Click chemistry in the design of AIEgens for biosensing and bioimaging

The development of rapid, selective, and sensitive fluorescent sensors is essential for visualizing and quantifying biological molecules and processes in vitro, ex vitro, and in vivo, which is important for not only fundamental biological studies but the accurate diagnosis of diseases. The emerging field of activity‐based sensing (ABS), a sensing method that utilizes molecular reactivity for analyte detection possesses many advantages including high specificity, sensitivity and accuracy. The aggregation caused quenching phenomenon which occurs in most conventional fluorophores results in reduced labeling efficiency of the target analytes and low photobleaching resistance, therefore limiting the applications of the ABS strategy. In contrast, aggregation induced emission (AIE) active luminogens (AIEgens) provide exceptional molecular frameworks for ABS. Of the many reaction classes utilized in the AIEgen ABS approach, click chemistry has become increasing popular. In this review, the sensing concepts of the ABS approach with AIEgens and the principles of click chemistry are discussed, followed by a systematic summary of the application of specific click chemistry reactions in AIEgen ABS protocols for the detection of an array of target analytes. Furthermore, the utility of click chemistry in the construction of AIEgens for bioimaging will also be showcased throughout the review.

exceptional biocompatibility. [1][2][3] In order to achieve accurate analyte detection, the design of the fluorescence probe is of paramount importance. [4,5] However, the luminescence of many traditional organic fluorogens (e.g. rhodamine, cyanine, fluorescein, etc.) are quenched at high concentrations due to the formation of aggregates in a phenomenon termed aggregation-caused quenching (ACQ). The ACQ phenomenon greatly restricts the amount of fluorophore labeling of the analyte possible, as they must be used in dilute solutions or dispersed throughout matrix materials. This can often result in severe photobleaching, low target-to-background ratio (TBR), and significantly reduced sensitivity limiting their utility. [6] In contrast to the ACQ effect, the aggregation-induced emission (AIE) concept describes a unique phenomenon in which a selection of luminogens are nonemissive or weakly emissive in dilute solutions but become highly luminescent when the molecules form aggregates in concentrated solutions or in the solid state. [7] The development of luminogens with AIE characteristics (AIEgens), such as the well-known AIEgens hexaphenylsilole tetraphenylethene (TPE), quinoline malonitrile, and 9,10-distrylanthracene, addresses the deficiencies of ACQ fluorophores for diverse applications. When compared to traditional ACQ fluorophores, AIEgens often display improved photostability, excellent signal reliability, and high TBR. Additionally, the "turn on" design of AIEgen sensors lessens the likelihood of false positive or negative signals in contrast to their "turn off" counterparts. Accordingly, AIEgens have been extensively applied in fluorescent detection techniques. [8] AIEgens in the design activity-based sensors has been demonstrated to combine the advantages of the AIE phenomenon and activity-based sensing (ABS), which has resulted in the application of a varied collection of sensors for the successful detection of many diverse biological and chemical species. [9,10] Click chemistry has developed into an increasingly popular approach in AIEgen design for ABS due to the reliability, specificity and biocompatibility of click reactions. Click chemistry, pioneered over 20 years, serves as a powerful organic chemistry strategy to create molecular connections in a highly efficient and orthogonal manner in a quest for easily accessible functional molecules to meet the demands of modern day chemistry (see Section 1.3). The click chemistry strategy can be summarized by one fundamental rule: "all searches must be restricted to molecule that are easy to make," and while initially conceived to aid in drug discovery, click chemistry has been transformative in many areas of the chemical sciences.
The design of AIEgens for ABS using click chemistry for the detection of small molecules (e.g., toxic metal ions and gas, amino acids, and small neutral molecules) highlighted in this review aligns with the concept of fluorescent chemodosimeters, [11] Chemodosimeters are a class of sensing system based upon irreversible chemical reactions induced by the target analyte. [12] Many fluorophores, including AIEgens, have been successfully incorporated into fluorescent chemodosimeters based on a variety of mechanisms, such as photo-induced electron transfer (PET) and excited-state intramolecular proton transfer (ESIPT). [13] The most reported luminescent chemodosimeters have been based on fluorescence turn-on, or emission wavelength shifts, which are induced by analyte detection. As the binding between the analyte and chemodosimeter is irreversible, this strategy offers the advantages of high sensitivity and selectivity and thus the design of fluorescent chemodosimeters has attracted great interest.
In addition to ABS, the construction of AIEgens using different classes of click chemistry has also become a prominent strategy. Many of the most common methods to construct AIEgen cores previously mentioned can be limited by narrow functional group tolerance, the need for expensive catalysts, poor regioselectivity, and tedious purification procedures. Furthermore, the synthesis of these well-known AIEgens is often complicated due to the difficulty in forming C-C bonds in conjugated planes. Thus, there is a need for innovative synthetic approaches to easily access diverse AIEgen cores in order to meet the ever-growing demands for new fluorescence materials. The application of click reactions, which preferentially form heteroatom-carbon or heteroatom-heteroatom bonds, in the construction of AIEgens for biosensing and bioimaging can circumvent the aforementioned limitations of traditional synthesis, by adhering to the click chemistry cri-teria (vide infra). By utilizing reactions which comply with the click chemistry criteria, which is discussed in more detail in Section 1.3, to construct novel AIEgen cores can simplify their synthesis as the synthetic procedures are more straightforward and less labor intensive by being selective for a single product, reducing the required purification, and are high compatible with an aqueous environments.
In this review, we will begin with a brief description to the advantages of ABS and principles used in the design of AIE sensors for activity-based detection followed by an introduction to click chemistry. In the subsequent sections, we will focus on click chemistry reactions used in AIEgen ABS strategies and the construction of AIEgen cores, including cycloadditions, 1,4-Michael addition chemistry, and Diels-Alder reactions. Finally, a summary and outlook of AIE research will be demonstrated for guiding the future design of click chemistry ABS probes.

"Always on" versus ABS
The success of fluorescence imaging techniques is completely reliant on a high TBR. A commonly used strategy to enhance the target signal based on conventional "always on" fluorophores is to adjust their pharmacokinetics and biodistribution in order to maximize the accumulation of fluorophore at the detection target. Nevertheless, "always on" fluorophores constantly emit a fluorescence signal regardless of whether the probe reaches the target tissues and therefore still suffers from low TBR because, although their signal increases upon interacting with the target, the background signal is still high. [14] As a result, the specificity and sensitivity of "always on" fluorophores requires significant improvement. In comparison, activatable fluorescence probes rely on the target to activate the signal, which maximizes the target signal but also minimizes the background signal. [15] The strategy enhances the TBR and lowers the limit of detection (LoD) enabling real-time and sensitive imaging. Furthermore, unlike "always on" fluorescence probes there is no need to wait for clearance of the nonspecifically distributed fluorophores to minimize the background signal for improving the TBR but only the required probe activation. Activatable fluorophores can be characterized as either "activity-based" or "binding-based" probes. [16] Activity-based probes can be activated by the specific chemical reaction with a target analyte; a burgeoning approach that utilizes molecular reactivity as opposed to molecular recognition for detection of the target analyte. In contrast, binding-based probes are comprised of fluorophore and targeted ligand that is mainly dependent on the coordination of the ligand and target analyte, resulting in the change of fluorescence properties in a "lock and key" approach. Many fluorescence-based detection methods have been designed using a "lock and key" approach, which use noncovalent interactions such as ionic, hydrogen bonding, and hydrophobic interactions. Such interactions can be easily affected by cell micro-environmental parameters resulting in large signal fluctuation therefore limiting their practical applications. ABS offers several advantages over traditional "lock and key" molecular recognition approaches such as accurate signal outputs, and improved and sensitivity and specificity. [17]

Activity-based AIE sensor design strategies
The development of AIEgens has proved to be excellent fluorescent molecular frameworks for ABS. Activity-based strategies offers a reliable approach for analyte detection with high specificity when compared to "lock and key" approaches due to the focus on molecular reactivity in contrast to molecular recognition. In ABS, the AIEgens are only activated through the analyte of interest affecting the targeted chemical transformation and consequently causing detectable variations in emission wavelengths, fluorescence intensity, fluorescence lifetime, and/or fluorescence polarization. A variety of explanations have been proposed for the mechanism of AIE luminescence including the restriction of intramolecular motions (RIM) encompassing the restriction of intramolecular rotation (RIR) and/or restriction of intramolecular vibration (RIV). In addition, PET, [18] intramolecular charge transfer (ICT), [19] Förster resonance energy transfer (FRET), [20] and ESIPT, [21,22] are all relevant photophysical processes that can be harnessed in the design of AIE sensors. However, the molecular mechanism of AIE has been discussed extensity elsewhere, [23] and thus the design principles and utilization of click chemistry in activity-based AIEgens will be the focus of this review.
To achieve different sensing purposes, a specific reactive group must be incorporated into the AIEgen for analyte detection to create an AIE sensor for ABS. To date, many sensing principles have been applied using approach including (i) the incorporation of a specific functionality into the AIEgen that can be cleaved or deactivated in the presence of the analyte of interest causing in a change in solubility of the AIEgen in the detection media due to dye aggregation resulting in a strong fluorescence output ( Figure 1A); (ii) bioconjugation via of the reactive groups of a matrix and the AIEgen causing an accumulation of the AIEgen with the matrix of the target analyte triggering a fluorescent emission by the induction of RIV/RIR of the AIEgen ( Figure 1B); (iii) a chemical reaction of two orthogonal groups on molecules that form an AIEgen that result in a fluorescence activation or changes ( Figure 1C).

Click chemistry
Click chemistry was introduced in 2001 by Sharpless as a new organic chemistry strategy for the highly efficient linkage of modular units via heteroatom carbon bond formation to meet the demands of modern day chemistry. [24] While click chemistry was initially conceived to help streamline the drug discovery process, it is now used in almost every corner of chemistry, materials science, and biology. [25] In recognition of this contribution, the 2022 Nobel prize in Chemistry was awarded to scientists Carolyn R. Bertozzi, Morten Meldal and K. Barry Sharpless for their development of click chemistry and biorthogonal chemistry. Click chemistry describes reaction classes that are thermodynamically favorable using "spring-loaded" modules to form new heteroatom-carbon bonds. To further define click chemistry, a set of strict criteria were outlined for a reaction to be classed as "click reaction". A click reaction should be "modular, wide in scope, high-yielding, simple to per- form, and stereospecific, it must create only inoffensive by-products and require benign or easily removed solvent." While meeting the outline criteria can be challenging, a number of transformations were classified as click reactions by Sharpless. These included nonaldol carbonyl chemistry, the nucleophilic opening of strained rings, cycloadditions, and Michael addition reactions ( Figure 1D). [26] This was later expanded by Bertozzi and coworkers to include the strainpromoted [3 + 2] azide−alkyne cycloaddition (SPAAC) reaction which focused on the covalent modification of biomolecules in living systems. [27] In 2014, the next generation of click chemistry was introduced by Sharpless and coworkers termed sulfur-fluoride exchange (SuFEx). [28] SuFEx chemistry forges covalent links with complete reliability by exploiting the unique properties of the S-F bond. SuFEx chemistry enables the formation of new heteroatom-heteroatom bonds under metal free conditions through S-F exchange with amines or aryl silyl ethers in the presence of a suitable nitrogen Lewis base at discrete sulfur connectors ( Figure 1E). [29] Molecules containing the valuable S-F bond act as a "SuFExable molecular plugin" allowing multiple linkages formed around a central sulfur core, which to date include the connective gases SO 2 F 2 and SOF 4 , [28] and the carbon-based connectors ethenesulfonyl fluoride (ESF), [30] 1-bromoethene-1-sulfonyl fluoride, [31] and 2-substitutedalkynyl-1-sulfonylfluorides. [32,33] Although SuFEx has made considerable contributions across many areas of chemistry, [34] it has not been used broadly in the ABS approach or in the construction of AIEgens in comparison to other click chemistry reactions and will only be discussed briefly in this review.
Since being reported, click chemistry has become synonymous with the copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) reaction (vide infra). The popularity of the CuAAC reaction, which has become the "go to" click reaction, has often resulted in the reduced use of the broader family of selective, "spring-loaded" reactions first identified in the click chemistry manifesto. This review aims to introduce and highlight the advantages of click reaction classes most applied in the design and synthesis of AIEgens for ABS including CuAAC, SPAAC, 1,4-Michael additions, and inverse demand Diels-Alder reactions. Their subsequent application including metal ions detection, protein labeling and detection, tumor imaging and ablation, cellular unfolded protein load quantification, and mitochondrial imaging will also be showcased.

CuAAC
The Huisgen 1,3-dipolar cycloaddition of alkynes and azides to yield 1,2,3-triazoles has become the premier example of a click reaction. [35] The ease of synthesis and introduction of the alkyne and azide functionalities into a molecule, in addition to their kinetic stability and tolerance to a wide variety of functional groups and reaction conditions, make these complementary coupling partners particularly appealing as they can be installed and remain unaffected through several subsequent transformations. In addition, as neither azides nor alkynes are present in nature, the reaction offers a unique biorthogonality. However, it was not until the introduction of the copper (I) catalyzed variant of this reaction (CuAAC) was reported independently by the groups of Meldal and Sharpless that the benefits of the cycloaddition were realized. [36,37] The copper (I) catalyst results in excellent regioselectivity for the 1,4-disubstituted 1,2,3-triazole products and increases the reaction rate up to 10 7 times, enabling the reaction to proceed with high rate at room temperature. As a result of this discovery, applications of the Huisgen 1,3-dipolar cycloaddition now extend far beyond organic synthesis. The advantages and the biorthogonal nature of the CuAAC reaction mean it has been able to be successfully applied in the AIEgen ABS approach for the detection of various biomolecules and target analytes. First, through a bioconjugation strategy where an azido-or alkyne-functionalized biomolecule reacts with an AIEgen modified with the complementary reactive group. For example, based on the incorporation of an alkyne functionalized thymidine analogue, 5-ethynyl-2′-deoxyuridine (EdU) into newly synthesized DNA, Tang and coworkers developed  [38] two AIEgens (TPE-Py-N 3 , (1) and Cy-Py-N 3 , (2)) decorated with an azide group for labeling of EdU to detect the DNA synthesis during S-phase in the cell cycle ( Figure 2). [38] The ability of TPE-Py-N 3 (1) and Cy-Py-N 3 (2) to detect DNA synthesis was determined using proliferating HeLa cells. Firstly, the HeLa cells were incubated with EdU followed by the addition of the AIEgens, CuSO 4 , and ascorbic acid. As the incubation time with EdU increased, the HeLa cells nuclei became more emissive, indicating TPE-Py-N 3 (1) and Cy-Py-N 3 (2) were able to quantitatively monitor DNA synthesis via a CuAAC reaction between the AIEgens and the alkyne modified DNA. When compared to Alexa647-azide, an ACQ dye, AIEgens 1 and 2 were brighter, had improved photostability, and displayed a wider working concentration range between 10 to 100 µM. Thus, AIEgens 1 and 2 were demonstrated to be promising alternatives to the commercial Alexa-azide dyes for DNA synthesis detection using the CuAAC reaction. Liu and coworkers also applied the CuAAC bioconjugation approach in the design of a fluorescence "turn on" AIEgen for the biorthogonal conjugation to cancer cells and subsequent ablation ( Figure 3A). [39] Biorthogonal conjugation with fluorescence probes, where abiotic functional groups can be artificially incorporated into specific cancer cells to achieve labeling, has become a popular alternative to traditional approaches that depend on biological  [42] interactions, which often face challenges due to the heterogeneity of cancer cells or saturation of receptors, for cancer cell targeting. [40] While several biorthogonal "turn on" probes have developed for the covalent labeling of biomolecules in cells using this strategy, many are blue or green emissive or suffer from the ACQ effect, greatly limiting their application. To address these deficiencies, the researchers synthesized a red emissive alkyne func-tionalized TPE derivative TPETSAI (3). TPETSAI (3) displayed very weak fluorescence in aqueous media but, following the CuAAC reaction using the model azide, 1,3,5tris(azidomethyl)benzene, a red fluorescence was induced due to the restriction of molecular motion. The potential of TPETSAI (3) for biomolecular imaging in live cells was then demonstrated using human cervix carcinoma HeLa and breast cancer MDA-MB-231 cells incubated with preacetylated N-azidoacetylmannosamine (Ac4ManNAz) to give azidefunctionalized glycans on the cancer cell surface. The glycoengineered cancer cells were then incubated with TPET-SAI (3), CuSO 4 , tris(3-hydroxypropyltriazolylmethyl)amine, and sodium ascorbate. A red fluorescence was then observed in the membrane of both the HeLa cells and MDA-MB-231 that became brighter as the CuAAC reaction progressed. Additionally, AIEgen 3 was successfully used as a therapeutic agent following irradiation with visible light (π = 400-700 nm), which generated reactive oxygen species (ROS) causing in tumor cell necrosis while leaving the normal cells untouched, making this a promising approach for phototherapy.
Bacterial infections are of growing medical and public concerning particularly due to the emergence of resistant bacteria worldwide limiting the efficacy of antibiotics. Increasing population growth has driven an increasing demand for an accurate and rapid method for bacterium type identification to design suitable treatments and improve antibiotic stewardship. [41] Considering this, and the ability of TPETSAI (3) to induce efficient ROS generation, Liu and coworkers later reported a modified biorthogonal fluorescence turn on probe AIEgen, TPEPA (4), to accurately induce bacterial elimination without complicated procedures or the need for antibiotics ( Figure 3B). [42] Pathogenic bacteria are often categorized as either Gram-positive or Gram-negative based up on their bacterial envelope structures. The Gram-positive bacterial envelope comprises a peptidoglycan (PG) layer, in contrast to Gram-negative bacteria whose cell envelope consists of a thin PG cell wall inside the outer membrane of lipopolysaccharide (LPS) ( Figure 3C). The unnatural metabolic precursors D-alanine (D-Ala) D-Ala-N 3 and 3deoxy-D-manno-octulosonic acid (Kdo) Kdo-N 3 derivatives were selected to be incorporated into the peptidoglycan layer of Gram-positive bacteria and the LPS outer membrane of Gram-negative bacteria, respectively. Then, the subsequent introduction of TPEPA (4), whose structure is shown in Figure 3D, with two terminal alkyne groups could effectively label bacteria via the CuAAC reaction. The hydrophilic poly(ethylene glycol) groups endowed TPEPA (4) with good water solubility resulting in weak fluorescence in aqueous solutions, although the AIEgen 4 displayed strong fluorescence which was attributed to the RIM effect upon the CuAAC reaction with the D-Ala-N 3 metabolically modified Gram-positive bacteria and Kdo-N 3 , metabolically modified Gram-negative bacteria enabling selective imaging and differentiation between Gram-positive and Gram-negative bacteria. In order to demonstrate the ability of this biorthogonal method to identify and discriminate Gram-positive and Gram-negative bacteria, a coculture of Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus), pretreated with either Kdo-N 3 or D-Ala-N 3 , was incubated with TPEPA (4). When pretreated with Kdo-N 3 , the Gram-negative E. coli was identified by a strong fluorescence emission from the coculture as a result of the CuAAC reaction with the AIEgen 4. However, S. aureus was able to be identified from the coculture when pretreated D-Ala-N 3 following treatment with TPEPA (4). These results indicated that this approach provided superb discrimination ability even in the coculture of Gram-positive and Gram-negative bacteria. The mechanism for selective biorthogonal labeling was rationalized by the difference in the envelope structures between the differ-ent bacteria. Kdo is an essential component of LPS inserted into the outer membrane, which is only present on the surface of Gram-negative bacteria. Although both Gram-negative and Gram-positive bacteria can metabolize D-Ala-N 3 to the PG on their cell wall, the outer membrane of Gram-negative bacteria prevents AIEgen 4, whose molecular weight is 1235 Da, and D-Ala-N 3 from reacting with each other due to the poor permeability of the bacterial outer membrane to molecules with a weight larger than 650 Da. Moreover, TPEPA (4) could effectively produce ROS following irradiation by white light destroying the integrity of bacterial envelopes, enabling selective bacterial ablation with high specificity. TPEPA (4) displayed negligible toxicity toward normal mammalian cells such as 3T3 cells when using a 40 µM concentration, which is double the required concentration for antibacterial treatment, highlighting its potential as a valuable antibacterial agent.
Further to bioconjugation approaches, the CuAAC reaction has been employed in the design of AIEgens in ABS for the analyte detection of reagents needed to affect the reaction between the alkyne and azide functionalities. Sanji, Tanaka, and coworkers employed with strategy in the development of a procedure for the fluorescence detection of Cu II using the CuAAC reaction. [43] Although copper is an essential transition metal for life and the function of numerous enzymes, elevated levels of Cu II have been linked with many diseases such as neurodegenerative conditions and copper can also be an environmental pollutant when found in its oxidized state. [44] Furthermore, several previously reported Cu II ion fluorescent sensors function as "turn off" sensors due to the paramagnetic nature of Cu II meaning the development of a selective and sensitive "turn on" method for Cu II detection is of great importance. [45] The researchers synthesized an azide functionalized AIEgen (5), which was able to react with the terminal diyne diethylene glycol dipropiolate (6) in the presence of sodium ascorbate upon the addition of Cu II ( Figure 4A). This reaction yielded a series of covalently cross-linked networks resulting in a dramatic enhancement in fluorescence of the AIEgen 5, which was ascribed to the induction of RIR upon formation of the networks. The emission intensity at 490 nm increased almost linearly within the Cu II concentration range between 0-20 µM and the LoD was calculated to be approximately 1 µM, which is significantly lower than the action level of copper in drinking water thus enabling "naked eye" detection. Furthermore, this detection method displayed high selectivity for Cu II as no fluorescence changes were observed when the assay was carried out a variety of different metal ions (Li + , Na + , K + , Mg 2+ , Ca 2+ , Mn 2+ , Fe 2+ , Fe 3+ , Co 2+ , Ag + , and Zn 2+ ). Similarly, Banerjee, Chatterjee and coworkers exploited the requirement for a reducing agent to maintain the copper catalyst in the Cu I oxidation state in the development of a "turn on" fluorescent ABS method for ascorbate ion detection. [46] Ascorbic acid is an essential component of the diet for mammals which endorses healthy cell development and tissue growth, improves fertility, and prevents oxidative damage to body tissues. [47,48] Two TPE AIEgens functionalized with azide (7) and alkyne (8) moieties were first synthesized, which could then become linked via the CuAAC reaction using a Cu II catalyst and ascorbate to yield a AIE active TPE-based polymer ( Figure 4A). In the presence of ascorbate ions, the Cu II species was reduced to Cu I enabling the CuAAC reaction resulting in the formation of a TPE polymer, which was insoluble in the detection media of water-tetrahydrofuran (THF) (93/7 v/v) resulting in a blue fluorescence emission. The selectivity of the fluorescence detection method for ascorbic acid was demonstrated using several other ions as the fluorescence response only increase when ascorbate and the Cu II catalyst were used. This detection method was also shown to be applicable for quantitative detection of ascorbic acid in commercial Vitamin C tablets offering a quick, straightforward, cost-effective approach for ascorbic acid detection.
While the majority of CuAAC procedures use ascorbate ions as an in situ reducing agent, nitric oxide (NO) is also able to generate the required catalytic species by reducing Cu II to Cu I meaning NO can be detected using the CuAAC reaction. This was utilized by Costero and coworkers who reported a TPE derivative (9) functionalized with the alkyne functionality for the sensing NO ( Figure 4A). [49] NO has a diverse range of functions in the cardiovascular system and impairment of NO production or function has been associated in several cardiovascular diseases, including thrombosis, restenosis, and hypertension. Inhaled NO (iNO) has been shown to redirect blood flow from better aerated air spaces within the body to poorly ventilated areas to improve oxygenation. [50] The recommended initial iNO concentration for these treatments is 20 ppm as higher concentrations displayed no increase in the efficacy of the treatments but have been associated with a higher occurrence of methemoglobeinemia and nitrogen dioxide formation. Consequently, the monitoring and control of iNO concentration is of great importance for effective, safe treatments of cardiovascular diseases. In this detection system, the AIEgen 9 was reacted with 1-azidohexane in the presence of copper(II) acetacte and NO, which generated the necessary Cu I species from copper(II) acetate enabling the CuAAC reaction. The resultant triazole has greater steric hinderance causing an increase in fluorescent emission, which corre-sponded with the concentration of NO therefore enabling the detection of NO. The selectivity of the system was demonstrated using the gases NO 2 , CO 2 , CO, and SO 2 which were separately passed through the reaction media and gave no or only minimal changes in the emission in comparison to NO. A LoD was determined to be 15 ppm and the linearity range between 20 and 80 ppm suggesting that the probe could be useful to control the use of iNO.
In addition to ABS, the CuAAC reaction is an attractive approach for the construction of AIEgen probes due to the high yielding and orthogonal nature of the reaction. Furthermore, the triazole linkage offers several advantages including metabolic and chemical stability. Tang, Liu, and coworkers used the CuAAC reaction for the construction of the AIEgen oligonucleotide conjugate, TPE-DNA P (10) for the specific complementary DNA detection ( Figure 4B). [51] TPE-DNA P was synthesized via a CuAAC reaction between an azide functionalized TPE molecule and an oligonucleotide sequence with an alkyne moiety incorporated into the 5′ end of the sequence (5′-alkyne-AGC ACC CAC ATA GTC AAG AT-3′) in an 80% yield. Following photophysical characterization, TPE-DNA P was found to be only very weakly fluoresecne in aqueous media which was proposed to be due to the negatively charged phosphate backbone of the oligonucleotide sequenced which endowed the AIEgen conjugate 10 with excellent water solubility. However, once hybridization of TPE-DNA P with the complementary single stranded DNA sequence (DNA T ) had occurred, a distinct fluorescence "turn on" event was observed which exhibited three-fold higher brightness than nonhybridized TPE-DNA P (10). It was proposed the double stranded helix structure of the hybridized TPE-DNA P limited the free rotation of the AIEgen conjugate (10) thus inducing the observed fluorescence enhancement and enabling the detection of a specific DNA sequence. Further investigations were also performed to showcase the potential of the strategy to detect one-base and two-base mismatch sequences for the identification of single nucleotide polymorphisms. Two oligonucleotide sequences with either a one-base or two-base mismatch, termed DNA 1 and DNA 2 respectively were incubated with TPE-DNA p (10), and the resultant hybridized products displayed a weaker fluorescence emission in comparison to the perfectly matched sequence. While this approach offers a promising approach for detection of nucleic acid hybridization with the potential to discriminate completely complementary strands from those with single mutation points, with only a three-fold fluorescence emission increase upon hybridization further improvement for specific and sensitive detection was required. Accordingly, Tang, Liu, and coworkers synthesized a second AIEgen triazole conjugate functionalized with two oligonucleotides (AIE-2DNA, (11)), whose structure is shown in Figure 6, using the CuAAC reaction from a bisazide functionalized TPE (TPE-2N 3 ) and an excess of the same alkyne modified oligonucleotide used in the previous study with DNA P (10) in an 85% yield. [52] Consistent with first design, AIE-2DNA (11) processed excellent water solubility and therefore was only weakly fluorescent in aqueous media. However, in this study, the researchers reported that by conjugating two oligonucleotide chains to the TPE molecule an improved six-fold enhancement in fluorescence emission was observed following hybridization with the exact complementary oligonucleotides. Furthermore, TPE-2DNA (11) was found to be able to discriminate a one base pair mutation showing an improved selectivity to the target sequence than TPE-DNA P (10). Due the fidelity and reliable nature of the CuAAC reaction, the construction of the AIEgen conjugates (10 and 11) offers a straightforward hybridization detection approach which is less demanding and lower cost than traditional fluorescence approaches such as FRET-pairs or dual-labeled molecular beacons. Additionally, the chemical stability of the triazole moiety meant TPE-DNA P (10) and TPE-2DNA (11) are tolerant of the high ionic strength required for optimal hybridization conditions.
In 2020, Liu and coworkers showcased the usefulness of the CuAAC for the construction of an AIEgen with photosensitizer properties, TPEPy-D-Ala (12) for real time imaging and photodynamic ablation of bacteria in a simplified one-step approach compared to the two-step ligation labeling process previously described above ( Figure 4B). [53] The researchers employed the CuAAC reaction to link the AIEgen, TPE-Py, with the metabolic precursor, D-alanine (D-Ala), which enabled TPEPy-D-Ala (12) to participate in peptidoglycan metabolism and therefore be incorporated into the peptidoglycan of the bacterial cell envelope. The design of TPEPy-D-Ala (12) endowed the AIEgen with good water solubility due the presence of the hydrophilic pyridinium and D-Ala functionalities and therefore was only weakly emissive in aqueous media. However, once incorporated into the peptidoglycan layer of bacterial envelope, it was anticipated the intramolecular motion of TPEPy-D-Ala (12) would become inhibited resulting in a fluorescence "turn on" effect thus enabling the monitoring of bacteria with a low TBR. Furthermore, the donor-acceptor structure of TPEPy-D-Ala (12) meant that the AIEgen was likely to exhibit 1 O 2 generation capabilities and could therefore be applied for the photodynamic elimination of bacteria. This proposal was showcased through the incubation of TPEPy-D-Ala (12) with Methicillin-resistant Staphylococcus aureus (MRSA) cells, which were successfully labeled following an incubation time of only 20 min. It was also observed that the septal planes of the bacteria displayed the strongest fluorescence signals due to the ongoing peptidoglycan synthesis of the bacterial cell envelope. Furthermore, two additional Gram-positive bacterial strains, vancomycin-resistant enterococcus (VRE) strain Enterococcus faecium (Van A) and Bacillus subtilis (B. subtilis) were also successfully labeled with strong fluorescence signals of the septal plane observed. The photodynamic bacterial elimination potential of TPEPy-D-Ala (12) was then tested using MRSA. Following incubation with TPEPy-D-Ala (12), the MRSA cells were irradiated with white light which resulted in a significant decrease in the colony-forming unit of the MRSA cells compared to the colony kept in the dark, which indicated good photodynamic antibacterial activity in vitro. Furthermore, the MIC of TPEPy-D-Ala (12) toward MRSA was estimated to be 20 ± 0.5 μg mL −1 , which is significantly more efficient than the commonly used antibiotic, vancomycin. Cumulatively, this study demonstrated the potential of the CuAAC reaction to prelabel the desired AIEgen with a metabolic precursor thus bypassing the twostep bacterial structural envelope manipulation and the need for catalysts for ligation greatly simplifying the process.
In summary, the CuAAC reaction has been successfully utilized in the development of AIEgens for ABS for the detection of a wide variety of analytes and biological processes, including toxic metal ions, cancer and bacterial cells, and DNA synthesis. Furthermore, we have shown a selection of examples for the construction of novel AIEgens using the CuAAC reaction, which has been proven to be an effective approach due to the mild reaction conditions, tolerant of a wide range of functionality, and the orthogonal nature of the CuAAC reaction.

SPAAC
While the CuAAC reaction is a highly valuable approach in AIEgen ABS for a wide range of targets, the mandatory copper catalyst is toxic to both bacterial and mammalian cells, [54] thus precluding applications where the cells must remain viable. In 2004, Bertozzi and coworkers reported the copper free SPAAC reaction for the selective modification of biomolecules and living cells without apparent physiological harm. [27] The copper free variant exploits the strain release from the 163 • bond angle of the alkyne, which amounts to 18 kcal −1 of ring strain. [55] This destabilization of the ground state versus the transition state of the reaction provides a dramatic rate acceleration compared to unstrained alkyne meaning the cycloaddition can be performed at a lower temperature with no requirement for any catalysis. Similar to the application of the CuAAC reaction, the SPAAC reaction has been used in ABS through a strained alkyne modified AIEgen for bioconjugation with the target biomolecule functionalized with an azide moiety. Liu and coworkers employed the SPAAC reaction to advance the abovementioned research on the biorthogonal imaging and ablation cancer cells to an in vivo environment. [56] A "turn on" fluorescence AIEgen, bicyclo[6.1.0]nonyne (BCN)-TPET-triethylene glycol (TEG) (13), was designed and synthesized, comprising an AIE active core (TPET) linked a hydrophilic TEG chain and a  [56] (D) 2TPE-2T-BI (14). (E) Composition of DBCO-AIE dots. (F) Time dependent fluorescence imaging of tumor bearing mice with or without pretreatment of Ac4ManNAz followed by DBCO-AIE dots adminstration. Reproduced with permission: Copyright 2018, Wiley-VCH GmbH. [58] strained alkyne, BCN as shown in Figure 5A. The hydrophilic nature of TEG endowed the AIEgen 13 with excellent water dispersibility and therefore has a weak fluorescence emission giving low background signal. However, once the BCN moieties of BCN-TPET-TEG (13) underwent the SPAAC reaction with the with azide modified acetyl sialic acid (AzACSA) expressed on the tumor cells, the fluorescence of AIEgen 13 became significantly enhanced due to RIM ( Figure 5B). The in vivo tumour imaging ability of BCN-TPET-TEG (13) was examined compared to the "always on" commercial azide-reactive probe, dibenzocyclooctyne (DBCO)-Cy5, in 4T1-bearing mice. In the mice injected with BCN-TPET-TEG (13), the tumor sites could be differentiated from other organs within the body in just 30 min. In contrast, the DBCO-Cy5 injected mice displayed strong fluorescent signals across the whole body after 30 min due to the "always on" nature of the probe. It required approximately 24 h for tumor sites to become distinguishable once sufficient body clearance had occurred ( Figure 5C). The therapeutic efficacy of BCN-TPET-TEG (13) was also investigated. To determine potential of BCN-TPET-TEG (13) in image-guided photodynamic tumour therapy, tumour bearing mice were irradiated with white light (50 mW cm −2 ) for 10 min, 6 h after injection with AIEgen 13. Following evaluation, tumour growth was found to be greatly reduced in the BCN-TPET-TEG (13) click group in comparison to the BCN-TPET-TEG (13) only group, AzAcSA only group or phosphate buffered saline (PBS) group, which aligned well with the results from the in vitro experiments.
Tang and coworkers also applied the SPAAC bioconjugation strategy for the in vivo tumor imaging using AIE dots, which were reacted directly with metabolically labeled cancer cells. In comparison to traditional molecular dyes, AIE dots have many advantages including excellent brightness, large Stokes shift, and outstanding photostability. Furthermore, AIE dots provide excellent opportunities for clinical translation due to their improved biocompatibility with inorganic nanoprobes. [57] In this strategy, an AIEgen (2TPE-2T-BI, (14)) ( Figure 5D) with red/near-infrared (NIR) emission, due to the presence of a benzo[d]imidazole core, was encapsulated within DBCO modified lipid to create the DBCO-AIE dots which could readily react with an azide moiety in a biological environment using the SPAAC reaction ( Figure 7E). The DBCO-AIE dots exhibited red absorption and NIR emission and several advantages including a large Stokes shift of 138 nm, low excitation energy, limited selfquenching, and low autofluorescence. The DBCO-AIE dots were determined to be spherically shaped and approximately 85 nm in diameter using transmission electron microscopy imaging and dynamic light scattering measurements meaning they were suitable for in vivo blood circulation. The DBCO-AIE dots were then tested in an michigan cancer foundation-7 (MCF)-7 tumor bearing mouse model pretreated the unnatural monosaccharides, Ac4ManNAz, to give azide functionalized tumors. After DBCO-AIE dots were delivered systemically for 12 h, the fluorescence signal for Ac4ManNAz-treated mouse was located exclusively around the tumor sites. By contrast, the fluorescence in the control mouse with no Ac4ManNAz pretreatment was over the whole body, suggesting that the accumulated DBCO-AIE dots in tumor tissue was greatly increased in Ac4ManNAz treated mouse due to the SPAAC reaction ( Figure 5F). In comparison to short-wavelength emissive AIE dots, the NIR emissive DBCO-AIE dots displayed higher contrast and excellent TBR for in vivo imaging due to the lower autofluorescence from the body.
In this section, we have shown the SPAAC reaction can successfully circumvent the limitations imposed by CuAAC reaction by the need for the copper catalyst. A strained alkyne can be introduced into an AIEgen molecule for bioconjugation with the target biomolecule functionalized with an azide moiety, such as metabolically labeled cancer without the need for any metal catalysts. Despite the advantages of the SPACC reaction in the design of AIEgens for ABS, there are only examples available in the literature using this approach. Therefore, there is still opportunity for the further development of the SPAAC reaction in ABS.

Diels-Alder reactions
The Diels-Alder (DA) cycloaddition reaction has become one of the most powerful synthetic approaches to access sixmember rings due to its atom economical, stereoselective, and highly efficient nature. [59] The DA reaction describes a [4 + 2] cycloaddition reaction between an electron rich diene and an electron deficient dienophile was first discovered by Otto Diels and Kurt Alder who were awarded the Nobel Prize in appreciation of their discovery. The DA reaction was then identified as a click reaction by Sharpless as the reaction easily meets many of requirements of click chemistry including being high versatile, selective, atom economical, and efficient. Furthermore, many popular diene (e.g., furans, 1,3-cyclopentadiene) and dienophiles (e.g., maleimides) are readily available or can be easily accessed from reactive moieties. Despite its advantages, the DA reaction has previously been overlooked for the creation of novel AIEgens. In 2022, Hong and coworkers addressed this oversight and reported a novel AIEgen core, 1,3,4-triphenyl-1,4-dihydro-  (19). Reproduced with permission: Copyright 2022, Elsevier. [60] 1,4-epoxynaphthalene (ENAP), accessed via a catalyst free DA reaction. The library of ENAP AIEgens was constructed via a DA reaction between a selection of electron deficient alkynes (15) and the diene, 1,3-diphenylisobenzofuran (DPBF, (16)) in a simple, straightforward reaction procedure ( Figure 6A). [60] The use of DPBF (16), in addition to being a high reactive diene enabling the DA reaction to proceed efficiently, also endowed the ENAP products (17) with AIE characteristics due to the presence of two pendent phenyl rings. Following photophysical characterization, it was found that the ENAP cores displayed AIE features and their fluorescent properties could be fine-tuned through altering the substituents on the electron deficient alkynes and DPBF. For example, two of the ENAP cores, ENAP-CN (18) and ENAP-PyMeI (19) displayed a red shifted absorbance maxima of approximately 400 nm, which was attributed to the incorporation of strongly electron withdrawing group into the AIEgen cores ( Figure 6B). These spectrally redshifted AIEgens (18 and 19) were selected for investigations into their cell uptake and staining capabilities due to their favorable photophysical properties and to demonstrate the utility of the new ENAP AIEgen core. As shown in Figure 6C, the results showed that ENAP-CN (18) could successfully strain the cytoplasmic region of HeLa cells with only 30 min of staining time requirement and minimal cytotoxicity. Furthermore, ENAP-PyMeI (19) demonstrated an excellent capability to stain both Gram-positive and Gram-negative bacteria with no autofluorescence observed ( Figure 6D). These results demonstrate the potential of the DA reaction as a highly versatile, useful click reaction for the development of a novel AIE core.
To summarize, the first use of the DA reaction in the construction of a library novel AIEgens has been showcased. The researchers have exploited the advantages of the DA to enable the straightforward synthesis of the novel AIE core. Furthermore, a variety of applications for the ENAP cores were shown, which will most probably continue to grow due to the favorable photophysical properties of the AIEgens.

Inverse electron demand diels-alder reactions
Following the DA reaction, the inverse electron-demand diels-alder (iEDDA) of 1,2,4,5-tetrazines and strained dienophiles was later discovered and has since become increasing popular by virtue of its kinetics, excellent orthogonality and biocompatibility. [61] Initially applied for the synthesis of pyridazines, iEDDA was introduced as a new addition to click chemistry simultaneously by two research groups. [62] The chemoselectivity, substrate scope tolerability, biocompatibility, and the fast reaction kinetics of the the iEDDA means the reaction has become an established click chemistry approach in many fields, including in the design of ABS tetrazine fluorogenic probes. These probes are designed based on the utilization of tetrazine as a fluorescence quencher via FRET or a through-bond energy transfer (TBET). [63] Upon an iEDDA reaction, the tetrazine is converted to the corresponding pyridine, relieving the quenching effect resulting in a "turn on" fluorescence response.
In 2021, a tetrazine modified AIE fluorophore was reported for biorthogonal fluorogenic bioimaging using the iEDDA reaction by Kim et al. [64] The researchers found that incorporation of the tetrazine functionality into an indolinzine AIE core, [65] termed Kaleidalizine (KIz) gave a biorthogonal fluorogenic probe, KIz-tetrazine with synergistic fluorescence quenching effect from both the AIE phenomena and tetrazine substituent resulting in a low background signal and high turn on ratio ( Figure 7A). Through altering the electronic properties of the KIz core, a series of different colored KIz Tz were developed via the introduction of three different substitutes to the KIz core demonstrating the versatility of the KIz Tz system for multiplexing purposes. As expected, the three KIz Tz AIEgens (20)(21)(22) presented negligible emission without reacting the trans-cycloocetene (TCO) olefin in the solid state under 365 nm irradiation; however, they became highly emissive after reacting with TCO to give emission maxima of 470, 510, and 590 nm, with turn-on ratios of 46.7, 67.8, and 31.5-fold, respectively ( Figure 7B). Furthermore, an  (20, 21, and 22) and their corresponding solid films on glass substrate irradiated at 365 nm before and after treatment with trans-cycloocetene (TCO). Reproduced with permission: Copyright 2018, Elsevier. [64] (C) Triphenylphosphonium (TPP)-KIz Tz (23). (D) Images of overexpressing Mito-YFP Chang liver cells overexpressing following incubation with TPP-KIz Tz ; followed TPP-TCO addition. Reproduced with permission: Copyright 2018, Elsevier. [64] additional KIz Tz was developed for mitochondrial imaging, triphenylphosphonium (TPP)-conjugated KIz Tz (TPP-KIz Tz , (23), Figure 7C) and TPP-TCO were both incubated with Chang liver cells overexpressing Mito-YFP. As shown in Figure 7D, a negligible fluorescent signal was observed in cells incubated with TPP-KIzTz only. However, the fluorescent signal inside the mitochondria after TPP-TCO treatment was turned on and displayed a strong colocalization signal between the probe and fluorescent protein with an excellent TBR.
Recently, Tian and coworkers applied the iEDDA reaction in the design of a series of AIE-active naphthalimide tetrazine AIEgens (NP-Tz, (24)) for protein and mitochondria imaging. [66] It was envisioned that the synergistic fluorescence quenching effect from the AIE phenomena and tetrazine mediated TBET quenching would minimize the background signal and improve the turn on efficiency ( Figure 8A). Furthermore, the biorthogonal activated fluorogenic property ensure these probes avoids the production "false positive" fluorescent signals arising from unspecific aggregation, giving superior signal fidelity. The researchers found that altering the π-bridge linker between naphthalimide and tetrazine enabled fine tuning of the emission from blue to red in addition to affecting the AIE characters. Following the synthesis photophysical characterization of 13 napthalimide tetrazine fluorogenic probes and their BCN conjugated adducts (NP-TzBCN, (25)), seven were selected for protein labeling with BCN-conjugated bovine serum albumin (BSA) (BSA-BCN). As shown in Figure 8B, multicolor protein labeling was observed, including blue, green, cyan, and yellow fluorescence demonstrating the usefulness of NP-Tz AIEgens as multicolor AIEgens. Finally, the cyan and yellow emitting NP-Tz AIEgens were selected for fluorescence imaging of mitochondria in live cells ( Figure 8C). After an hour incubation in the presence or absence of BCN-TPP that targets mitochondria, HeLa cells were treated with the chosen NP-Tz AIEgens and MitoTracker Red (MTR), a commercially available mitochondria staining dye, for another hour. The BCN-TPP/NP-Tz treated cells displayed excellent colocalization of the signal between MTR and naphthalimide with high TBR.
Through these two examples, we have demonstrated that the use of the iEDDA reaction in the design of AIEgens for ABS. The incorporation of the tetrazine substituent into an AIEgen creates a cooperative fluorescence quenching effect from both the AIE phenomena and tetrazine, which is relieved following the iEDDA reaction with the target analyte. This approach enables superior signal fidelity and gives high TBR, in addition to the click reaction advantages of orthogonality and fast reaction kinetics. Furthermore, like the SPAAC reaction, the iEDDA reaction requires no metal catalyst and therefore offers excellent biocompatibility.

MICHAEL ADDITION REACTIONS
Michael addition reactions represents a powerful reaction class for the addition of a selection of nucleophiles to electron deficient olefin to yield the corresponding Michael adduct. [67] This thermodynamically favored methodology to generate carbon-carbon and carbon-heteroatom (e.g., C-S, C-N, and C-O) in the Michael adduct product has been widely applied across many areas of chemical synthesis more than 125 years. [68] The Michael addition reaction represents a modular class of click reaction, which gives highly regiospecific and stereospecific adduct products. Furthermore, the Michael addition is a robust, simple to perform and reliable reaction  [66] therefore qualifying as a click reaction. The Michael addition reaction classes includes the carbon-Michael reactions, [69] oxa-Michael reactions, [70] aza-Michael reactions, [71] and the thiol-Michael reactions, [72] all of which have been applied across many scientific areas. For example, the aza-Michael addition has recently been applied for the catalyst free surface immobilization of native proteins. [73] In this study, the reaction between an activated alkyne functionalized surface and a selection of target biomolecules, such as BSA, antibodies and fluorescent dyes, were completed in short reaction times using mild reaction conditions, highlighting the potential of Michael addition reactions in biosensing and imaging. The application of the Michael addition reaction in ABS offers additional advantages in comparison to the click cycloaddition reaction classes. Unlike the CuAAC reaction, Michael addition reactions are able to proceed without a metal catalyst and utilize an abundance of available substrates, such as amines, phenols, and thiols thus eliminating the requirement for premodification with azide and alkyne functionalities that can be difficult and time consuming to install and may change the biomolecule's primary function. While both the SPAAC and iEDDA reactions are catalyst free, the SPAAC reaction is still restricted by the requirement for expensive reagents, time consuming optimizations, and complex preparation procedures and the iEDDA reaction requires premodification of biomolecule with the strained olefin.
In 2018, Tang and coworkers presented a strategy for metal free click bioconjugation to highly diverse biomolecules based on the nucleophilic Michael addition to activated alkynes. [74] In the proof-of-concept study, it was shown that the highly abundant native groups (e.g., -SH, -OH, and -NH 2 ) can react directly with activated alkynes with no need for a metal catalysis or any premodification of the target biomolecules. The AIEgens, triphenylamine (TPA)-alkyne (26) and TPE-alkyne (27), were synthesized incorporating an electron withdrawing carbonyl group activated alkyne, which were able to function as Michal acceptors for the fluorescence labeling of a selection of biomolecules such as polysaccharides, bacteria, and proteins. This strategy was further applied to whole cell mapping, and rapid discrimination and labeling of Gram-positive bacteria ( Figure 9A). This approach has many advantages such as extremely mild reaction conditions, 100% atom economy, and catalyst free and consequently offers promising method for rapid fluorescence labeling of several biotargets and biotarget-related processes in vitro and in vivo. Despite the many advantages, target-and site-specific fluorescence labeling remains a challenge with this approach due to the lack of selectivity between the common native amine, thiol, and hydroxy moieties. This strategy was then showcased in the labeling of silk fibres with activated alkyne functionalized AIEgens. [75] The remarkable flexibility, excellent biocompatibility, and controllable biodegradability of silk make it favorable as the structural material in tissue engineering. Additionally, silk can be functionalized to show advanced properties, including fluorescence which exhibit great advantages in functional bio-optical devices and bioscaffolds. Therefore, there is a need for high performance fluorescent dyes and efficient labeling procedures for silk functionalization. To address this need, Tang and coworkers synthesized five AIEgens (27)(28)(29)(30)(31), including TPE-alkyne, for the purpose of constructing chemical conjugated fluorescent silk with bright emission and great stability ( Figure 9B). As there are many primary amine groups in the lysine residues of silk proteins, they were employed for the straightforward conjugation via a 1,4-Michael addition to the activated alkynes of the AIEgens 27-31 by immersing silk fibres into AIEgen solutions at room temperature overnight. As shown in Figure 9C, the AIEgen-silk threads with uniform fluorescence covering the entire visible light region were obtained. Furthermore, red emissive MTPABP-functionalized silk was successfully applied for real-time and long-term cell tracking ( Figure 9D). MTPABP-functionalized silk fabrics showed large two-photon absorption and demonstrated great potential for deep-tissue imaging.
Michael addition reactions has been expansively applied in protein labeling through the design of thiol-specific AIEgens. [76] Cellular thiols, including cysteine (Cys), homocysteine (Hcy), and glutathione (GSH), are central in several physiological and pathological processes, and have important roles in the stability and solubility of proteins. Changes in biothiol levels have been associated with various pathologies such as neurologic disorders, hepatic and renal failure, and reduced HIV disease survival. [77][78][79] Tang and coworkers reported the first example of a thiol-specific AIEgen, TPE-MI (32) in 2010. AIEgen 32 was constructed through the functionalization of TPE with a maleimide (MI) group for detections of thiols through a Michael addition reaction. [80] TPE-MI (32) was nonfluorescent in both solution and the solid state. It was postulated that the fluorescence of TPE-MI (32) was most likely quenched due to the maleimide (MI) moiety quenching the TPE emission through the n-π electronic conjugation of the carbonyl and olefinic groups. However, following a Michael addition reaction between the MI functionality of AIEgen 32 and a thiol group, the AIE activity was restored as the n-π electronic conjugation is disrupted ( Figure 10A). Therefore TPE-MI (32) was able to be utilized in the selective detection of L-cysteine (Cys) in comparison to other amino acids using a TLC plate. Under 365 nm UV illumination, the Cys spot emitted a bright blue light in the presence of TPE-MI (32) while other nonthiol functionalized amino acid spots were non emissive. This approach for Cys was shown to be sensitive from 1 to 100 ng/mL. The biosensing method was shown to be a straightforward and convenient approach for the detection of free thiols in organic solvents and in the solid state. Hong, Hatters, and coworkers later used TPE-MI (32) to establish a strategy to differentiate and evaluate folded and unfolded proteins during the disruption of cellular protein homeostasis (proteostasis) via the detection of free thiols. [81] In many diseases, particularly neurodegeneration, proteostasis can become chronically imbalanced resulting in excessive protein aggregation. Cys residues are usually buried inside the folded proteion structure, but upon protein unfolding, buried free Cys residues can become surface-exposed and thus can be used as a proxy reporter for the levels of unfolded proteins. The researchers showed TPE-MI (32) could be used to report cysteine units in the solvent-exposed unfolding areas, but not the cysteine units buried in the folded region. The ability of AIEgen 32 to measure cellular unfolded protein load was first determined with a selection of model proteins beginning with three proteins unfolded with guanidine hydrochloride, all of which displayed far greater reactivity to TPE-MI (32) when unfolded with guanidine hydrochloride than when in their native state. Furthermore, the probe was used to report the unfolding of proteins in living cells, and to detect induced protein damage in malaria parasites.
A TPE-MI derivative, TPE-NMI (33), was later reported by Hong and coworkers as part of a follow up study. TPE-NMI (33) processed the same "turn on" effect and thiol  [75] specificity as TPE-MI (32) upon reacting with unfolded proteins both in vitro and in live cells. However, the AIEgen (33) also displayed enhanced water miscibility and redshifted photophysical properties, meaning TPE-NMI was compatible with 405-nm laser used in most confocal microscopes and flow cytometers ( Figure 10B). [82] TPE-NMI (33) was then further optimized to give a solvatochromic probe, termed NTPAN-MI (34). NTPAN-MI (34) was successfully developed to evaluate the polarity of the local environment surrounding unfolded proteins in cells. To achieve the environmental sensitivity, a phenyl ring of the TPE molecule was replacement with an electronegative cyano moiety resulting in a push-pull AIEgen. [83] Push-pull dyes, functionalized with both electron donor and acceptor moieties, experience excited-state charge transfer, which is stabilized through interactions with the dipoles presence in the specific solvent, resulting in emission redshift in more polar solvents. Unlike both TPE-MI and TPE-NMI (32 and 33), which only reacts with unfolded proteins in the cytoplasm, NTPAN-MI (34) offers the additional advantages for the direct visualization and quantification of unfolded proteins in the nucleus as well as cytoplasm. Through spectral phasor analysis, Hong and coworkers could map and quantify the polarity of the intracellular environment surrounding the labeled unfolded proteins in terms of dielectric constant (ɛ) in subcellular resolutions. The NTPAN-MI (34) labeled unfolded proteins experienced ɛ in a range of 22-32 in the hydrophobic environment of the endoplasmic reticulum, 32-36 at the interface of the endoplasmic reticulum and cytoplasm, and 36-45 inside the nucleus where the environment is more hydrophilic. The ability of NTPAN-MI (34) to detect potentially unfolded proteins in cellulo was assessed. Upon treatment with a range of proteostatic stressors, puromycin, tunicamycin, brefeldin A, and MG132, the environment of the labeled unfolded protein in the ER can be more hydrophobic or hydrophilic depending on the specific stressor, whereas in the nucleus, all stress conditions lead to a more hydrophilic environment. NTPAN-MI (34) was also utilized to show proteostasis collapse in cells upon influenza virus infection demonstrating that such a process disrupts the protein quality control of the host cells.
The latest addition to the TPE-MI fluorescence toolbox for labeling and tracking unfolded proteins in cells was reported in 2021, named MI-2,1,3-benzothiadiazole (BTD)-P (35). [84] Following the incorporation BTD, a strongly electron-withdrawing group, the TPE-based AIEgen MI-BTD-P (35) exhibited a red-shift in the absorption spectra in comparison to TPE-MI (32). The maximum absorption of MI-BTD-P (35) was shown to peak at 405 nm, making the AIEgen 35 highly compatible with the light source settings currently used for flow cytometry and confocal microscopy while retaining its AIE properties. In this study, an ER targeting hydrophilic peptide was also incorporated into the design of the AIEgen ( Figure 10B). Functionalized with the peptide, AIEgen 35 retains outstanding cell permeability and water solubility, in addition to possessing highly bright emission and low background noise up on the reaction with unfolded proteins in live cells. In vitro experiments with model proteins showed that the fluorescence of AIEgen 35 could only be turned on when reacted with the thiol group of free Cys residues on either unfolded or aggregated proteins. The researchers also demonstrated that MI-BTD-P (35) can successfully measure the unfolded protein load in cells that have been exposed to a variety of stress conditions including heat shock and the use of proteostatic stressors. Furthermore, using fluorescence lifetime imaging microscopy and phasor plots, in control and stressed cells stained by MI-BTD-P (35), it was proved possible to differentiate intracellular proteins at different folding states using fluorescence lifetime.
Distinguishing between different biothiols such as Cys, Hcy and GSH using ABS can be extremely difficult due to their similar reaction activities and structures. For example, Hcy is a Cys homologue containing only one additional methylene (CH 2 ) unit and consequently it can be very challenging to differentiate between them especially as most methods previously reported were performed in organic solvent dramatically limiting their application in biological systems. Abnormal levels of Hcy have been linked to several diseases including neural tube defects, Alzheimer's disease, and osteoporosis. The selective detection of Hcy is therefore crucial for early detection and treatment of those diseases. [85] Tang and coworkers reported a red-emissive hemicyanine dye (TPE-Cy, (36)) with AIE properties for the selective detec-tion of Hcy over Cys, GSH and other amino acids in weakly basic buffer solution ( Figure 10C). [86] Upon addition Hcy to AIEgen 36 in pH 8 buffer, the red emission was suppressed to be replaced with a strong blue emission. In contrast to Hcy, when Cys and GSH was added to AIEgen 36, either a very weak blue emission, following the addition of Cys, or no change in emission, in case of GSH, was seen. Nuclear magnetic resonance (NMR) analysis suggested that the thiol group attacked the double bond of AIEgen 36 between the TPE unit and the cyanine unit via a 1,4-Michael addition reaction causing the ratiometric shift . The Hcy selectivity of TPE-Cy (36) was ascribed to the presence of the additional methylene unit present in Hcy resulting in reduced steric hinderance in comparison to the other biothiols facilitating the 1,4-Michael addition. In addition to the reactivity differences, as Hcy has increased hydrophobicity in comparison to Cys, and therefore the Michael adduct product of TPE-Cy (36) and Hcy would be more likely to aggregate in the aqueous detection media giving a greater fluorescent emission compared to the other biothiol Michael adducts. A novel α,β-unsaturated ketone functionalized TPE AIEgen, termed TPE-Py, (37) for the selective detection of Hcy was also reported by Li, Tang and coworkers ( Figure 10C). [87] The α,β-unsaturated ketone unit of AIEgen 37 was able to undergo a Michael addition reaction with biothiols, which caused in a disruption of molecular conjugation and consequently a fluorescence change. TPE-Py (37) displayed a strong yellow emission although, following the addition of Hcy, the yellow emission was replaced with blue. The researchers hypothesized that the observed fluorescence change arose from the 1,4-Michael addition of the biothiol to TPE-Py (37), which was confirmed by NMR and mass spectra analysis. Furthermore, the researchers showed only the addition of Hcy could trigger the ratiometric fluorescence shift as fluorescence quenching occurred following the addition of Cys or GSH to TPE-Py (37). Similar to TPE-Cy (36), the poor solubility of the Hcy-TPE-Py adduct product and subsequent propensity to form aggregated in the aqueous detection media in comparison the analogous Cys and GSH adducts was attributed to the Hcy selectivity of TPE-Py (37).
Tang and coworkers reported a series of malonitrilefunctionalized TPE derivatives, TPE-DCV (38), DMTPS-m-DCV (39), and DMTPS-p-DCV (40) for selective biothiol detection ( Figure 10C). [88,89] Firstly, the AIEgen, TPE-DCV (38), was shown to be able to selectivity detect GSH over Cys, Hcy and other amino acids via a Michael addition reaction. The researchers proposed the ability of GSH to initiate a fluorescence response from TPE-DCV (38) was due to the varied solubility of the corresponding Michael adducts as the higher hydrophilicity of the GSH-TPE-DCV adduct product in comparison to other biothiol adducts would result in decreased solubility in the aqueous detection mixture leading to the formation of aggregates giving rise to a fluorescence response. The GSH selectivity of TPE-DCV (38) was exploited for the real time fluorescence monitoring of glutathione reductase (GR) activity, an enzyme that cleaves glutathione disulfide (GSSG) to GSH in the regulation of the redox balance. Both GSSG or GR are enable to induce the "turn on" fluorescence response of TPE-DCV (38), the fluorescence response of TPE-DCV (38) could result only from GSH generated from the cleavage of GSSG by GR. The malononitrile-containing silole derivative, DMTPS-p-DCV (40) was shown to be able to differentiate GSH from Cys and Hcy via distinctive differences in the reaction kinetics. Through modifying the malononitrile functionality to the meta position, DMTPS-m-DCV (39) showed high selectivity to Cys with a detection limit as low as 0.5 µM. The underlying mechanism can be attributed to their molecular structures and the solubility of the AIEgen Michael adducts.
An AIE and ESIPT based kinetically fluorophore, termed AIE-S (41), was designed by Cui et al., which could distinguish between the thiols Cys, GSH, Hcy, and Na 2 S. [90] The design of AIE-S (41) was based upon the "AIE + ESIPT" fluorophore salicylaldazine functionalized with an acrylate moiety, which serves simultaneously as both the thiol recognition moiety and fluorescence quencher. When the hydroxyl group in AIEgen 41 was protected, the ESIPT from the hydroxyl group (proton donor) to nitrogen (proton acceptor) was blocked and therefore AIE-S (41) displayed only a weak fluorescence emission. However, when AIE-S (41) was incubated with Cys at pH 7 for 30 min a strong green fluorescence signal with the maximum wavelength was observed. The observed increase in fluorescence signal was attributed to a two-step reaction outlined in Figure 11A where the Cys molecule first reacted with the acryloyl functionality in a Michael addition reaction followed by a spontaneous intramolecular cyclization to give the salicylazdazine AIEgen 42 restoring the ESIPT process and consequently induc-ing a strong fluorescence signal. This proposed reaction mechanism was also confirmed by 1 H NMR, 13 C NMR, and high-resolution mass spectrometry (HRMS). Next, timedependent fluorescence response of AIE-S (41) to Hcy, GSH, and Na 2 S were also measured in comparison to Cys to investigate the ability of AIEgen 41 to detect other biothiols. The results showed strong fluorescence emissions after 40 min for both Cys and GSH and similar intensities were detected for Cys, GSH and Hcy after 4 h. However, no significant change in fluorescence of AIE-S (41) was observed following Na 2 S as it is unable to induce the intramolecular cyclization, and thus the ESIPT effect is not restored. It was proposed by the researchers that the differences in the reaction kinetics between Cys and Hcy with AIE-S (41) was due to the rate of the Michael adduct formation and subsequent intramolecular cyclization. In the case of the intramolecular cyclization of Cys Michael adduct, the seven-membered ring formation is kinetically favored over the eight-membered ring formation required in the Hcy addition reaction. Finally, AIE-S (41) was applied to the fluorescence imaging biothiols in living cells. Following the addition of Cys or GSH to HeLa cells pretreated with AIE-S (41), a strong green fluorescence was detected after 40 min. However, when Hcy was added no fluorescence signal was observed which agreed with the previous investigations in the reaction kinetics.
An additional AIE-ESIPT combination maleimideappended fluorophore, named ABTT-MA (43), for the highly selective discrimination of Cys over other biothiols was reported by Feng et al. [91] The researchers proposed the fluorescence signal of ABTT-MA (43) was quenched prior to incubation with biothiols due to d-PET from the fluorophore to the maleimide moiety. However, following Cys addition to AIEgen 43, via a Michael addition reaction, generated a 500-fold enhancement in fluorescent emission at 502 nm with stokes shift greater than 200 nm ( Figure 11B). Meanwhile, the addition of Hcy resulted in a 61-fold fluorescence enhancement while the addition of GSH triggered a weak fluorescence peak at 475 nm. In order to gain mechanistic insight into the selectivity of ABTT-MA (43) for Cys, a number of mechanistic investigations were performed including NMR, and HRMS analysis, and density functional theory (DFT) and time-dependent DFT calculations. Following these investigations, the initial weak fluorescence peak at 475 nm observed was attributed to the Michael addition to afford the corresponding thio-adduct which undergoes an intramolecular S,N-rearrangement to give amino-adducts which exhibited a strong fluorescence emission accompanied by a bathochromic shift. In the case of Cys, the S,N-rearrangement from the thio-to amino-adduct is thermodynamically favorable due to the geometric arrangement between the amino group of Cys with thioether, which forms a five-membered cyclic transition state. However, the rearrangement of the Hcy Michael adduct was slow in comparison to Cys by the additional carbon-chain extension resulting in a weaker fluorescence signal. In the case of GSH, the S,N-rearrangement would require the formation of a highly unfavorable 10-membered cyclic transition state. Finally, given the specificity toward Cys, the researchers investigated the practical potential of AIEgen 43 for Cys in a paper-based point-of-care test. Upon dropping Cys solution of different concentrations between 1-20 µM, the test papers emitted bright fluorescence under UV light within  (44) to BSA. Reproduced with permission: Copyright 2019, American Chemical Society. [92] (E) Confocal images of HeLa cells stained with CPV-ESF (45). Reproduced with permission: Copyright 2022, CSIRO Publishing. [98] 15 min and correlated well with the concentration of the Cys solution.
Recently, Hong and coworkers reported the first use of β-arylethenesulfonyl fluoride functionalized AIEgens in ABS. [92] β-Arylethenesulfonyl fluorides can function as bifunctional electrophiles due to the presence of both the electron deficient olefin and sulfonyl fluoride functionalities, which can be selectivity modified using Michael addition and SuFEx click reactions respectively. [93][94][95] The first reported β-arylethenesulfonyl fluoride-functionalized AIEgen termed HPC-ESF (44) for trypsin detection through the formation of a sensing complex with natural trypsin substrate, BSA, was described by the researchers. Trypsin is a highly important proteases produced by the pancreas which regulates pancreatic exocrine function and changes in trypsin activity have been associated with pancreatitis and pancreatic cancer resulting in dramatically increased levels of trypsin in urine. [96,97] Due to the incorporation of a hydroxyethylamino moiety, HPC-ESF (44) was endowed with excellent water solubility and consequently was nonemissive in aqueous solution. However, the fluorescence of HPC-ESF (44) can be switched on by the induction of RIM. Furnished with a highly electrophilic double bond, HPC-ESF (44) can conjugate to BSA, a natural trypsin substrate, through a 1,4-Michael addition reaction with amino acid possessing nucleophilic side chains such as lysine and cysteine ( Figure 11C). The resultant HPC-ESF:BSA complex was found to be highly fluorescent in the aqueous detection media until the BSA matrix was hydrolysed following the introduction of trypsin, resulting in a decrease in fluorescence which correlated with the concentration of trypsin therefore enabling trypsin detection. The sensing mechanism was demonstrated to be through a covalent reaction between BSA and HPC-ESF (44) using SDS-PAGE and a series of surrogate compounds (P1, P2, and P3) where the reactive recognition moieties were blocked ( Figure 11D), which offers several advantages for previous methods for trypsin detection. Moreover, during investigation into the sensing mechanism, it was found the sulfonyl fluoride group of HPC-ESF (44) can react with BSA via a click SuFEx reaction with lysine and tyrosine residues. HPC-ESF (44) can therefore react with several different amino acid side chains of BSA, enabling high dye loading of the HPC-ESF:BSA complex to achieve maximum fluorescence enhancement. The sensing capability of AIEgen 44 in real urine sample spiked with trypsin was also investigated. A linear relationship was established between fluorescence intensity and trypsin concentration between 0−100 μg/mL demonstrating the potential of HPC-ESF (44) for pancreatitis detection in a clinical setting (μg/mL range up to 84 μg/mL). Furthermore, it is highly significant as this was the first reported use of SuFEx click chemistry in ABS and demonstrates its potential for future application. Shortly after, Hong and coworkers reported a modified β-arylethenesulfonyl fluoride functionalized AIEgen, which functioned as a color-switch fluorescence probe (CPV-ESF, (45)) for the detection of BSA. [98] BSA is often applied as a HSA homologue in different fields as they share 76% amino acid sequence homology and due to its availability and lower cost. [99,100] Additionally, due to its stability and noninterference, BSA has been used in biochemical applications as a protein concentration standard, enzyme stabilizer, ion and protease detection etc. [101] Due to its wide range of applications, the determination of BSA concentration has received considerable research interest. By replacing the hydrophilic hydroxyethylamino group with an ethylamino group, CPV-ESF (45) remains strongly emissive in aqueous media and therefore is able to function as a color-switch fluorescent probe ( Figure 11A). Similar to HPC-ESF (44), AIEgen 45 can bind to BSA via a 1,4-Michael addition reaction with nucleophilic amino acid side chains. Following incubation with BSA, CPV-ESF (45) displayed a blue-shifted spectral profile which can be explained by the extension of the π-conjugate of the vinyl group and the contribution of the electron withdrawing sulfonyl fluoride of CPV-ESF (45), which was removed following conjugation to BSA resulting in a hypsochromic shift observed and thus enabling the detection of BSA. The LoD of the CPV-ESF (45) for BSA was calculated to be 7.26 mg/L, which would be suitable for applications like urinary albumin detection. Furthermore, good uptake of CPV-ESF (45) in cells observed in confocal laser scanning microscopy experiments highlighting the promise of CPV-ESF (45) as a chemical tag for future experiments in biological systems ( Figure 11E). Both these studies demonstrate the potential of β-arylethenesulfonyl in the design and application fluorescent sensors using a click chemistry covalent labeling strategy.
Of all the click reactions, the Michael addition reaction has been the most widely used in the design of AIEgens for ABS for the detection and labeling of a variety of analytes, such as biological thiols, enzymes, and bacteria. The use of the Michael addition reaction for ABS offers many advantages of the other classes of click reactions. For example, similar to the SPAAC and DA reactions, the Michael addition reaction requires no metal catalysts, and therefore offers superior biocompatibility. Furthermore, the Michael addition reaction exploits a wealth of natural substrates negating the requirement for premodification of the biological analyte, simplifying the detection process. However, target-and sitespecific fluorescence labeling can be challenging with this strategy due to the lack of selectivity between common native functionalities.

STAUDINGER LIGATION
The Staudinger ligation is a bioconjugation method reported by Bertozzi and coworkers who described a modification of the classical Staudinger reaction published over a hundred years ago in 1919. [102,103] In their studies on the metabolic engineering of cell surfaces, Bertozzi and coworkers identified the restrictions of the previously reported hydrazone ligation method, in which the formation of conjugates between a hydrazine probe and a ketone-modified sample could be unreliable owing to competition reactions with endogenous keto-metabolites. [104] The search for a milder reaction of two truly biorthogonal functionalities led the researchers to the Staudinger ligation. In Staudinger ligation a triaryl phosphine reagent functionalized with an ester in the ortho position to the phosphine is employed as an electrophilic trap to react with the phospha-aza ylide in order to cleave the ester and form an amide bond releasing the corresponding alcohol. [105] The reaction has come to be regarded as one of the most important click chemistry techniques, as it combines the advantages of biorthogonality and selectivity, while being simultaneously rapid and high yielding. It has been used for a wide variety of applications including peptide or protein synthesis, cell surface engineering, and labeling with dyes. [106] Wang, Tang, and coworkers employed the Staudinger ligation to developed a ratiometric AIEgen (TCFPB-HNO, (46)) for nitroxyl (HNO) detection and real time fluorescence imaging in vitro and in vivo which could be easily synthesized in two step process ( Figure 12A). [107] HNO has been reported to serve several critical roles in biological functions and pharmacological properties, including the regulation of thiol-containing enzymes such as glyceraldehyde-3-phosphate dehydrogenase and aldehyde  (46) in live mice (top) control group (first row) and experimental group (second row). Copyright 2020, Royal Society of Chemistry. [107] dehydrogenase. [108,109] Furthermore, HNO can be used as a vasodilator and positive inotropic drug to treat heart failure and exhibits anti-tumor activity. [110] Due to the short lifetime and highly reactive nature of HNO, a rapid, selective, and sensitive method for HNO visualization was urgently needed. The researchers found that TCFPB-HNO (46) possessed weak ICT effect due to the presence of the weakly electron-donating ester group connected the luminescent and recognition moieties. Furthermore, following photophysical investigation, TCFPB-HNO (46) was found to be AIE active therefore reducing the occurrence of autofluorescence. The reaction between the AIEgen 46 and HNO generated the intermediate phospha-aza species immediately followed by a Staudinger ligation with the ester functionality yielding tricyanofuranyl iminosalicylaldehyde (47), which displayed strong ICT effects as shown in Figure 12A. As predicted, TCFPB-HNO (46) gave only a weak emission at 670 nm in PBS solution but, following incubation with HNO, TCFPB-HNO (46) showed a ratiometric shift (I 618 /I 670 ). The LoD was calculated to be 157.6 nM demonstrating the great potential of AIEgen 46 for quantitative and specific detection of HNO with excellent photostability. The proposed reaction mechanism was also confirmed using HRMS analysis. The probe could also be used for real-time monitoring of HNO in living cells. The unchanged cell viabilities indicated the excellent biocompatibility of AIEgen 46 for biological applications. MCF-7 cells pretreated with HNO were then incubated with the AIEgen for 30 min. The results displayed an enhanced emission intensity in the green channel with increasing HNO concentration with a high signal-to-noise ratio ( Figure 12B). To further investigated the potential of TCFPB-HNO (46), imaging of HNO in live mice was performed. As seen in Figure 12C, only a slight increase in fluorescence intensity was observed in mice treated with the TCFPB-HNO probe alone (A). In contrast, fluorescence signals in mice treated both the AIEgen and an HNO donor solution displayed an over 10-fold increase in fluorescence emission (B).
The research by Wang, Tang, and coworkers has highlighted the utility of the Staudinger ligation in the design of AIEgens for ABS. The researcher's strategy offered the selective and sensitive detection of HNO. Furthermore, this work opens up new avenues to access easy to handle AIEgens for real time monitoring in biological system.

CONCLUSION
In this review, the advancement of click chemistry in the design and application of ABS AIEgens and the construction of AIEgens has been summarized ( Table 1). The working principles of AIE, ABS, and click chemistry were introduced in addition to their advantages. A thorough summary of the application of specific click chemistry reactions in AIEgen ABS protocols was discussed. These AIEgens rely on selective click chemistry reactions to achieve specific detection and imaging of analytes of interest (e.g., toxic metal ions and gas, biothiols, and tumour cell imaging). We have shown that the biocompatible and orthogonal features of numerous click reactions make them perfectly suited to be used for the design of AIEgens for ABS through numerous examples. Furthermore, we have highlighted the advantages and potential of the use of click reactions beyond the CuAAC reaction. This review has demonstrated the considerable advancements made in the development of activity-based AIEgens using click chemistry. However, there are still many aspects to be considered for further development. Several click reactions remain under exploited in the development of AIEgens for ABS. For instance, to our knowledge, there is only one reported use of the Staudinger ligation in the development of AIEgens for ABS despite the advantages of AIEgens over conventional fluorophores and the truly orthogonal and biocompatible nature of the Staudinger ligation. Furthermore, the potential of next generation click chemistry, SuFEx, in the use of AIEgens for ABS protocols is yet to be exploited. The availability to proceed without a metal catalyst, the use of abundant native groups such as amine and phenols, and the range of available synthetic methodologies for sulfonyl fluoride installations demonstrates the potential benefits of application of SuFEx in this area. There are also limitations, which limit the clinical translation of activity-based AIEgens. These include: (1) the optimal emission wavelengths of bioimaging materials are in the NIR range due to requirements for deep tissue penetration and low autofluorescence and therefore the development NIR activity-based AIEgens should be further investigated; and (2) target-and site-specific fluorescence labeling without the requirement for premodification of the biological target requires further improvement. Through this review, we hope to inspire the implementation a wide range of click chemistry reaction in the development of novel ABS techniques using AIEgens. TA B L E 1 Summary of the application of click chemistry reaction in design of ABS AIEgens.

Click chemistry reaction Application
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C O N F L I C T O F I N T E R E S T S TAT E M E N T
The authors declare no conflict of interests.