Applications of QDs in ECL Biosensing: History
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Electrochemiluminescence (ECL) is the chemiluminescence triggered by electrochemical reactions. Due to the unique excitation mode and inherent low background, ECL has been a powerful analytical technique to be widely used in biosensing and imaging. As an emerging ECL luminophore, semiconductor quantum dots (QDs) have apparent advantages over traditional molecular luminophores in terms of luminescence efficiency and signal modulation ability. Therefore, the development of an efficient ECL system with QDs as luminophores is of great significance to improve the sensitivity and detection flux of ECL biosensors. 

  • electrochemiluminescence
  • quantum dots
  • mechanism
  • coreactant
  • biosensor

1. Immunoassay

1.1. Immunoassay Based on Antigen-Antibody Recognition

Specific antigen-antibody recognition is the basis of immunoassay. Due to the high affinity and specificity of antigen-antibody interaction proved by various characterization techniques [1][2], it has been widely used in the construction of ECL-based immunosensors with QDs as luminophores. According to the difference of immune structures, immunosensors based on antigen-antibody recognition can be divided into “antibody-antigen” and “antibody-antigen-antibody” formats, and the latter one is generally referred to as sandwich structure.
Due to the simplified structures and no requirements for complex antibody labeling techniques, “antibody-antigen” type immunosensors were first proposed and widely used for sensitive detection of lipoprotein, human prealbumin, human IgG and carcinogenic antigens [3][4][5][6]. QDs are first bound to the electrode through surface ligand functionalization and then antibodies are covalently conjugated to the electrode coated with QDs, followed by the block of non-specific binding sites with Bovine Serum Albumin (BSA) to reduce background interference. In the absence of antigens, QDs exhibit intense ECL because of the effective charge transfer. In the presence of target antigens, the immunocomplex formed by the specific recognition between antigen and antibody increases the steric hindrance and inhibits the charge transfer from coreactant radicals to QDs, resulting in a decrease in ECL intensity [5]. In addition, a variety of nanomaterials such as TiO2 nanotubes and metal-organic frameworks (MOFs) were used to form composites with QDs to accelerate the electron transfer between QDs and electrodes, further improving the sensitivity of detection [7][8].
With the rapid development of antibody-labeling techniques, immunosensors with sandwich structures have gradually become the mainstream of QD-based ECL immunosensors. They can be further divided into “signal-on” and “signal-off” immunosensors according to the variation of ECL intensity with the concentration of targets. Among them, the former with a sandwich structure is the most classic ECL biosensing strategy, which has been successfully commercialized in the field of immunodiagnosis using [Ru(bpy)3]2+ as ECL labels. In view of the significantly superior optical properties of QDs over [Ru(bpy)3]2+, a variety of sandwich-structured “signal-on” immunosensors with QDs as labels were constructed to improve the analytical performance of ECL immunoassay [9][10][11][12][13][14][15]. In the construction of “signal-on” immunosensors, primary antibodies are typically bound to the solid substrate to capture antigens and QDs are used as signal labels for the conjugation with secondary antibodies. In the presence of antigens, secondary antibodies labelled with QDs are captured to the solid substrates through the formation of immunocomplexes and then the concentration of antigens can be correlated with the ECL intensity of QDs to realize the quantitative detection of antigens. It should be noted that the solid substrates employed to fix primary antibodies can be either electrodes or magnetic beads. Due to the high surface area to volume ratio, the ease of bioconjugation and the convenience of magnetic separation, magnetic materials are the ideal solid carriers for the construction of ECL immunosensors. 
In addition, due to the extremely low concentration of antigens in biological samples and the limited number of QDs labelled on secondary antibodies, various signal amplification strategies have been developed to improve the detection sensitivity of trace antigens [13][16]. Specifically, MWCNTs are first modified to the glassy carbon electrodes (GCE) to bind with the primary antibody and then CdTe QDs-functionalized SiO2 nanoparticles are combined with GO to conjugate the secondary antibody. MWCNTs can accelerate electron transfer between CdTe QDs and electrodes. The combination of CdTe QDs-functionalized SiO2 nanoparticles and GO can not only increase the ECL intensity of a single label but also provide sufficient binding sites for the conjugation of secondary antibodies. Dual signal amplification strategies significantly improve the sensitivity of AFP immunoassay. In addition, QDs can be enriched by encapsulating them in porous nanostructures, thereby amplifying ECL signals and improving detection sensitivity. For example, Liu et al. combined SnS2 QDs and MOFs into the construction of ECL immunosensors to achieve synergistic amplification of ECL signals with K2S2O8 as a coreactant [17]. The MOFs served not only as carriers to enrich ECL luminophores but also as catalysts for oxygen reduction to promote the ECL generation of SO4•−/O2 system, eventually realizing the ultrasensitive detection of the carbohydrate antigen 24-2 with a detection limit of 0.015 mU/mL. Deng et al. developed an ECL immunosensor for the detection of procalcitonin using manganese dioxide nanoflowers and ZnS QDs as dual ECL emitters [18]. In this strategy, abundant Au nanoparticles (AuNPs) and ZnS QDs were incorporated into the nanoflowers to improve the conductivity and ECL efficiency. Meanwhile, manganese dioxide nanoflowers could act as ECL emitters and co-reaction accelerators to further amplify the ECL signal, achieving the sensitive detection of procalcitonin with a detection limit of 0.033 pg/mL.
In contrast to “signal-on” immunosensors, ECL intensity of the “signal-off” immunosensors is negatively correlated with the concentration of targets, which is mainly achieved by quenching ECL signals of QDs in various ways after the formation of sandwich immunocomplexes. Currently, there are mainly two ECL quenching strategies reported in the literature, namely resonance energy transfer (RET) and competitive consumption of coreactants [19][20]. The former usually requires a donor-acceptor pair, in which the ECL emission spectrum of the donor partially overlaps with the ECL absorption spectrum of the acceptor [21]. Since donors and acceptors are fixed on the electrodes and the secondary antibodies, respectively, and far away from each other, the ECL-RET cannot occur in the absence of immunoreactions. After the capture of target antigens, the formation of immunocomplexes narrows the distance between donors and acceptors, meeting the distance requirement for RET. Then ECL emission of the donor fixed on the electrode is significantly quenched, establishing a quantitative relationship between ECL intensity and the concentration of antigens. Typical donor-acceptor pairs include two QDs with different ECL emission wavelength, or QDs and corresponding quenching molecules such as benzoquinone [22][23].
In addition, the “signal-off” immunosensors based on the competitive consumption of coreactants are mainly constructed by labeling electrocatalysts with oxygen reduction catalytic activity on the secondary antibodies, which can selectively catalyze the reduction of dissolved oxygen to produce H2O instead of H2O2, thus reducing the ECL intensity of QDs with H2O2 as coreactants [19][24]. In this way, the ECL intensity of QDs is negatively correlated with the concentration of antigens, enabling the quantitative detection of trace antigens

1.2. Immunoassay Based on DNA/RNA Aptamers

Aptamers are DNA/RNA oligonucleotide fragments or short peptides with a high affinity for specific targets [25]. Compared with antibodies, aptamers have the advantages of low cost, being easy to synthesize and having good stability, showing the potential to replace antibodies as the emerging target receptors [26]. In recent years, the application of aptamers in QD-based ECL immunoassay has been explored due to its high specificity and selectivity to the target [27][28][29][30]. For example, Zhu et al. developed a QD-based ECL biosensor for the detection of lysozyme through the recognition of lysozyme by aptamers [27]. Due to the competitive binding of complementary DNA labeled by QDs with lysozyme to the aptamer fixed on the electrode, the ECL intensity of QDs is negatively correlated with the concentration of lysozyme, thus enabling the quantitative detection of lysozyme. Later, Jie et al. proposed a multiple DNA cycle amplification strategy for thrombin assay based on ECL quenching of QDs [31]. In this strategy, QDs was first fixed to the electrode, followed by the conjugation to the hairpin DNA labeled with AuNPs on the other end. In this case, the ECL signals of QDs could be efficiently quenched by AuNPs in the absence of thrombin. In the presence of thrombin, the recognition of thrombin by aptamers led to the release of complementary DNA (cDNA), which then hybridized with the loop of hairpin DNA and induced the recognition of this region by endonuclease, resulting in the break of the hairpin DNA and the release of AuNPs. ECL signals were greatly amplified after multiple cycles of the cleavage reaction, thus improving the detection sensitivity of thrombin. In addition, Feng et al. constructed a dual-stimuli responsive ECL biosensor for the detection of pathogenic bacterial [32]. In the absence of bacterial, silver nanocluster-labeled hairpin DNA quenched ECL emission from CdS QDs by resonance energy transfer. In the presence of bacteria, the hairpin DNA was cleaved and silver nanoclusters were released from the surface of CdS QDs, and then probe DNA labeled with AuNPs was introduced to pair with the residual sequence of hairpin DNA, resulting in the enhancement of ECL emission by the surface plasmon resonance effect.
In addition, target-triggered ratiometric sensing strategies have also been used to construct aptamer-based ECL immunosensors. For example, Han et al. prepared a novel MOFs/Au/G-quadruplex as both quencher and enhancer to fabricate a target-triggered ratiometric ECL sensor for accurate detection of prostatic specific antigen (PSA) [33]. In this work, CdSe/ZnS QDs and luminol were used as dual-potential-dependent ECL emitters for ratiometric sensing. After the sequential hybridization of cDNA labelled with QDs, PSA aptamer and probe DNA linked with Au/hemin@MOFs-DNAzyme and the further competition of PSA, the probe DNA would keep away from the electrode, causing a switchover of ECL signals of QDs-luminol pairs from an “off-on” state to an “on-off” one. Therefore, PSA was sensitively quantified by an ECL intensity ratio of two emitters.

1.3. Multiplex Immunoassay

The ECL immunosensor discussed above can only achieve the detection of a single antigen in a single run. In fact, the demand for simultaneous detection of multiple targets in immunodiagnostics has increased rapidly, which puts forward higher requirements for the signal-resolving strategies of multiple ECL emitters [34]. According to the difference in signal-resolving strategies, the multiplex immunosensors based on the ECL of QDs can be divided into potential-resolved and wavelength-resolved formats, both of which depend on the adjustment of the band gap by tuning the size and composition of QDs [35][36][37][38].
Yang et al. first constructed ECL immunosensors based on potential-resolved strategies for simultaneous determination of triple latent tuberculosis infection (LTBI) markers [36]. In the presence of LTBI markers, the formation of the immunocomplex generates three potential-resolved ECL signals during one potential scanning and the ECL intensities reflect the concentrations of three LTBI markers, respectively. Recently, Zeng et al. constructed a dual-signal ECL immunosensor based on sandwich structure and a magnetic separation technique for the simultaneous detection of carbohydrate antigen 125 (CA125) and human epithelial protein 4 (HE4) markers of ovarian cancer [39]. Eu MOFs loaded with isoluminol and AuNPs generated a strong anodic ECL signal, and the composite of CdS QDs and a Cu single-atom catalyst could act as a cathodic ECL emitter and catalyze the reduction of H2O2 to produce a large amount of OH and O2•−, therefore achieving remarkable bipolar ECL signals. With two potential-resolved ECL luminophores as labels, this platform successfully performed simultaneous detection of ovarian cancer markers with detection limits of 0.37 pg/mL and 1.58 pg/mL for CA125 and HE4, respectively.
Guo et al. first proposed the ECL-immunosensing strategy with multicolor QDs as labels for the simultaneous determination of two different tumor markers, AFP and CEA [35]. Subsequently, Zou’s group has conducted several explorations on wavelength-resolved ECL immunosensors with QDs as labels [37][40]. For example, Zou and co-workers proposed a spectrum-resolved triplex-color ECL multiplexing immunoassay for the simultaneous determination of three different tumor markers, CEA, PSA and AFP [40]
It can be seen that the construction of multiplex ECL immunosensors significantly depends on the development of QD synthesis strategies. QDs with narrow emission spectra and continuously tunable wavelength are more favorable for ECL multiplex immunoassay. Therefore, it is still of great importance to develop high-quality synthesis strategies for QDs in aqueous systems.

2. Nucleic Acid Analysis

2.1. Nucleic Acid Analysis Based on Sandwich Structures

In recent years, QD-based ECL biosensors with sandwich structures have been widely explored in nucleic acid analysis. QD-based DNA/RNA biosensors with sandwich structures generally consist of a capture probe, a target and a detection probe. Capture probes are attached to the solid substrates, which can be electrodes or magnetic beads, for the specific recognition of targets [41][42]; QDs as ECL emitters are conjugated to detection probes or electrodes [43][44].
The “signal-on” ECL biosensors for nucleic acid analysis are constructed by labeling QDs on the detection probes, in which ECL intensity is positively correlated with the concentration of targets. For example, Jie et al. constructed a “signal-on” ECL biosensor with dendritic QDs nanocluster as emitters for the detection of target DNA [45]. After being hybridized with the target microRNA and oligonucleotide-encapsulated Ag nanoclusters sequentially, the hairpin structure opens up and then Ag nanoclusters are in close proximity to CdS QDs on the modified GCE. Ag nanoclusters can not only quench the ECL emission of CdS QDs by RET but also catalyze the electrochemical reduction of K2S2O8 to promote the consumption of coreactants. Based on the dual quenching effects, a sensitive ECL biosensing of microRNA is achieved with a wide linear range and acceptable selectivity.
In addition, various signal amplification strategies have been widely explored to further improve the sensitivity of nucleic acid analysis, such as target-induced recycling amplification, cascade amplification strategies and strand displacement reactions [46][47][48][49][50][51][52]. For example, Yuan et al. constructed a novel ECL biosensor to achieve the ultrasensitive detection of microRNA by combining target recycling amplification and double-output conversion strategies [46]. In this case, the ECL efficiency of QDs was improved by conjugation with ruthenium complexes, in which ECL-RET took place efficiently because of the short path of energy transfer. Target-induced DNA polymerization and the subsequent release of synthesized reporter DNA enabled a small number of microRNA to be successfully transferred into a large number of reporter DNA strands, which could capture numerous QD-labeled signal probes on the sensing surface to realize the sensitive ECL response to target microRNA. Song et al. developed the “signal-on” ECL biosensor for the detection of microRNA-141 based on a dual isothermal enzyme-free strand displacement reaction [50]. In the presence of trace target microRNA, the strand displacement reaction was triggered and abundant mimic targets were released, thus achieving amplification of the target. In the detection process, capture probes were fixed on a AgInZnS QD-modified electrode and paired with ferrocene-labeled probes, causing the ECL signal to be in an “off” state. However, the competitive binding of mimic targets and ferrocene-labeled probes to capture probes led to the release of ferrocene from the electrode and the recovery of ECL signals, thus enabling the sensitive biosensing of microRNA-141 with a low detection limit of 33.3 aM.
In recent years, the DNA walking machine has aroused increasing interest for its efficient self-assembly and signal amplification abilities, and it has also been used in QD-based DNA/RNA biosensing [53][54]. Researchers have made some explorations of QD-based ECL biosensors amplified by a DNA walking machine for ultrasensitive detection of microRNA, including dual-legged DNA walkers and a three-dimensional (3D) DNA walking machine [55][56][57]. Once paired with protecting DNA, walking DNA is locked and cannot work. In the presence of target microRNA, walking DNA is released by the competitive pairing of target microRNA and protecting DNA, followed by pairing with supporting DNA to form the recognition site to be released again under the shearing of a Nt.BsmAl nicking endonuclease. Subsequently, the released walking DNA moves along the surface of Au@Fe3O4 and repeats the process, generating a large number of intermediate DNA strands in the presence of trace target microRNA. Finally, intermediate DNA is used to open the hairpin structure on the electrode, generating LSPR enhancement effect on ECL intensity of QDs. Dual signal amplification strategies significantly improve the sensitivity of microRNA detection. Therefore, the 3D DNA walking machine has higher efficiency of payload release and superior signal amplification than those of the traditional DNA walking machines, allowing ultrasensitive detection of microRNA.

2.2. Nucleic Acid Analysis Based on Recognition of Complementary Sequences

Another type of QD-based ECL biosensor for nucleic acid analysis is constructed through the specific recognition of the target by hairpin DNA. In this strategy, the film of QDs with stable ECL emission is generally coated on the electrodes, followed by target-induced ECL enhancement or quenching to achieve a sensitive response of the ECL intensity to the target. Currently, ECL quenching strategies reported in the literature include RET effects and competitive consumption of coreactants, while ECL enhancement strategies are mainly based on the LSPR effect [58][59][60][61]. Among them, the RET quenching effect and LSPR enhancement effect are both derived from the interaction between noble metal nanocrystals and ECL emitters. The dominant effect mainly depends on the distance between the noble metal nanocrystals and ECL emitters. Generally, the RET effect dominates at short range, while the LSPR effect dominates at long range. For instance, Xu et al. first proposed a distance-dependent ECL quenching or enhancement strategy based on the interaction between AuNPs and CdS:Mn QDs for DNA detection [62]
In addition, ECL quenching strategies based on competitive consumption of coreactants are also used to construct biosensors for nucleic acid detection. Ju et al. developed a label-free QD-based ECL system for DNA assay based on the consumption of coreactants by the electrocatalytic reduction of dissolved oxygen with DNAzyme [60]. In this system, the label-free hairpin DNA was attached to the electrode modified with QDs. Upon the hybridization of the hairpin with target DNA in the presence of hemin, the specific sequence conjugated with hemin to form a G-quadruplex architecture, which showed a high catalytic activity for electrochemical reduction of dissolved oxygen, leading to a decrease in the ECL signal.
By introducing QDs with high ECL efficiency into the construction of nucleic acid biosensors, the sensitivity of DNA/RNA detection has been significantly improved, but there is still a lack of studies on high-throughput DNA/RNA detection. More efforts need to be devoted to develop QD-based nucleic acid biosensing strategies with both high sensitivity and high throughput.

3. Small Molecules and Ions Detection

3.1. Target-Induced ECL Quenching

Since many molecules and ions have quenching effects on ECL emission of QDs, target-induced ECL quenching has been the most classical sensing strategy for the detection of small molecules and ions. The quenching mechanisms of ECL include quenching of excited states of QDs [63][64][65][66], quenching of coreactant radicals [67][68][69][70], inhibition of the electrochemical process [71][72][73][74], competitive consumption of coreactants [75] and destruction of QD structures [76][77]. Sensing strategies for small molecules and ions based on the various ECL quenching mechanisms are described in this section.
Dopamine and benzoquinone are common model analytes that can quench QD*. For example, Ag2Se QDs with near-infrared ECL emission were used to construct the sensor for dopamine based on its quenching effect on the excited state [63]. The energy level of dopamine was between the valence band and conduction band of Ag2Se QDs, so the quenching effect on the near-infrared ECL emission of QDs resulted from the process of electron transfer. In addition, the energy transfer between excited ZnSe QDs and benzoquinone produced by the oxidation of hydroquinone was used for the bio-detection of hydroquinone [65]. As an efficient coreactant promoting ECL emission of ZnSe QDs, K2S2O8 with strong oxidizing properties could oxidize hydroquinone to benzoquinone, which thus displayed a strong inhibition on ECL emission.
Thiol compounds and ascorbic acid have a strong quenching effect on coreactant radicals by the electron-transfer process and thus inhibit ECL emission of QDs, which has been used to construct ECL biosensors for these molecules. For example, based on the quenching effect of ascorbic acid on SO4•−, the activity of alkaline phosphatase was determined indirectly according to the concentration of ascorbic acid, which was generated in the hydrolysis reaction of L-ascorbic acid 2-phosphate sesquimagnesium catalyzed by alkaline phosphatase [68]. In addition, homocysteine was a potent radical quencher for near-infrared ECL of CdSeTe/ZnS QDs [69].
The quenching effect of nitrites on the ECL emission of QDs followed an “electrochemical oxidation inhibition” process [71]. The presence of nitrites produced a large voltage drop and made the practical potential less than the applied potential, leading to a weak ECL emission. A hydroquinone/horseradish peroxidase (HRP)/H2O2 system was used as a model system to construct an ECL biosensor for the detection of hydroquinone [75]. HRP catalyzed the enzymatic reaction of hydroquinone and H2O2, leading to the consumption of coreactants and thus the quenching of ECL. In addition, ECL sensors for metal ions with stronger metal-S interaction than a Cd-S bond were constructed based on the structure destruction of CdSe QDs [76]. The competitive binding of Cu2+ to the stabilizer led to the precipitation of QDs and thus the quenching of ECL emission, establishing a negative correlation between the ECL intensity of QDs and the concentration of Cu2+. This strategy could be extended to the rapid detection of other cations with strong metal-S interactions [77].

3.2. Coreactant Concentration-Dependent Biosensing Strategy

In this strategy, the concentration of target molecules is correlated with the concentration of coreactants and thus with the ECL intensity of QDs, which can be achieved either by considering target molecules as coreactants or by the target-induced consumption or production of coreactants. For example, DBAE and TPrA were sensitively detected as ECL coreactants of CdTe and ZnSe QDs, respectively [78][79]. In addition, an ECL biosensor for the detection of glucose was constructed based on the competitive consumption of dissolved oxygen, which acted as a coreactant in the QD-based ECL process [80]

3.3. DNA Aptamer-Based Biosensing Strategy

DNA aptamers are efficient probes for the specific recognition of some molecules and metal ions, which have also been extensively explored for ECL biosensing of these analytes [81][82][83][84]. The aptamer-based ECL biosensor for adenosine 5′-triphosphate (ATP) detection was developed based on the aptamer-ATP specific affinity and the rule of Watson–Crick base pairing [82]. After the formation of aptamer-ATP complexes on the electrode, cDNA was hybridized with the remaining free probes. Subsequently, QDs were labeled through the biotin-avidin reaction in the existence of biotin-modified cDNA. Therefore, the ECL intensity of QDs showed sensitive response to ATP. In addition, DNA aptamers with a hairpin structure were also used to construct an ECL biosensor for the detection of Pb2+ [83]. In the presence of Pb2+, the “stem-loop” structure of hairpin aptamer opened up, followed by the formation of a G-quadruplex and the conjugation of QDs to the terminal amino, thus ECL emission was significantly enhanced by the addition of Pb2+. In addition, the DNAzyme-triggered ECL ratiometric biosensing strategy was developed for the sensitive detection of Mg2+ using CdS QDs and luminol as dual-potential ECL emitters [81]

This entry is adapted from the peer-reviewed paper 10.3390/bios13070708


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