Framework-Enhanced Electrochemiluminescence in Biosensing: Comparison
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Please note this is a comparison between Version 2 by Sirius Huang and Version 1 by Jianping Lei.

Electrochemiluminescence (ECL) has attracted increasing attention owing to its intrinsic advantages of high sensitivity, good stability, and low background. Considering the fact that framework nanocrystals such as metal–organic frameworks and covalent organic frameworks have accurate molecular structures, a series of framework-based ECL platforms are developed for decoding emission fundamentals. The integration of fluorescent ligands into frameworks significantly improves the ECL properties due to the arrangement of molecules and intramolecular electron transfer. 

  • electrochemiluminescence
  • frameworks
  • biosensing
  • nanoemitters

1. Introduction

Electrochemiluminescence (ECL) is a classic and powerful analytical technique involving a redox process at electrodes where excited states are electrochemically generated and emit light [1,2][1][2]. Benefitting from its unique light-free luminescence mechanism, ECL has many advantages for an analysis, such as a high sensitivity, good stability, and low background [3]. Nowadays, ECL is widely applied in the areas of environmental monitoring [4], cell sensing [5], imaging [6], food [7], and water safety [8]. ECL-driven tumor photodynamic therapy (PDT) was proposed through the effective energy transfer from ECL emission to photosensitizer chlorin e6 [9]. With a high spatiotemporal controllability, stable luminescence, and high photon flux of ECL, ECL microscopy may be more fascinating than fluorescence [10], bioluminescence [11], and surface-enhanced Raman scattering [12]. Over several decades of research, the variety of ECL emitters has substantially increased, and they can be broadly classified into an inorganic system (such as Ir or Ru complexes), an organic system (such as luminols), and semiconductor nanomaterials [13]. Recently, several novel nanomaterials have been used as luminophores in ECL, such as Au nanomaterials [14], quantum dots [15], and frameworks [16]. The combinations of ECL techniques and these new materials broaden the scope of ECL applications.
Frameworks containing metal–organic frameworks (MOFs), covalent–organic frameworks (COFs), and hydrogen-bonded organic frameworks (HOFs) have been developed rapidly since the 21st century [17,18][17][18]. Owing to their flexible, synthetically controllable, and adjustable structure, frameworks have been utilized in various areas such as energy storage, sewage treatment, gas separation, catalysis, and biosensing [19]. Although there are some deficiencies for electrochemical reactions in frameworks, such as an intrinsic poor electroconductivity and low mass permeability [20], frameworks have been gradually regarded as one of the most promising nanomaterials in ECL assays. Different from other ECL luminophores like metal complexes and quantum dots, predesignable structures of frameworks make them more suitable and efficient in ECL processes. For example, Yin’s group designed an ECL-active MOF by using a ruthenium complex as a ligand and more intense ECL emission was observed with the aid of grapheme oxide [21]. By combining a predesigned structure with post-modification, frameworks provide various strategies to regulate their ECL signals for adapting the requirement. Furthermore, the structures of frameworks are utilized dexterously to create novel ECL enhancement mechanisms for developing sensitive and stable analytical methods. Overall, the variation of an ECL signal highly depends on the optoelectronic properties of frameworks, which are adjustable with designation or post-modification [22].
In order to design self-luminescent reticular nanoemitters, three types of methods have been developed. First, frameworks can be synthesized with ECL-active luminophore ligands, such as porphyrin [23], pyrene [24], and aggregation-induced emission luminogen [25], for constructing emitters with an improved ECL efficiency. Second, the doping of transition metal elements is a promising way to deal with an intrinsic low conductivity of MOFs in ECL reactions. Classic Ru complexes [26] and novel lanthanide ions [27] are already applied in biosensing with this method. Third, non-emitting monomers can be endowed with intense ECL emission through a rational design. Typically, by utilizing well-designed donor/acceptor units, a high ECL efficiency of COFs could be realized [28,29][28][29].
Therefore, integrating the frameworks and ECL methods is of great significance to construct high-performance biosensing platforms. In recent years, the roles of frameworks in ECL processes develop rapidly with multifunctions in biosensing. Initially, frameworks were used as carriers of classic luminophores or catalysts to accelerate the ECL reactions, but they later became ECL emitters for biosensing platform establishment, achieving a successful analysis of proteins, nucleic acids, small molecules, and cells.

2. Framework-Enhanced ECL for Biosensing

The sensitive, specific, and reliable detection of tumor markers is vital for the early diagnosis of cancer, which brings hope to human patients for cancer prevention. Based on unique physical properties, chemical compositions, and functional methods, framework-enhanced ECL may provide an ultrasensitive and comprehensive assay for monitoring these markers. By combining with biological tools, framework-based biosensors can distinguish various biomarkers such as proteins, nucleic acid, cells, and small molecules in a clinical analysis.

2.1. Proteins

Proteins are typical biomacromolecules that are generally analyzed through immunoassays. Once a protein is clinically certified as a disease-related biomarker for a diagnosis, it will receive much attention in ultrasensitive detection. For example, to improve the survival of patients, cancer markers are of great significance in the guidance of an early tumor diagnosis and introducing appropriate targeted therapies [71][30]. Integrating with highly specific immunoreactions, ECL immunoassays are powerful tools for protein detections. Alpha-fetoprotein (AFP) is a well-known biomarker for the diagnosis of a liver malignant tumor [72][31]. Zhao et al. synthesized bimetallic NiZn MOF nanosheets to amplify cathodic luminol ECL through the synergistic effect of the bimetallic catalyst in AFP immunodetection [73][32]. Li et al. designed a signal-off ECL biosensor for AFP detection by utilizing the MnO2 nanosheet/polydopamine dual-quenching effect towards a Ru(bpy)32+-functionalized MOF [74][33]. Cytokeratin 19 fragment 21–1 is recognized as an essential biomarker of non-small-cell lung cancer with a high specificity. Wei’s group constructed a “signal-on” ECL immunosensor for this biomarker detection by using a copper-doped terbium MOF as a luminescent tag, which exhibited a strong ECL emission with K2S2O8 as a coreactant through electrocatalyzing the reduction of S2O82− [75][34]. In the same group, a biocompatible tris(4,4′-dicarboxylic acid-2,2′-bipyridyl)ruthenium(II) [Ru(dcbpy)32+]-functionalized γ-cyclodextrin MOF not only served as a carrier to immobilize the detection antibody via a Pd-N bond but also facilitated the electron transfer rate to amplify the ECL signal [76][35], providing the ultrasensitive method for an early diagnosis of lung cancer. Through potential-resolved ECL, a reticular biosensor could detect multiple protein biomarkers in a single run. Zhang et al. developed a MOF-based ECL tag with both anodic and cathodic emission [77][36]. A useful strategy with the isolated anodic and cathodic coreactants was applied to improve the analytical performance of this potential-resolved ECL sensor, leading to a successful analysis of a carcinoembryonic antigen (CEA) and neuron-specific enolase (NSE) simultaneously. According to the different roles of frameworks in ECL processes, various signal transductions can be realized in an analysis of the same targets. For example, a hollow hierarchical MOF was employed as a carrier to graft Ru complexes as a signal amplification with the catalytic hairpin assembly strategy [78][37], showing an excellent selectivity and high sensitivity for thrombin determination. By tuning the reaction time, a series of porphyrin Zr-MOFs (PCN-222) with different specific surface areas, pore sizes, structures, and surface charge states were synthesized, which served as an ECL emitter, coreactant promoter, and connection in the ECL immunoassay [79][38]. Furthermore, Xiao’s group designed a COF-based ECL biosensor with conductivity- and pre-reduction-enhanced ECL, which overcame the intrinsic poor conductivity of COF [80][39]. With the aid of the signal amplification of the aptamer/protein-proximity-binding-induced 3D bipedal DNA walker, the constructed ECL sensor realized the supersensitive detection of thrombin. In addition, some proteins can be detected by utilizing their bioactive properties. For example, telomerase can extend the length of specific DNA, indicating its possible role as a signal switch. By monitoring bioactivity, telomerase was already analyzed with several well-designed ECL methods [81,82][40][41]. In Lei’s group, an ECL telomerase biosensor was proposed with a BODIPY-based MOF nanoemitter composed of pyridine-substituted BODIPY, a terephthalic acid ligand, and Zn nodes [83][42]. The BODIPY-based MOF showed the P6/m trigonal crystal system, reducing the over-aggregation of BODIPY for enhanced optical signals. After an elaborative design, the BODIPY-based MOF ECL sensors reached a good sensitivity under different telomerase concentrations. The mechanism of this sensor was that the DNA hairpin opened when telomerase appeared, allowing the MOF to approach the electrode surface for ECL signal generation. Integrating with unique immunoreactions, framework-based ECL biosensors become powerful for protein detection (Table 1).
Table 1.
A summary of framework-enhanced ECL for detection of proteins.
Targets Frameworks Linear Range LOD Ref.
AFP NiZn MOF 0.00005 to 100 ng/mL 0.98 fg/mL [73][32]
AFP Ru(bpy)32+@TMU-3 0.01 pg/mL to 5 ng/mL 10.7 fg/mL [74][33]
AFP Magnetic MOF@CdSnS 1 fg/mL to 100 ng/mL 0.2 fg/mL [84][43]
CYFRA21-1 Pd-ZIF-67 0.01 to 100 ng/mL 2.6 pg/mL [75][34]
CYFRA21-1 Ru@ γ-CD-MOF 0.1 pg/mL to 50 ng/mL 0.048 pg/mL [76][35]
Thrombin Ru-UiO-66-NH2 100 fM–100 nM 31.6 fM [78][37]
Thrombin PCN-222 50 fg/mL to 100 pg/mL 2.48 fg/mL [79][38]
Thrombin Conductive COF 100 aM to 1 nM 62.1 aM [80][39]
Telomerase BODIPY MOF 8.0 × 10−4 to 8.0 ng/mL 0.43 pg/mL [83][42]
PSA Ru-MOF 5 pg/mL to 5 μg/mL 1.78 pg/mL [85][44]
PSA MOF/Au/DNAzyme 0.5 to 500 ng/mL 0.058 ng/mL [86][45]
CEA N,B-doped Eu MOF 0.1 pg/mL to 1 μg/mL 0.06 pg/mL [87][46]
NSE J-aggregated MOF 10 pg/mL to 50 ng/mL 7.4 pg/mL [88][47]
Peptide Cu:Tb-MOF 1.0 pg/mL to 50 ng/mL 0.68 pg/mL [89][48]
ALP π-conjugated COF 0.01 to 100 U/L 7.6 × 10−3 U/L [90][49]
D-dimer RuZn MOFs 0.001~200 ng/mL 0.20 pg/mL [91][50]

2.2. Nucleic Acids

In the analysis of nucleic acids, signal amplification techniques such as a catalytic hairpin assembly (CHA) [92][51], rolling circle amplification [93][52], and hybridization chain reaction [94][53] have been widely used for a long time. The Crisper/Cas12a technique is also utilized for an enhanced ECL signal in DNA biosensing [95][54]. Combined with these powerful tools, a series of framework-based ECL genosensors are being developed rapidly for ultrasensitive nucleic acid detection. As noncoding RNAs, microRNAs (miRNAs) regulate the expression of messenger RNA by binding to complementary sequences. Once alterations in miRNA expression happen, messenger RNA expression is disrupted, which leads to potential oncogenic changes [96][55]. Therefore, it is crucial to construct reliable and sensitive biosensors for miRNA detection. With the structural development of frameworks, framework-based ECL genosensors for a miRNA analysis were extensively investigated. For instance, Wang et al. synthesized a Zn MOF as a self-enhanced ECL emitter with dual ligands of DPA and N,N-diethylethylenediamine for miRNA-21 detection [97][56]. DPA is a typical luminophore in ECL while DEAEA could be used as both a coreactant and a morphologic regulator, which leads to a strong and stable ECL emission with the efficient intramolecular electron transfer process. Based on CHA and ECL resonance energy transfer, this sensor realized ‘signal-off’-mode signal amplification in the presence of miRNA-21. Similarly, Xue et al. developed a microRNA-141 ECL bioassay by using a dual-ligand MOF, which simultaneously contained a luminophore TPE derivative and a coreactant ligand (1,4-diazabicyclo[2.2.2]octane) in the structural unit [98][57]. Using a DNA triangular prism as a signal switch to detect microRNA-141, this ECL biosensor achieved a low detection limit at the level of 22.9 aM. Furthermore, a dual-wavelength multifunctional ECL biosensor was established for the rapid simultaneous detection of dual targets miRNA-141 and miRNA-155 [99][58].  To overcome an intrinsic low conductivity in MOFs, a conductive NiCo bimetal–organic framework nanorod was successfully applied in miRNA-141 detection, broadening the horizon of conductive MOFs in ECL sensing applications [101][59]. Furthermore, with the long-range orderly arrangement and effective intramolecular charge transfer, a pyrene-based sp2 COF was synthesized as an efficient ECL emitter via the polycondensation of tetrakis(4-formylphenyl)pyrene and 2,2′-(1,4-phenylene)-diacetonitrile. Because of topologically linking pyrene luminophores and aggregation-induced emissive luminogens, the luminescent COF showed a strong and stable ECL emission [102][60], leading to a highly sensitive microRNA-21 biosensor. As a great threat to health, viruses also receive much attention in ultrasensitive detection. For instance, the Zika virus, a member of the Flaviviridae virus family, is suspected to be associated with severe congenital malformations [103][61]. Mao’s group quantified the Zika virus based on Zr-based metal–organic gel and Fe-MIL-88 MOFs as an electrode matrix and nanotag, respectively [104][62]. The double quenching effect originated from Fe-MIL-88 MOFs as both an ECL acceptor and metal active centers to consume the coreactant, resulting in a distinct turn-off signal in the presence of the virus. On the other hand, Shan’s group designed a 2D MOF with an excellent ECL performance by combining the photosensitizer ZnTCPP and electroactive [Co2(-CO2)4] secondary building units for a Sars-CoV-2 gene analysis [105][63]. The ECL sensor achieved a rapid nonamplified detection of the RdRp gene of SARS-CoV-2 with an extremely low limit of detection (30 aM). Furthermore, Wu et al. designed an ECL biosensor using PCN-224/ZnO/polyacrylamide as signal tag for an accurate analysis of the HPV-16 virus [106][64]. With the aid of multiple target-cycling amplification technologies and HCR reactions, this method achieved a rapid and effective “signal-off” detection of the target with the detection limit of 0.13 fM. Overall, by integrating appropriate frameworks with well-designed DNA sequences, these above methods show a great performance in a nucleic acid analysis, which expands the application of frameworks in biosensing.

2.3. Small Molecules

Compared to traditional analytical methods like chromatography and enzyme catalysis, framework-based ECL methods are more sensitive and convenient for small molecule detection. For the determination of small molecules, utilizing specific recognition between an aptamer and target is the most common strategy. For instance, the transduction of aptamer configurations alters the distance between a signal promoter and ECL luminophores, resulting in a signal change by introducing target molecules. A plasmon-enhanced ECL aptasensor displayed highly sensitive detection for lincomycin [107][65]. Based on a suitable aptasensor, a wide range of molecules can be efficiently detected, such as kanamycin [108][66], sulfadimethoxine [109][67], and isocarbophos [110][68]. Apart from the aptamer, a competition-type ECL immunosensor using Pt NPs@MOFs for the quantitative detection of trenbolone was successfully constructed, demonstrating the simplicity of framework-based ECL systems [111][69]. Based on the quenching effect between MOF radicals and oxidized dopamine, dopamine can be analyzed without the aid of an aptamer [55][70]. This hindrance to ECL was highly relevant to the dopamine concentration, and then was applied to construct an ECL method for the highly sensitive detection of dopamine in serum samples. Similarly, uric acid [112][71], rutin [113][72], and deoxynivalenol [114][73] can also be directly measured with framework-based ECL sensors. In addition, a MOF/COF-mixed emitter with dual-color ECL was prepared [115][74]. Based on a π–π interaction between targets and a MOF/COF, diclazepam can not only be absorbed but also selectively quench ECL, achieving sensitive detection. Furthermore, metal ions with potential harm to human health are generally analyzed with inductively coupled plasma mass spectrometry, ion chromatography, and atomic absorption spectroscopy, requiring expensive instruments and staff costs. The inhibition effect of metal ions towards ECL makes them detectable through well-designed framework-based ECL sensors [116][75].

2.4. Cellular Analysis

ECL-based cellular analyses [117][76], such as circulating tumor cells (CTCs) [118][77] and the cell matrix [119][78], have been developed for several decades. With the combination of ECL biotechnology, framework-based ECL sensors for a cell-related analysis gradually emerge. In a typical manner, Liu’s group realized single-molecule movement visualization at the cellular membrane through capturing photoluminescence signals of the designed Ru(bpy)32+-embedded MOF complex (RuMOF) [120][79]. With the aid of the nanoconfinement effect within frameworks, RuMOFs had a splendid ECL intensity at the single-molecule level, which was conducive to visualize the distribution of RuMOF-labeled-membrane PTK7 proteins at low-expressing cells, demonstrating a great potential of framework-based ECL systems in cellular monitoring. Bacteria may cause great harm to health while existing in the human circulatory system, indicating the importance of sensitive detection. Utilizing steric hindrance on electron transfer, Vibrio parahaemolyticus [121][80] and Escherichia coli [122][81] can be successfully analyzed with ECL sensors based on Ru-MOF and NH2-MIL-53(Al) signal reporters, respectively. In addition, an exosome as a subcellular structure is also accurately detected using well-designed ECL sensors with a different signal transduction. For example, Cui’s group constructed a label-free HepG2-derived exosome ECL sensor based on the selectivity of the CD63 peptide in recognizing CD63 proteins on the exosome surface and strong coordination interactions between the Zr4+ of Zn-TCPP/UiO-66-NH2 and the phosphate head of exosomes. The ECL biosensor exhibited a good sensitivity with a detection range from 1.00 × 104 to 3.16 × 106 particles/μL, which is better than most of the existing label-free methods for detecting exosomes [123][82], showing the great prospects of framework-based ECL in sensitive bioassays.

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