A different approach was used by Deng et al., who developed an ECL immunoassay for CEA detection using a signal-amplification strategy based on the adsorption-induced catalytic reduction of dissolved oxygen at the side wall of nitrogen-doped single-walled carbon nanotubes (N-CNTs), which promoted ECL emission of CdS quantum dots [65]. In this work, the authors revealed that N-SWNTs have a strong effect on the chemisorptions of O2, changing it from the usual monoatomic end-on adsorption at CNTs to a diatomic side-on adsorption at sidewalls. This facilitated the breaking of the O– O bonding, bringing about the reduction of O2 to a radical superoxide intermediate, O2•−, that acted as a coreactant for the ECL emission of CdS QDs. First, polystyrene-sulfonated (PSS)-functionalized N-CNTs were used for labeling with the signal antibody (Ab2) and forming a sandwich immunocomplex on the CdS-QDs–chitosan and a captureantibody (Ab1)-modified electrode. The generation of the superoxide radical specie, O2•−, was crucial to generate a strong ECL signal without the addition of any other strong

P. Bertoncello et al.

Fig. 6 Schematic diagram of the preparation of the ECL immunosensor for CEA detection, and proposed mechanism. Reprinted from Ref. [60] with permission of Elsevier (2013)

oxidant as coreactant. The as-prepared CdS–N–CNT ECL immunosensor had very good sensitivity in the concentration range 50 pg mL−1 to 5 μg mL−1, and a detection limit for CEA of 2.4 pg mL−1 [65]. Interestingly, the proposed method could reduce the false positives that usually occur because of the steric hindrance resulting from the formation of the immunocomplex and the consumption of the coreactant in the ECL reaction. As mentioned previously, aptamers as molecular-recognition substances for proteins seem to be an excellent alternative to antibodies because of their ease of production in vitro, wide target range, reversible thermal denaturation, and unlimited shelf life. An ECL aptasensor that incorporates SWNTs in its design has been developed for the detection of thrombin [66]. In this work, thrombin-binding aptamer (TBA) was used as a molecular-recognition element, and Au NPs and SWNTs were used as a carrier of the ECL capture–signal probe. The principle of the so-called “signal off/on” ECL aptasensor is shown in Fig. 7. In the “signal off” configuration, a thiolated capture probe (ss-DNA, 12-mer) was attached to Au NPs that were deposited on the Au electrode surface using a self-assembly method. The capture probe was then hybridized with a six-base segment of the ss-DNA sequence (Tgt-aptamer, 21-mer)

containing a TBA-I (ss-DNA, 15-mer) previously tagged with a ruthenium complex. The probe tagged with the Ru complex produced a high ECL signal. The introduction of thrombin induced the dissociation of the Tgt-aptamer tagged with the ruthenium complex from the aptasensors, leading to significant quenching of the ECL signal. The quenching of the ECL signal was proportional to the concentration of thrombin in the range 2.7 pmol L−1 to 2.7 nmol L−1, with a detection limit of 0.8 pmol L−1. In the “signal on” configuration, the thiolated TBA-I was self-assembled on the Au electrode surface. Then the TBA-II (ss-DNA, 29-mer) labeled with a SWNT-ECL tag was bound with an epitope of thrombin, producing a high ECL signal. The enhancement of the ECL intensity was linear with the concentration of thrombin in the range 0.01–10 pmol L−1, with a detection limit of 3 fmol L−1 [66]. Composite materials based on MWNTs are a useful base for the development of sandwich-type ECL immunosensors. Wu et al. recently reported an ECL-based immunosensor for the detection of retinol-binding protein (RBP) [67]. In this work, a primary antibody (anti-RBP) was immobilized on to MWCNTs. A RBP antigen and a [Ru(bpy)3]2+-Nafion deposited on a SiO2-nanosphere-labeled secondary antibody were then successively conjugated to form a sandwich-type

Analytical applications of nanomaterials in electrogenerated chemiluminescence

Fig. 7 Design of thrombin aptasensor. (a) Au NPs are taken as a capture-probe carrier. (b) SWNTs are taken as a signal-probe carrier. Reprinted from Ref. [66] with permission of Elsevier (2010)

immunocomplex through the specific interaction between antigen and antibody. The as-prepared ECL immunosensor was extremely specific and selective and worked linearly in the concentration range 78–5000 ng mL−1, with a detection limit of 26 ng mL−1. The sensor was successfully used with a sample of urine, with recoveries and RSDs in the range 98– 112 % and 0.6–5.9 %, respectively [67]. In a similar approach, but using graphene oxide rather than carbon nanotubes, Cheng et al. developed an ECL immunosensor for the detection of CEA [68]. In this work, they fabricated a luminol ECL immunosensor using ZnO NPs and glucose-oxidase-enhanced graphene as labels. The hydrogen peroxide generated in situ by the enzymatic reaction with glucose oxidase was used as a coreactant for the ECL reaction, and the CEA antibody was anchored on Au NPs electrodeposited on to glassy carbon electrodes. The proposed approach enabled the detection of CEA at ultratrace concentrations, with a detection limit of 3.3 pg mL−1 and a linear concentration range of 10–80 pg mL−1 [68]. Ink-jet carbon-nanotube forest arrays have recently been developed in the Forster’s lab to detect picomolar concentrations of immunoglobulin G (IgG) using ECL [69]. Arrays of vertically aligned nanotube forest were grown on indium-tinoxide electrodes. The vertical alignment configuration

significantly increased the conductive surface area of carbon nanotubes available for functionalization. Then, via peptidebond formation, capture anti-IgG was coupled to the carboxyl groups of SWNTs. A ruthenium luminophore was then used to functionalize SiO2 nanoparticles and IgG-labeled G1.5 acid-terminated poly(amido amine) (PAMAM) dendrimers. In the presence of sodium oxalate as a coreactant, a significant ECL signal was obtained (Eqs. 6–12) that enabled the detection of IgG concentrations in the range 20 pmol L−1 to 300 nmol L−1 , with a detection limit of 1.1 pmol L−1 [69], based on the proposed mechanism reported below.  2þ  3þ RuðbpyÞ3 −e− → RuðbpyÞ3 ð6Þ 

RuðbpyÞ3

2þ

 þ þ C 2 O42− → RuðbpyÞ3 þ C 2 O:4−

C 2 O:4− →CO:2− þ CO2



RuðbpyÞ3

3þ

 2þ þ CO:2− → RuðbpyÞ3 þ CO2

ð7Þ

ð8Þ

ð9Þ

P. Bertoncello et al.





RuðbpyÞ3

RuðbpyÞ3



2þ

3þ

RuðbpyÞ3

 þ þ CO:2− → RuðbpyÞ3 þ CO2

 þ  2þ  2þ þ RuðbpyÞ3 → RuðbpyÞ3 þ RuðbpyÞ3

2þ

 2þ → RuðbpyÞ3 þ hν

ð10Þ

ð11Þ

ð12Þ

Here we see oxidation of [Ru(bpy)3]2+ (Eq. 6), followed by reduction of oxalate in the presence of [Ru(bpy)3]3+ (Eq. 7) to create the oxalate anion radical that rapidly decomposes to carbon dioxide and a carbon-dioxide anion radical (Eq. 8). This species, crucial to ECL production, can then be oxidized through interaction with either [Ru(bpy)3]3+, to directly create the excited-state ruthenium molecule (Eq. 9), or [Ru(bpy)3]2+, to create [Ru(bpy)3]+ (Eq. 10) which can then react with [Ru(bpy)3]3+ to produce the excited state (Eq. 12). Recently, carbon nanocrystals (CNCs) have been revealed to be a very useful material for ECL sensing because of their stable and strong ECL emission and their easy synthesis, low cytoxicity, water solubility, and easy labelling. These properties make CNCs a valid alternative to conventional luminophores including [Ru(bpy)3]2+ [70]. For example, CNCs have successfully been used in detecting K562 leukemia cells, using ECL detection based on aptamers and ZnOfunctionalized carbon nanocrystals [71]. The aptamers were used for cell capture and, after conjugation of Concanavalin A with the ZnO-nanosphere-functionalized CNCs, the composite material was used for selective recognition of the cellsurface carbohydrate. The functionalization of ZnO nanospheres with CNCs improved the ECL signal by approximately one order of magnitude compared with pristine ZnO nanospheres and ca. fourfold compared with pristine CNCs. The high surface-to-volume ratio of ZnO nanospheres enhanced the loading of CQDs and Concanavalin A, leading to a significant ECL signal amplification. The as-prepared ECL sensor had high specificity towards the detection of K562 cells in the range 1×102 cells mL−1 to 2×107 cells mL−1, with a detection limit of 46 cells mL−1 [71]. In another work, Wang et al. developed an ECL aptasensor for the detection of thrombin using graphene oxide with an intercalated Ru(phen)32+ probe [72]. Graphene oxide is an important graphene derivative that has great potential for the production of chemically-modified graphene composites on the ton scale. This is because of its essentially infinite possibility of functionalization and suitability for cost-effective and large-scale production. Graphene oxide (GO) was deposited on conducting electrode surfaces (glassy carbon and/or Au electrodes) through physical adsorption, and the amino-tagged aptamers were immobilized on the electrode surface via amide

linkage between the amino group of the aptamer and the carboxyl groups of GO. Then a functional oligonucleotide containing two parts, a complementary strand and an intermolecular duplex for the intercalation of [Ru(phen)3]2+ as ECL probe, was introduced. The hybridization between the aptamer and its complementary part at the functional oligonucleotide containing the [Ru(phen)3]2+ probe enabled detection of the ECL emission. The hybridization between the aptamer and thrombin led to the release of the functional oligonucleotide containing the intercalated [Ru(phen)3]2+ probe, causing the quenching of the ECL signal. The quenching of the ECL signal was directly proportional to the concentration of thrombin. The as-prepared ECL aptamer sensor had a linear response for the detection of thrombin in the range 0.90–226 pmol L−1, with a detection limit of 0.40 pmol L−1 [72]. Composite materials based on the combination of graphene and CdSe QDs have found applications in ECL immunosensing. Li et al. developed a novel strategy to fabricate a polymer-based (poly(diallyldimethylammonium chloride) (PDDA)-protected graphene–CdSe (P-GR–CdSe) composite for ECL detection of human IgG [73]. GQD composites were prepared using electrostatic interactions between negatively charged thioglycolic-capped CdSe QDs and positively charged PDDA-graphene. The latter was prepared via noncovalent functionalization of graphene oxide with PDDA. The ECL immunosensor had enhanced sensitivity for human IgG detection: in the range 0.02–2000 pg mL−1, with a detection limit of 5 fg mL−1 [73]. This high sensitivity is particularly important because it reveals how ECL-based immunosensors could be a potential replacement for the ELISA-based methods currently used for detecting proteins. Jie et al. fabricated a very sensitive ECL immunoassay for the detection of human IgG using MWNT–CdSe-quantumdots composites [74]. First, MWNTs were functionalized by polymer wrapping with poly(diallyldimethylammonium chloride, PDDA). Then, thioglycolic-acid-capped CdSe QDs (CdSe–TGA) were dispersed into PDDA-functionalized MWNTs and deposited on a gold electrode. The immunosensor was completed by adding citrate-capped Au NPs, and dipped into the antibody (Ab) solution. In this work, the ECL sensing was based on the fact that the increment of the steric hindrance that occurred after the immunoreaction resulted in quenching of the ECL signal. The ECL immunosensor had high sensitivity for IgG in the linear range 0.002–500 ng L−1, with a detection limit of 0.6 pg mL−1 [74]. Similarly, Wang et al. developed a “signal-on” ECL biosensor for the detection of choline and acetylcholine based on CdS– MWNT composites [75]. Carboxylated MWNTs were treated with NaOH, and after addition of CdCl2 Na+ ions were exchanged with Cd2+ ions. By reaction with tioacetamide, CdSQD-functionalized MWNTs were formed. Choline oxidase (ChO) and acetycholine esterase (AChE) were deposited by cross-linking with glutaraldehyde on CdS-QD-functionalized

Analytical applications of nanomaterials in electrogenerated chemiluminescence

MWNT-modified electrodes. Using H2O2 as a coreactant, a strong and stable ECL signal was generated. The resulting ECL sensor had linearity in two different concentration ranges, 1.7 μmol L−1 to 332 μmol L−1 and 3.3 μ mol L−1 to 216 μmol L−1, with detection limits of 0.8 μmol L−1 and 1.7 μmol L−1 for choline and acetylcholine, respectively, even in the presence of common interferents including ascorbic and uric acids [75]. Very recent works have seen the use of luminescent graphene quantum dots (GQDs) for ultrasensitive ECL immunosensing. For example, a substantial improvement in sensitivity has recently been obtained by using luminescentblue graphene quantum dots. Lu et al. developed a simple approach to prepare water-soluble GQDs using exfoliating and disintegrating treatments for graphene oxide, followed by hydrothermal synthesis [76]. The as-prepared GQDs were used to fabricate an ultrasensitive ECL immunosensor for the detection of ATP using the procedure summarized in Fig. 8. ssDNA1 was adsorbed on a clean Au electrode surface and then, by using SiO2 nanospheres previously functionalized with an amino group as signal carrier, novel SiO2–GQD ECL signal-amplification labels were synthesized and bioconjugated with a ssDNA2 for ultrasensitive ECL detection. Water-soluble GQDs produced bright blue emission under irradiation at 365 nm (ultraviolet irradiation). Notably, in the presence of hydrogen peroxide as a coreactant the GQDs provided a strong anodic ECL signal at relatively low potentials (ca. 0.4 V vs. Ag–AgCl), and the following mechanism was proposed [76]: GQDs−e− →GQDs•þ

ð13Þ

H2 O2 →Hþ þ HOO−

ð14Þ

Fig. 8 Schematic representation of the ECL aptamer ATP sensor. (a) Immobilization of ssDNA1 on the surfaces of Au electrode. (b) Treated with MCH to obtain a well-aligned DNA monolayer. (c) Hybridization of the two fragments in the presence of ATP. Reprinted from Ref. [76] with permission of Elsevier (2013)

HOO− −e− →HOO• ↔O2 −•

ð15Þ

GQDs•þ þ O2 −• →GQDs

ð16Þ

GQDs →GQDs þ hν

ð17Þ

The formation of the superoxide radical O2−• (Eq. 15) is crucial for the ECL emission, because this specie reacting with the radical cation GQDs•+ (Eq. 16) leads to the formation of GQDs* and consequent emission of light (Eq. 17). Under optimized experimental conditions, the as-prepared ECL aptamer sensor had excellent analytical performance for adenosine triphosphate (ATP) detection within the linear concentration range of 5 pmol L−1 to 5 nmol L−1, with a detection limit of 1.5 pmol L−1. Because of their low cytotoxicity and excellent biocompatibility, GQDs are highly useful for applications in ECL immunosensing [76]. In another application, Wu et al. used carbon quantum dots as labels to develop an ECL immunosensor for detecting tumor markers, for instance prostate-specific antigen [77]. In this work, graphene was conjugated with Au NPs and deposited on the surface of a glassy carbon electrode, which provided a good matrix for the antibody immobilization (Ab1). Luminescent carbon quantum dots (CQDs) were synthesized by electro-oxidation of graphite and immobilized on to highly porous Ag, which provided a highly accessible surface area. CQDs were labelled with Ab2 and immobilized on to the nanoporous Ag surface. CQDs were used as an ECL reagent. The ECL immunosensor had good analytical performance for

P. Bertoncello et al.

the detection of PSA in the concentration range 1 pg mL−1 to 50 ng mL−1, with a detection limit of 0.5 pg mL−1 [77] Composite materials obtained by the combination of graphene and magnetic nanoparticles, polymers, and redox probes have been particularly effective for the development of highly sensitive ECL immunosensors. Liao et al. recently reported the development of a reagentless ECL immunosensor for the detection of human total 3,30,5-triiodothyronine (T3), a biomarker involved in thyroid disease [78]. In this work, they used a poly(L-lysine)-[Ru(bpy)3]2+ composite material as a coreactant for signal amplification, and magnetic Fe3O4-functionalised graphene sheets as a nanoprobe. The probe was prepared by immobilizing -[Ru(bpy)3]2+ and T3 antibody on the surface of Fe3O4-functionalized graphene sheets, and the T3 capture antibody was immobilized on an Au-NP-functionalized poly(L-lysine)-modified electrode. The as-prepared ECL immunosensor worked in the linear concentration range of T3 of 0.1 pg mL−1 to 10 ng mL−1, with a detection limit of 0.03 pg mL−1 [78]. Other works have combined magnetic nanoparticles and quantum dots to generate magnetic quantum dots that have both magnetic and optical properties. Jie et al. fabricated magnetic Fe3O4–CdSe quantum dots that were further labelled with an a1/bbc-QD signal probe [79]. The as-prepared signal probe was immobilized on graphene-modified capture DNA (c-DNA1) deposited on to a gold electrode. The whole procedure is summarized in Fig. 9.

Fig. 9 Schematic representation of the ECL aptasensor for cell detection by DNA cyclic amplification technique, using Fe3O4–CdSe-QD composite as signal probe. Reprinted from Ref. [79] with permission of Elsevier (2013)

An endonuclease-assisted amplification technique was used to amplify the change in the ECL signal induced by target cells. Specifically, the bi-functional Fe3O4–CdSe QDs, composite QDs with excellent magnetic properties, can be conveniently labelled to obtain an ECL signal probe with high specificity and sensitivity. To evaluate the analytical performance of the ECL immunosensor for early diagnosis of cancer, a target and a control-cell sample with a concentration of 2000 cells mL−1 were spiked into two different clinical samples. The results revealed that the ECL signals of the target cells in the clinical sample and in cell media were almost the same. Also, the ECL signal of the cell media was greater than that obtained from the control cells, indicating the suitability of the proposed ECL immunoassay. The proposed ECL method had linearity in the range 300–24,000 cells mL−1, with a detection limit of 98 cells mL−1 [79]. Another recent ECL immunosensor application used graphene and luminol for cancer-biomarker detection. Xu et al. developed a multiple-signal-amplification strategy using functionalized graphene and Au nanorods multi-labelled with glucose oxidase and secondary antibody (Ab2) [80]. Graphene was used to increase the electron transfer and to attach the primary antibody (Ab1), and Au nanorods acted as a carrier of the secondary antibody and catalyzed the ECL reaction of luminol in the presence of glucose oxidase and oxygen. The as-prepared ECL immunosensor had high

Analytical applications of nanomaterials in electrogenerated chemiluminescence Fig. 10 Schematic representation of the inkjet printing to produce paper microfluidic substrates for ECL sensing using camera phones. Reprinted from Ref. [82] with permission of American Chemical Society (2011)

sensitivity and selectivity towards the detection of prostatespecific antigen (PSA) with a linear concentration range of 10 pg mL−1 to 8 ng mL−1, and a detection limit of 8 pg mL−1 [80]. A similar approach, but without the use of graphene, was later used by Cheng et al. to develop an ECL resonance energy transfer (ERET) based on CdTe QDs and Au nanoclusters [81]. Au nanoclusters were first labelled with hairpin DNA and then attached to carboxylated CdTe QDs on glassy carbon electrodes via amide reaction. The interactions between Au nanorods and CdTe quenched the ECL signal. Upon addition of assistant DNA and miRNA, the ligase selectively bound both of them to the strand of the hairpin DNA to form DNA– RNA heteroduplexes. This caused the recovery of the ECL as a result of the blocking of the ERET signal. In comparison, the ECL emission signal was weak when the hairpin DNA was

directly opened by the target. Based on the distance-dependent ERET, a “signal on” ECL system was used for the detection of miRNA with the advantages of a six orders of magnitude linear range and excellent sequence specificity. For instance, the ECL signal scaled linearly with the concentration of miRNA in the range 100 fmol L−1 to 100 nmol L−1, with a detection limit of 21.7 fmol L−1 [81]. By substituting the hairpin DNA with different sequences, this novel strategy can be extended to detection of other short miRNA and DNA.

Fig. 11 Design of microfluidic ECL array: (1) syringe pump, (2) injector valve, (3) switch valve to guide the sample to the desired channel, (4) tubing for inlet, (5) outlet, (6) poly(methylmethacrylate) (PMMA) plate, (7) Pt counter wire, (8) Ag–AgCl reference wire (wires are on the underside of the PMMA plate), (9) polydimethylsiloxane (PDMS) channels, (10) pyrolytic graphite chip (PG) (2.5×2.5 cm) (black), with hydrophobic polymer (grey) to make microwells. Bottoms of microwells (red

rectangles) contain primary-antibody-enhanced SWCNT forests, (11) ECL label containing RuBPY-silica nanoparticles with cognate secondary antibodies is injected to bind to the capture-protein analytes previously bound to cognate primary antibodies. ECL is detected with a CCD camera. Reprinted from Ref. [83] with permission of Springer (2013)

Future trends ECL, as discussed, can be readily detected with a photomultiplier tube (PMT). However, despite PMTs being widely available even in miniaturized form, they are

P. Bertoncello et al.

expensive. Future trends in the development of ECL sensors may focus on the use of cheaper alternatives to PMTs to make commercialization more attractive. Photodiodes might be one avenue of future investigation, being small and inexpensive. Simple hand-held detection systems could easily incorporate photodiodes [82]. Another possibility is the use of camera phones [82], which have been revealed to detect ECL from paper-based ECL sensors (Fig. 10). The fact that luminescence can be detected and analyzed by a camera phone reveals how the development of ECL sensors could be feasible in contexts where scientific and trained personnel are in short supply. The potential of ECL detection for biomedical diagnostics is clearly evident from its integration into microfluidic devices. Sardesai et al. recently incorporated an ECL immunoassay into a prototype microfluidic device for highly sensitive protein detection [83]. The prototype was used for detection of prostate-specific antigen (PSA) and interleukin-6. The design of the microfluidic device is shown in Fig. 11. Interestingly, whereas the microfluidic system was built using standard procedures on conducting pyrolytic graphite, the wells into the PDMS channels were functionalized with capture antibody enhanced with SWNT forests. The antigen was captured by these antibodies on the well bottoms. A [Ru(bpy)3]2+-SiO2-secondary antibody (Ab2) label was then injected to bind to antigen on the array. Then, to produce ECL, tri-n-propylamine (TPrA) was added and used as a sacrificial reductant. For the ECL detection, the chip was placed into an open-top measuring cell, with the channels in contact with the electrolyte in the chamber. A suitable potential was used to oxidize TPrA and the [Ru(bpy)3]2+ species in the particles, and the ECL light was collected by a charge-coupled device camera. This novel microfluidic device was able to achieve detection limits at ultratrace levels up to 100 fg mL−1 for PSA and 10 fg mL−1 for IL-6 in calf serum. This setup improved the analytical signal by ca. 10–25-fold compared with nonmicrofluidic arrays and enabled the detection of PSA and IL-6 in serum samples in ca. 1 h [83].

Conclusions This review describes the use of nanomaterials (quantum dots and carbon nanomaterial) in electrogenerated chemiluminescencebased medical diagnostic applications for the detection and diagnosis of cancers and the detection of other biomarkers of clinical relevance. Recent ECL work using quantum dots and carbon nanomaterials reveals the possibility of productive use of such systems in multi-analyte and multi-variable assays. In this respect, coreactant ECL is being widely used in a variety of analytical applications in clinical, environmental, and biodefense fields. There are currently hundreds of biomarker assays, with applications ranging from thyroid disease to

tumor and cardiac markers, cell-signaling pathways, nucleic acids, and genetic diseases. ECL is set to have a crucial function in these fields, but with particular emphasis on the development of novel portable and high sensitivity devices, e.g., biomedical point-of-care devices. Many of these applications rely on the simple use of such instrumentation and on their high sensitivity and selectivity. In particular, the excellent chemical and ECL properties of [Ru(bpy)3]2+, water-soluble quantum dots, and carbon nanocrystals immobilized on different conducting substrates make such systems particularly attractive for biomedical applications, because of the variety of analytes that can be detected using different electrogenerated species. Currently, the trend is to use such ECL probes as a composite with other nanomaterials (MWNTs, SWNTs, graphene derivatives, and metal NPs) and biomolecules to confer high sensitivity and selectivity. Another field that is expected to grow rapidly is ECL immunoassay detection using enzymatic reactions. The integration of ECL with microfluidic techniques is leading to the development of lab-on-a-chip systems at ultra-trace detection levels (fg mL−1 or even ag mL−1) for the early detection of diseases and for other biomedical or environmental applications that could soon replace current analytical techniques based on the ELISA method.

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