Recent advances in new luminescent nanomaterials for electrochemiluminescence sensors

Abstract

Electrochemiluminescence (ECL) has received considerable interest for broad applications due to the potential remarkably high sensitivity and extremely wide dynamic range. In recent years, due to the unique physical (structural, electronic, magnetic and optical) and chemical (catalytic) properties of nanomaterials (NMs), great efforts have been made to investigate their application in ECL. In this review, we present a general description of ECL related to NMs as the emitter. It mainly focuses on the basic mechanisms and the development of an ECL sensing platform fabricated from semiconductor nanocrystals and metal nanoclusters. Finally, we conclude with a look at the future challenges and prospects of the development of ECL.

---

Jing Li

Dr Jing Li was born in Shandong Province, China. She received her PhD degree from the Changchun Institute of Applied Chemistry under the direction of Professor Erkang Wang in 2009. In 2010, she joined the Changchun Institute of Applied Chemistry as a Research Associate. Her scientific interests focus on functionalized nanomaterials for electrochemiluminescence and electroanalysis.

Shaojun Guo

Dr Shaojun Guo was born in Shandong Province, China. He received his BS degree in Jilin University in 2005. Then, he moved to the Changchun Institute of Applied Chemistry as a PhD student, and majored in analytical chemistry and materials chemistry, and received his PhD degree in 2010. He is currently a Postdoctoral Research Associate in the Department of Chemistry at Brown University. He has published over 90 papers in peer-reviewed international journals. His papers have been cited more than 1700 times and he has an h-index of 27. His scientific interests focus on carbon and metal nanomaterials for electrochemical, analytical and energy applications.

Erkang Wang

Erkang Wang is a Professor of Chemistry at Changchun Institute of Applied Chemistry, Chinese Academy of Sciences (CAS). He received his PhD degree in 1959 from the Czechoslovak Academy of Sciences directly under Professor J. Heyrovsky, the Nobel Prize Laureate. He is an academician of both the CAS and the Academy of Sciences of the Developing World. He has been on the Editorial and Advisory Board of nine international journals. His research interests focus on bioelectrochemistry, electrochemiluminescence and environmental analysis. He has published over 900 papers and monographs in international journals with the SCI cited over 10 000 times.

---

Introduction

Electrochemiluminescence (ECL) is the optical emission from the excited states of an ECL luminophore produced at an electrode surface via electrochemical high-energy electron transfer reaction.¹ At present, different chemiluminescent reagents are applied in ECL reactions and can be divided into organic systems (e.g., luminol)² and inorganic systems (e.g., metal complexes).^3–8 Among them, ruthenium complexes using amine-containing compounds as coreactants^9,10 and a luminol–H₂O₂ system^11,12 have been extensively investigated. As an important analytical method, ECL shows many advantages over photoluminescence such as simplicity, high sensitivity, rapidity and no background form unnecessary photoexcitation and has been widely used in a large number of environments, ranging from fundamental studies to practical applications.^13,14

In recent years, due to the unique physical (structural, electronic, magnetic and optical) and chemical (catalytic) properties of nanomaterials (NMs), great efforts have been made to investigate their application in ECL. These NMs employed in ECL include nanoparticles (NPs),^15–17 nanotubes,^18,19 nanobelts,²⁰ nanorods^21,22 and nanocrystals^23,24 of virtually any chemical composition. The roles the NMs play can be summarized as follows: (a) NMs as the amplification element can provide an ideal ECL platform for enhancing the signal of a sensor due to the high surface area-to-volume ratio and good electrical conductivity, such as the introduction of conductive carbon nanotubes (CNT) and metal nanoparticles (NPs) into biosensors; (b) NMs as appropriate immobilization matrices can retain ECL reagents for developing good robust solid-state ECL sensors. The materials used for immobilization are mainly focused on silicon NPs, silicon sol–gel and magnetic NPs; (c) NMs can act as energy acceptors to quench the ECL, such as gold NPs and functionalized semiconductor nanocrystals; (d) NMs as the new luminescence material can be used for fabricating novel ECL sensors and biological labels for ECL detection. Many NM-based ECL emitters including II–VI, III–V and IV–VI nanocrystals, carbon dots and metal nanocluster, etc. have been used for fabricating ECL sensors because of their unique electronic and optical properties. Thus, traditional ECL analysis is facing new opportunities and challenges with the rapid development of nanotechnology.

In this review, we will discuss the use of NMs as emitters for fabricating the various ECL sensors. More attention is paid to the mechanism and their sensing application. Finally, we conclude with a brief look at the future challenges and prospects in the development of ECL based on the NMs.

Semiconductor nanocrystals as the emitter of ECL

The ECL properties of highly luminescent semiconductor nanocrystals, or quantum dots (QDs) have been extensively studied since 2002.²³ Compared with conventional organic luminescence material, QDs as ECL luminophors show excellent properties, such as high quantum yields, size-dependent luminescence due to the quantum size effect and good stability against photobleaching. More importantly, the light scattering produced due to their small size can be completely eliminated when used in ECL devices since there is no need for a radiation resource. Previously, the ECL from QDs was mainly obtained in nonaqueous media due to the instability of the excited QDs,²³ this restricted their further application in biosensors and more attention was paid to examining the relationship between ECL and photoluminence.²⁵ Later, the ECL emissions of silicon nanocrystals²⁶ and CdS spherical assemblies²⁷ in aqueous solution were observed, however, the strong alkaline media (pH 13) greatly limited the application of the QDs when incorporated with biomelecules. In 2004, the first sensing applications of QDs ECL were studied for H₂O₂ detection by depositing CdSe QDs on a paraffin-impregnated graphite electrode (PIGE) at physiological pHs,²⁸ which increased the use of QD based ECL in the analytical field. In the following section we will discuss the basic mechanisms of QD based ECL and recent advance in ECL-based sensing.

Mechanism of QD based ECL

Many types of QD can undergo redox chemistry and their ECL properties were investigated, such QDs include elemental and compound semiconductors, such as Ge,²⁹ carbon dots,^30,31 CdS,²⁷ CdTe,³² CdSe,^25,33 PbS,³⁴ Mn²⁺ dopped ZnS³⁵ and CdSe/ZnS.³⁶ Generally, the ECL mechanism of QDs mainly involves the annihilation and coreactant ECL reaction pathways. In the annihilation mechanism, the reduced and oxidized species are both generated in the vicinity of the electrode surface upon hole or electron injection in solution during potential cycling or pulsing. The electron-transfer annihilation of the eletrogenerated anion and cation radical results in the production of excited states, which is described as follows (eqn (1)–(4)). In order to make ECL occur efficiently through the annihilation mechanism, the QDs must be chemically stable and maintain their charged states long enough to transfer the charge upon colliding with an oppositely charged species in solution. In addition, the applied electrode potential must be sufficient to generate both the negatively and positively charged species.¹⁴

QD − e⁻ →QD^+• (1)

QD+ e⁻ → QD^−• (2)

QD^+• + QD^−• → QD^* + QD (3)

QD^* → QD + ħv (4)

An alternative mechanism of generating QD ECL is through the use of a coreactant, where the ECL emission can be generated by a single direction potential sweep in the presence of both the luminophore and corectant. The introduction of a coreactant in ECL can overcome the limited potential window of a solvent or the poor stability of radical anions or cations, but also can produce more intense ECL emission compared with an annihilation reaction.²³ Thereafter, the coreactant pathway can be obtained conveniently and is often adopted in biosensing applications. The coreactants commonly used in ECL include peroxydisulfate (S₂O₈²⁻), H₂O₂, O₂, oxalate (C₂O₄²⁻), SO₃²⁻, tripropylamine (TPA) and 2-(dibutylamino)ethanol (DBAE). Among these coreactants, some ECL systems were based on the “oxidation–reduction” reaction mechanism, such as the TPA, DBAE, C₂O₄²⁻ and SO₃²⁻ systems. Taking C₂O₄²⁻ as an example, upon oxidation, a strong reducing agent (CO₂⁻) is formed, which can react with an ECL luminophore by electron transfer to produce an excited state and then generate light. The corresponding mechanism is expressed in eqn (5)–(8).

QD − e⁻ → QD^+• (5)

C₂O₄²⁻ − e⁻ → [C₂O₄^−•] → CO₂^−• + CO₂ (6)

QD^+• + CO₂^−• →QD^* + CO₂ (7)

QD^* → QD + ħv (8)

Unlike in the above reaction mechanism, “reductive–oxidative” coreactants can also be employed to generate ECL by applying a negative potential. A typical example of this ECL reaction model is the QD/S₂O₈²⁻ system, the light emission processes are as follows (eqn (9)–(12)). The reduction of S₂O₈²⁻ releases a strong oxidizing agent, which can interact with the negatively-charged QDs, and finally leads to an ECL signal.

QD + e⁻ → QDs^−• (9)

S₂O₈²⁻ + e⁻ → SO₄^−• + SO₄²⁻ (10)

SO₄^−• + QD^−• → QD^* + SO₄²⁻ (11)

QD^* → QD + ħv (12)

Other coractants often used in the “reductive–oxidative” mechanism include O₂ and H₂O₂. In most cases, the resulting ECL emissions observed in aqueous media are produced in a cathodic process and the dissolved oxygen can be electrochemically reduced to H₂O₂ and then acts as the coreactant for ECL generation. However, the anodic ECL of mercaptopropionic acid (MCA) modified CdTe QDs in the presence of O₂ has also been observed in recent work.³⁷ In that case, O₂ and the electrode material (ITO) played an important role in the ECL process. The ECL emission involved the collision between the superoxide ion produced at the ITO surface and the oxidation products of the QDs. The whole process can be expressed as follows:

In/SnO_x + QD → QD^+• + In/SnO_x(e⁻) (13)

O₂ + In/SnO_x(e⁻) → O₂⁻ + In/SnO_x (14)

QD + O₂⁻ → QD^−• + O₂ (15)

QD^•+ + QD^−• → 2QD^* (16)

QD^* → QD + ħv (17)

However, the more negative or positive the ECL peak potential and weaker ECL intensity than the Ru(bpy)₃²⁺ or luminol ECL restricted their wide analytical applications. Various approaches have been developed to improve the ECL behavior of QDs. The ECL behavior of QDs displayed a size-dependency as reported by Hua³⁸ and Liu et al.³⁷ It was found the ECL intensity gradually increased as the particle size increased. On the other hand, the ECL peak potential and onset potential shifted more positively in the cathodic ECL due to the decreased band gap. The development of the NMs provides good opportunities for effectively improving the ECL performance of QDs. A series of NMs, such as Au NPs,^39,40 CNT,^41–44 silica NPs,⁴⁵ graphene oxide⁴⁶ and graphene^47,48 were employed to enhance the ECL emission efficiency. For instance, Chen et al.⁴¹ illustrated that the introduction of CNT into CdS nanocrystals enhanced the ECL intensity ca. 5.3-fold compared to pure CdS. Moreover, the corresponding onset potential can shift positively by about 400 mV, which reduced H₂O₂ decomposition and improved the detection sensitivity. Similar phenomena were also obtained using CdSe⁴² or ZnO⁴⁴ as the illuminant with nitrogen doped CNTs or CNTs as the enhanced conducting materials. Recently, Li's group⁴⁶ reported that the graphene oxide amplified ECL of QDs by facilitating QD oxidation and accelerating the output of O₂^•, which improved the shortcomings of QD ECL, such as low emission efficiency and unstable radical species. To further improve the ECL performance, Zhu's group⁴⁷ presented a novel strategy using the poly(diallyldimethylammonium chloride)-protected graphene–CdSe composites instead of the electrically insulating graphene oxide. Due to the extraordinary electron transport and the large specific surface area of graphene, a more positive onset potential and higher ECL intensity were observed compared with that of the free CdSe film. Except for tunneling, the size of the QDs and introducing NMs into the QD ECL system, surface modification can significantly improve the quantum yield and lower the applied potential for ECL emission. For example, Wang et al.⁴⁹ found the ECL of CdS nanocrystals showed enormous enhancement in ECL (ca. 310 times) after heating in the presence of ammonia due to easy reduction process of CdS QDs. Ju's group⁵⁰ used lower solubility meso-2,3-dimercaptosuccinic acid as a stabilizer for the preparation of CdTe QDs by the electrolysis method. The as-prepared QDs could be facilely immobilized on an electrode surface using the drop-coating method. Furthermore, the rigid structure and steric hindrance of the short chain bithiol compound provided an avenue for obtaining low-potential ECL emission (cathodic ECL emission peak at −0.87 V). Mercaptosuccinic acid-protected CdTe QDs for anodic ECL research has also been studied by Dong's group and lower potential was needed for the ECL production in the presence of DBAE.³² In addition, Xu's group³⁵ explored the ECL behavior of Mn²⁺ doped ZnS QDs and a new ECL emission peak was observed, which was lower than that obtained with the pure ZnS QDs.

ECL sensing of QDs

Since the first sensing application based on QD ECL was studied by depositing CdSe QDs on a paraffin-impregnated graphite electrode (PIGE),²⁸ advances in the development of novel ECL sensors based on the highly luminescent QDs have gained increasing attention, especially in bioanalysis. For some analytes usually involved in the ECL reaction, the ECL sensors fabricated can be divided into two categories as follows according to the influence on the original ECL signal produced by the analyte.

One is based on the enhancement of the ECL intensity, mainly focused on the well-studied coreactant system, such as in the determination of H₂O₂,^28,35,43 C₂O₄²⁻²³ and amines.³² Plenty of ECL sensors of this type were fabricated for H₂O₂ sensing using different kinds of QDs (Table 1).^{28,35,42,43,51–53}

Table 1 H₂O₂ sensors fabricated using different kinds of QDs

QD^a	Stabilizer^a	Electrode^a/pH	ECL peak	Ref.
a TGA: thioglycolic acid; P-gly: polyglycol; Glu: glutathione; CdSHS/CNF: CdS hollow spheres/carbon nanofibers; GCE: glassy carbon electrode; ECL peak is given vs. Ag/AgCl.
CdS/CNT	TGA	GCE (pH = 9)	Not Given	43
CdSHS/CNF	P-gly	GCE (pH = 8)	−1.02 V	51
Mn^2+-doped ZnS	None	GCE (pH = 9)	−1.5 V	35
CdSe	None	PIGE (pH = 9.3)	−1.2 V\−1.5 V	37
CdSe–N doped CNT	TGA	GCE (pH = 9)	Not given	42
ZnSe	Glu	GCE (pH = 7.4)	−1.3 V	52
(CdS/Hb)n	TGA	GCE (pH = 9.6)	Not given	53

---

As is well known, H₂O₂ is a product of multifarious oxidase catalytic reactions, where the corresponding substrates are oxidized by O₂. Therefore, some biological compounds can also be measured coupled with enzymatic reactions by monitoring the concentration of H₂O₂. The first enzyme biosensor was reported using glucose oxidase (GOD) for glucose determination with O₂ as the coreactant.³³ In a typical GOD catalytic cycle, O₂ is consumed to produce H₂O₂. As an efficient coreactant, O₂ can capture more electrons than H₂O₂ from the electrochemically reduced QDs. So the ECL response decreased upon addition of glucose. However, Wang et al.⁴¹ recently described a sensitive “signal-on” enzyme sensors for choline (Ch) and acetylcholine (ACh) using a CdS–MWCNT nanocomposite. Unlike the glucose sensor based on the CdSe NC film,³³ O₂ therein did not influence the ECL intensity on the CdS–MWCNT nanocomposite film and the ECL resulted from the H₂O₂ generated by the choline oxidase (ChO) and acetylcholine esterase (AChE) enzymatic reactions, which became the base of “signal-on” enzyme-based biosensors. The possible mechanism of the ECL generated from CdS reacted with H₂O₂ could be expressed as follows (eqn (18)–(25)):

QD + e⁻ → QD^−• (20)

QD^−• + H₂O₂ →QD + OH^• + OH⁻ (21)

OH^• + QD → QD^+• + OH⁻ (22)

QD⁺• + QD^−• → 2QD^* (23)

QD^−• + OH^• → OH⁻ + QD^* (24)

QD^* → QD + ħv (25)

The other category are sensors based on the “signal-off” strategy. In this detection process, some analytes quenched the QD ECL by the inhibition effect. For example, thiols⁵⁴ can scavenge the intermediate OH^•, which is crucial for producing hole-injected QDs, and thus lead to the decrease of ECL intensity. On basis of this mechanism, glutathione and L-cysteine as model reagents were investigated. Recently, Dong's group⁵⁵ reported ECL quenching using anodic ECL based on the competition from Cu²⁺ for the stabilizer due to the stronger metal–S interaction with the stabilizer than the Cd–S bond. A similar phenomenon was also observed with the cathodic ECL.⁵⁰ This was further extended to detect other metal ions, such as the detection of Hg²⁺ due to the lower K_sp of HgS (4.0 × 10⁻⁵³).⁵⁶ Besides the inhibition effect, some analytes can result in the signal of ECL emission being reduced via energy transfer between the excited molecule and the byproduct of the analyte, for example, the detection of catechol derivatives using anodic ECL emission from CdTe QDs with the coreactant O₂ (Fig. 1).³⁷ Taking dopamine (DA) as a model compound, in the cyclic sweep process, DA was oxidized to produce the o-benzoquinone species, which collided with the excited state of the QDs and led to ECL quenching. Thus, some quencher-related analytes can also be measured following this quenching principle, such as ECL analysis of L-adrenalin.³⁷

Fig. 1 The anodic ECL mechanism of QDs (A) and its quenching procedure by oxidation product of DA (B). Reprinted with permission from Ref. 37. Copyright (2007) American Chemical Society.

In addition, enzymatic reactions were also used to induce the generation of a quencher, such as the detection of hydroquinone (HQ) in the presence of horseradish peroxidase (HRP).⁵⁷ After HRP was assembled on the electrode surface, the cathodic scan in air-saturated detection solution would induce an enzymatic reaction between H₂O₂ and HQ, which would consume coreactant H₂O₂ and cause the decrease of ECL intensity. Other coreactant systems were also proposed for detecting the quenchers of excited QDs or their precursors by Ju's group,⁵⁸ for instance, the sulfite-coreactant ECL emission. To further amplify the sensitivity, they designed an enzymatic cycle to produce the oxidized product using tyrosinase and achieved an extremely sensitive method for ECL detection of tyrosine with a sub-picomolar limit of detection.

Notably, the above mentioned analytes are mainly involved in the ECL emission process directly or indirectly. Nevertheless, for the analytes, which do not participate in the ECL reaction, such as proteins and DNA, etc. the sensing assays were often carried out using QDs as the emitter by linking them to one of the species involved in an affinity binding reaction. A number of interactions can be used for biorecognization including antibody/antigen, enzyme/inhibitior, Watson–Crick base pairing and aptamer/target. For example, a label-free ECL immunoassay sensor⁵⁹ has been developed for the sensitive detection of human IgG (HIgG) based on the co-immobilization of an antibody with CdSe QD/CNT–chitosan(CHIT)/3-aminopropryl-triethoxysilane (APS) on the electrode surface (Fig. 2). APS, as the cross-linker, was covalently conjugated to the CdSe/CNT–CHIT film, which greatly enhanced the ECL of the composite film (ca. 20-fold). The antibody was bound to the functionalized film via glutaric dialdehyde (GLD). The specific immunoreaction between HIgG and the antibody resulted in the increasing steric hindrance upon formation of the immunocomplex, which inhibited the transfer of the coreactant to the surface and thus caused a decrease in ECL intensity. This low-cost, sensitive and specific strategy shows vast potential in other biological assays, such as, the determination of low-density lipoprotein³⁹ and human prealbumin.⁶⁰ Recently, a novel hybrid gold/silica/CdSe–CdS QD superstructure⁶¹ was also successfully applied in the development of an ultrasensitive immunosensor for a protein tumor marker. The superstructure enhanced the ECL intensity 17 fold in magnitude compared to that of the pure QDs, but also significantly improved the biocompatibility. The proposed sensor can detect carcinoembryonic antigens down to 0.064 pg mL⁻¹ and a wider linear range from 0.32 pg mL⁻¹ to 10 ng mL⁻¹ was obtained.

Fig. 2 Fabricating steps of the ECL immunosensor for HIgG. Reprinted with permission from Ref. 59. Copyright (2008) American Chemical Society.

As an alternative to the charger transfer induced by steric hindrance, ECL detection via energy transfer is also employed for bioassays. For instance, Shan and coworkers¹⁷ developed a simple sensing platform for target DNA with CdS:Mn QDs as the luminophores and Au-NPs as the quenchers. The quenching process depends on the distance between Au-NPs and excited QDs. Upon hybridization with target DNA, ECL enhancement occurs due to the larger separation between the luminophore and quencher (Fig. 3).

Fig. 3 Electrochemiluminescence (ECL) sensing strategy with energy transfer between CdS:Mn (QDs) and Au NPs. Reprinted with permission from Ref. 17. Copyright (2009) Royal Society of Chemistry.

To obtain high quenching efficiency, the use of a highly effective and stable energy transfer ECL quencher⁶² was explored by activation of surface carboxyl groups of CdTe QDs (A-QDs) using EDC/NHS, which changed the QDs from photon emitters to strong photon absorbers without emission (Fig. 4C). Using the A-QDs, an ultrasensitive immunosensing platform for antigen detection was constructed based on the sandwich type with a detection limit down to 1 fg mL⁻¹. Recently, the combination of superparamagnetic Fe₃O₄ nanoparticles with Mn doped CdS QDs was investigated to develop an enhanced immunosensing platform for mouse IgG antigen via energy transfer induced by the opto-magnetic interaction.⁶³

Fig. 4 Normalized UV-Vis absorption and photoluminescence spectra (A,C) and transmission electron microscopy (TEM) images (B,D) of CdTe QDs (A,B) and A-CdTe QDs (C,D). Insets in (A,C): the photographs of CdTe QDs and A-CdTe QDs. Insets in (B,D): the selected area electron diffraction (SAED) patterns of CdTe QDs and A-CdTe QDs. Photoluminescence spectra were recorded with an excitation at 400 nm. Reprinted with permission from Ref. 62. Copyright (2010) Royal Society of Chemistry.

ECL resonance energy transfer (ECL-RET), as another powerful technique for probing changes in the distance between donors and acceptors, has also been exploited for the sensitive detection of molecular interactions with a particular target molecule.^64,65 In these ECL-RET methods, the ECL spectra were recorded by a series of optical filters and a suitable donor–acceptor pair was crucial for sensitive detection. Take the QD/Ru(bpy)₃²⁺ system for determination of β2 M expressed cells as an example,⁶⁴ the mechanism of this ECL-RET strategy is illustrated in Fig. 5. In the absence of Ru(bpy)₃²⁺-streptavidin(Ru–SA) labeled cells, the CdS QDs acted as donor exhibited only emission at 450–550 nm. Once the specific combination of antibody on glassy carbon electrode and antigen on the cell surfaces takes place (Fig. 5), a new emission peak occurred at 620 nm, which is attributed to the emission of Ru(bpy)₃²⁺. The ECL-RET ratio (the increment at 620 nm: the decrement at 500 nm) was 5.78, indicating that this ECL-RET system efficiently amplified the signal due to the high quantum yields and strong luminescence of Ru(bpy)₃²⁺. In addition, a luminol/QD system was also investigated for monitoring the interactions and conformational changes of proteins with luminol as the donor and the luminescent QDs as the acceptor.⁶⁵

Fig. 5 ECL biosensor based on ECL-RET for determination of β2 M expressed cells. Reprinted with permission from Ref. 64. Copyright (2011) Royal Society of Chemistry.

In the ECL process discussed above, QDs as the emitters were often immobilized on the surface of the electrode in advance and the presence of targets influence the original ECL signal via charge transfer or energy transfer. Unlike this process, some of analytes detected only affect the amount of QDs that are integrated into the affinity binding reaction. For example, a series of aptasensors were developed for the detection of thrombin,⁶⁶ lysozyme⁶⁷ and adenosine 50-triphosphate⁶⁸etc. As shown in Fig. 6, probe I with a thiol-terminated aptamer was first immobilized on an Au electrode, then thrombin and another 5′-biotin modified aptamer (29 nucleotides, probe II) was incubated with the above electrode. To gain the sandwich structure, streptavidin modified QDs (avidin–QDs) were bound to probe II via the biotin–avidin system. Thrombin was measured by monitoring the ECL intensity of the bound QDs. Based on a similar detection mode, a recent investigation on the use of CdTe QD coated silica nanosphere for the detection of a biomarker was reported by Liu's group.⁴⁵ In this case, Rabbit IgG was used as the model and was sensitively detected with amplification of the silica nanosphere due to the high loading of CdTe QDs. To extend QD ECL application in cell detection, Zhang's group⁶⁹ combined specially designed capture DNA as a high-affinity aptamer to the target cell with the fifth-generation dendrimer nanoclusters to label CdSe–ZnS QDs as amplified ECL signal probes to develop versatile ECL assays for cancer cells. To further improve sensitivity and simplify the separation procedure, a DNA device on magnetic beads was employed in the ECL assay of cancer cells.

Fig. 6 Schematic diagram for the biosensor fabrication. Reprinted with permission from Ref. 66. Copyright (2009) Elsevier.

Metal nanocluster as luminophors and their sensing abilities

Analogous to semiconductor nanocrystals, metal nanoclusters (NCs) composed of very few atoms with sizes comparable to the Fermi wavelength of the electron (0.7 nm), exhibit molecule-like properties of discrete electronic states and are promising candidates in optical systems, biosensors and catalysis compared to highly toxic semiconductor QDs such as CdSe, CdS, CdTe. However, only few reports on ECL of metal NCs have been published.^70–72 Ras's group⁷⁰ illustrated the cathodic hot electron-induced ECL behavior of Ag NCs in the presence of S₂O₈²⁻via an “oxidation–reduction” excitation pathway for the first time. However, the obtained ECL is only observed under strong cathodic polarization (a pulse voltage of −30 V), which can evoke some unpredictable reactions and thus limits its application. In addition, the relatively poor stability of Ag NCs was also unfavorable for their application. Recently, the ECL of Au NCs with excellent stability under attainable operating conditions was investigated and a potential application was illustrated.^71,72 The Au NCs were protected by bovine serum albumin and different ECL phenomena were obtained using different electrodes and co-reactants. Li and coworkers⁷¹ reported that the cathodic ECL originated via electron-transfer annihilation of negatively charged Au₂₅^−• and SO₄^−• radicals produced by the electroreduction of S₂O₈²⁻ by immobilizing Au NCs on the ITO electrode. Notably, the ITO electrode employed in this process as an effective reductant for Au NCs played an important role in transferring electrons from the conduction-band of ITO to the LUMO of the Au NCs, while other electrodes such as, Au and the glassy carbon electrode can not. Finally, Au NCs modified ITO electrode as a sensing platform was applied to the detection of DA based on the enhanced ECL mechanism by formation of a charge transfer complex between DA and ITO. Later, Chen's group⁷² observed the ECL of Au NCs dispersed in aqueous solution with bare Pt electrode by employing triethyamine (TEA) as the coreactant. They found that the anodic ECL observed was attributed to the electrochemical oxidation of TEA rather than the Au NCs . The different influence of heavy metal ions on ECL suggested that the ECL emission had a major bearing on the surface states due to the different binding interactions with S²⁻. The whole mechanism can demonstrated as follows (Fig. 7). Briefly, the Au₂₅ was firstly oxidized to Au₂₅⁺ by the electrochemical oxidation product (TEA^−•), then the oxidation product can be deprotonated to form a very reductive radical (TEA^•), which can reduce the Au₂₅ to Au₂₅⁻. The light emission occurs when the Au₂₅⁺ collides with Au₂₅⁻ which produced excited states (Au₂₅^*). In this ECL system, only amines can provide the pathway to inject holes at the S 3p band via reaction with the electrochemical oxidation product, other coreactants such as C₂O₄²⁻ and SO₃²⁻ can not. Based on the quenched ECL from Au NCs by Pb²⁺, a novel method for the selective Pb²⁺ determination was developed with L-cysteine as the masking agent.

Fig. 7 Schematic ECL mechanism from the Au₂₅ clusters. Reprinted with permission from Ref. 72. Copyright (2011) Royal Society of Chemistry.

Conclusion and outlook

Due to the high fluorescence quantum yields, size or surface trap-control luminescence, and good stability against photobleaching, luminescent nanomaterial based ECL shows vast potential in analytical applications.^73,74 Combining bioreorganization with nanomaterials provided new opportunities for ECL in biorelated species analysis via charge transfer and energy transfer, such as the determination of DNA, proteins and cells. Through surface functional modification and the introduction of NMs in ECL systems, an amplified ECL signal was obtained, for instance, the sensitive determination of carcinoembryonic antigen with a low detection limit of 0.064 pg mL⁻¹ and a wider linear range from 0.32 pg mL⁻¹ to 10 ng mL⁻¹ was achieved.⁶¹ Moreover, because heavy metal-based QDs are toxic, using low-toxicity environment friendly alternatives as light-emitting molecules (e.g., silicon, carbon dots and metal clusters) with highly efficient ECL systems should be developed and the corresponding mechanisms and applications should be further understood.

Acknowledgements

We greatly appreciate the support of the National Natural Science Foundation of China (21190040 and 21105094). This work is also supported by the National Basic Research Program of China (2009CB930100 and 2010CB933600).

References

1. A. J. Bard, Marcel. Dekker, New York, Electrogenerated Chemiluminescence, 2004.
2. Z.-F. Zhang, H. Cui, C.-Z. Lai and L.-J. Liu, Anal. Chem., 2005, 77, 3324–3329.
3. Y. Zu and A. J. Bard, Anal. Chem., 2000, 72, 3223–3232.
4. L. Dennany, E. J. O'Reilly, P. C. Innis, G. G. Wallace and R. J. Forster, Electrochim. Acta, 2008, 53, 4599–4605.
5. L. Dennany, E. J. O'Reilly, T. E. Keyes and R. J. Forster, Electrochem. Commun., 2006, 8, 1588–1594.
6. L. Dennany, T. E. Keyes and R. J. Forster, Analyst, 2008, 133, 753–759.
7. L. Dennany, R. J. Forster and J. F. Rusling, J. Am. Chem. Soc., 2003, 125, 5213–5218.
8. L. Dennany, P. C. Innis, G. G. Wallace and R. J. Forster, J. Phys. Chem. B, 2008, 112, 12907–12912.
9. W. Zhan and A. J. Bard, Anal. Chem., 2006, 79, 459–463.
10. W. Miao, J.-P. Choi and A. J. Bard, J. Am. Chem. Soc., 2002, 124, 14478–14485.
11. D. Tian, C. Duan, W. Wang and H. Cui, Biosens. Bioelectron., 2010, 25, 2290–2295.
12. R. Wilson, C. Clavering and A. Hutchinson, Anal. Chem., 2003, 75, 4244–4249.
13. M. M. Richter, Chem. Rev., 2004, 104, 3003–3036.
14. W. Miao, Chem. Rev., 2008, 108, 2506–2553.
15. L. Y. Fang, Z. Z. Lu, H. Wei and E. Wang, Anal. Chim. Acta, 2008, 628, 80–86.
16. S. Guo, J. Li and E. Wang, Chem.–Asian J., 2008, 3, 1544–1548.
17. Y. Shan, J.-J. Xu and H.-Y. Chen, Chem. Commun., 2009, 905–907.
18. J. Li, Y. H. Xu, H. Wei, T. Huo and E. K. Wang, Anal. Chem., 2007, 79, 5439–5443.
19. Z. H. Guo and S. J. Dong, Anal. Chem., 2004, 76, 2683–2688.
20. J. Yu, F.-R. F. Fan, S. Pan, V. M. Lynch, K. M. Omer and A. J. Bard, J. Am. Chem. Soc., 2008, 130, 7196–7197.
21. K. M. Omer and A. J. Bard, J. Phys. Chem., 2009, 113, 11575–11578.
22. M. Chen, L. Pan, Z. Huang, J. Cao, Y. Zheng and H. Zhang, Mater. Chem. Phys., 2007, 101, 317–321.
23. Z. Ding, B. M. Quinn, S. K. Haram, L.E. Pell, B.A. Korgel and A.J. Bard, Science, 2002, 296, 1293–1297.
24. S. K. Poznyak, D. V. Talapin, E. V. Shevchenko and H. Weller, Nano Lett., 2004, 4, 693–698.
25. N. Myung, Z. Ding and A. J. Bard, Nano Lett., 2002, 2, 1315–1319.
26. Y. Bae, D. C. Lee, E. V. Rhogojina, D. C. Jurbergs, B. A. Korgel and A. J. Bard, Nanotechnology, 2006, 17, 3791–3797.
27. T. Ren, J.-Z. Xu, Y.-F. Tu, S. Xu and J.-J. Zhu, Electrochem. Commun., 2005, 7, 5–9.
28. G. Zou and H. Ju, Anal. Chem., 2004, 76, 6871–6876.
29. N. Myung, X. Lu, K. P. Johnston and A. J. Bard, Nano Lett., 2003, 4, 183–185.
30. L. Zheng, Y. Chi, Y. Dong, J. Lin and B. Wang, J. Am. Chem. Soc., 2009, 131, 4564–4565.
31. H. Zhu, X. Wang, Y. Li, Z. Wang, F. Yang and X. Yang, Chem. Commun., 2009, 5118.
32. L. Zhang, X. Zou, E. Ying and S. Dong, J. Phys. Chem. A, 2008, 112, 4451–4454.
33. H. Jiang and H. Ju, Chem. Commun., 2007, 404.
34. L. Sun, L. Bao, B.-R. Hyun, A. C. Bartnik, Y.-W. Zhong, J. C. Reed, D.-W. Pang, H. c. D. Abrunña, G. G. Malliaras and F. W. Wise, Nano Lett., 2008, 9, 789–793.
35. X.-F. Wang, J.-J. Xu and H.-Y. Chen, J. Phys. Chem., 2008, 112, 17581–17585.
36. L. Dennany, M. Gerlach, S. O'Carroll, T. E. Keyes, R. J. Forster and P. Bertoncello, J. Mater. Chem., 2011, 21, 13984–13990.
37. X. Liu, H. Jiang, J. P. Lei and H. X. Ju, Anal. Chem., 2007, 79, 8055–8060.
38. L. Hua, H. Han and X. Zhang, Talanta, 2009, 77, 1654–1659.
39. G. Jie, B. Liu, H. Pan, J.-J. Zhu and H.-Y. Chen, Anal. Chem., 2007, 79, 5574–5581.
40. L. Qian and X.-R. Yang, Adv. Funct. Mater., 2007, 7, 1353–1358.
41. X.-F. Wang, Y. Zhou, J.-J. Xu and H.-Y. Chen, Adv. Funct. Mater., 2009, 19, 1444–1450.
42. L. Guo, X. Liu, Z. Hu, S. Deng and H. Ju, Electroanalysis, 2009, 21, 2495–2498.
43. S.-N. Ding, J.-J. Xu and H.-Y. Chen, Chem. Commun., 2006, 3631–3633.
44. R. Zhang, L. Fan, Y. Fang and S. Yang, J. Mater. Chem., 2008, 18, 4964–4970.
45. J. Qian, C. Zhang, X. Cao and S. Liu, Anal. Chem., 2010, 82, 6422–6429.
46. Y. Wang, J. Lu, L. H. Tang, H. X. Chang and J. H. Li, Anal. Chem., 2009, 81, 9710–9715.
47. L.-L. Li, K.-P. Liu, G.-H. Yang, C.-M. Wang, J.-R. Zhang and J.-J. Zhu, Adv. Funct. Mater., 2011, 21, 869–878.
48. K. Wang, Q. Liu, X.-Y. Wu, Q.-M. Guan and H.-N. Li, Talanta, 2010, 82, 372–376.
49. H. Wang, F. Zhang, Z. Tan, Q. Chen and L. Wang, Electrochem. Commun., 2010, 12, 650–652.
50. L. X. Cheng, X. Liu, J. P. Lei and H. X. Ju, Anal. Chem., 2010, 82, 3359–3364.
51. Q. Zhu, M. Han, H. Wang, L. Liu, J. Bao, Z. Dai and J. Shen, Analyst, 2010, 135, 2579–2584.
52. C.-G. Shi, J.-J. Xu and H.-Y. Chen, J. Electroanal. Chem., 2007, 610, 186–192.
53. X. Hu, H. Han, L. Hua and Z. Sheng, Biosens. Bioelectron., 2010, 25, 1843–1846.
54. H. Jiang and H. Ju, Anal. Chem., 2007, 79, 6690–6696.
55. L. Zhang, L. Shang and S. Dong, Electrochem. Commun., 2008, 10, 1452–1454.
56. D. R. Lide, CRC Handbook of Chemistry and Physics, 88th ed., CRC Press, Inc., Boca Raton, FL, 2008.
57. X. Liu and H. Ju, Anal. Chem., 2008, 80, 5377–5382.
58. X. Liu, L. Cheng, J. Lei, H. Liu and H. Ju, Chem.–Eur. J., 2010, 16, 10764–10770.
59. G. Jie, J. Zhang, D. Wang, C. Cheng, H.-Y. Chen and J.-J. Zhu, Anal. Chem., 2008, 80, 4033–4039.
60. G. Jie, H. Huang, X. Sun and J.-J. Zhu, Biosens. Bioelectron., 2008, 23, 1896–1899.
61. G.-F. Jie, P. Liu and S.-S. Zhang, Chem. Commun., 2010, 46, 1323–1325.
62. Y. Shan, J.-J. Xu and H.-Y. Chen, Chem. Commun., 2010, 46, 5079–5081.
63. Y. Shan, J.-J. Xu and H.-Y. Chen, Chem. Commun., 2010, 46, 4187–4189.
64. M.-S. Wu, H.-W. Shi, J.-J. Xu and H.-Y. Chen, Chem. Commun., 2011, 47, 7752–7754.
65. L. Li, M. Li, Y. Sun, J. Li, L. Sun, G. Zou, X. Zhang and W. Jin, Chem. Commun., 2011, 47, 8292–8294.
66. H. Huang and J.-J. Zhu, Biosens. Bioelectron., 2009, 25, 927–930.
67. H. Huang, G. Jie, R. Cui and J.-J. Zhu, Electrochem. Commun., 2009, 11, 816–818.
68. H. Huang, Y. Tan, J. Shi, G. Liang and J.-J. Zhu, Nanoscale, 2010, 2, 606–612.
69. G. Jie, L. Wang, J. Yuan and S. Zhang, Anal. Chem., 2011, 83, 3873–3880.
70. I. Díez, M. Pusa, S. Kulmala, H. Jiang, A. Walther, A. S. Goldmann, A. H. E. Müller, O. Ikkala and R. H. A. Ras, Angew. Chem., Int. Ed., 2009, 48, 2122–2125.
71. L. Li, H. Liu, Y. Shen, J. Zhang and J.-J. Zhu, Anal. Chem., 2011, 83, 661–665.
72. Y.-M. Fang, J. Song, J. Li, Y.-W. Wang, H.-H. Yang, J.-J. Sun and G.-N. Chen, Chem. Commun., 2011, 47, 2369–2371.
73. J. Lei and H. Ju, TrAC, Trends Anal. Chem., 2011, 30, 1351–1359.
74. H. P. Huang, J. J. Li and J. J. Zhu, Anal. Methods, 2011, 3, 33–42.

---