Matthew effect: General design strategy of ultra‐fluorogenic nanoprobes with amplified dark–bright states in aggregates

Fluorescence imaging, a key technique in biological research, frequently utilizes fluorogenic probes for precise imaging in living systems. Tetrazine is an effective emission quencher in fluorogenic probe designs, which can be selectively damaged upon bioorthogonal click reactions, leading to considerable emission enhancement. Despite significant efforts to increase the emission enhancement ratio (IAC/IBC) of tetrazine‐functionalized fluorogenic probes, the influence of molecular aggregation on the emission properties has been largely overlooked in these probe designs. In this study, we reveal that an ultrahigh IAC/IBC can be realized in the aggregate system when tetrazine is paired with aggregation‐induced emission (AIE) luminogens. Tetrazine amplifies its quenching efficiency upon aggregation and drastically reduce background emissions. Subsequent click reactions damage tetrazine and trigger significant AIE, leading to considerably enhanced IAC/IBC. We further showcase the capability of these ultra‐fluorogenic systems in selective imaging of multiple organelles in living cells. We term this unique fluorogenicity of AIE luminogen‐quencher complexes with amplified dark‐bright states as “Matthew effect” in aggregate emission, potentially providing a universal approach to attain ultrahigh IAC/IBC in diverse fluorogenic systems.


INTRODUCTION
In recent decades, fluorescence imaging has emerged as a pivotal technique in life science for directly visualizing dynamic living processes.Given the crowded biological environments, achieving precise imaging with a high signal-to-noise ratio is of utmost importance.Therefore, significant efforts have been devoted to developing practical methodologies to obtain bright and high-resolution images.These strategies span from innovations in microscope design [1][2][3][4][5][6] and imaging system optimization [7] to intricate molecular-level [2,[8][9][10][11] and nano-interface [12,13] engineering of fluorescent probes.[20][21] For example, by linking an emission quencher to a fluorophore, the resulting fluorophore-quencher complex exhibits negligible emission.When the quencher is removed in response to specific biological events, the fluorophore's emission is restored, resulting in turn-on emission. [21,22]This characteristic defines the fluorophore-quencher complex as a fluorogenic probe.Therefore, the identification of easily removable, strong emission quenchers is of utmost importance for enhancing the functionality of these fluorogenic probes. [20]etrazine, a benzene-like molecule with four embedded nitrogen atoms, has been identified as an ideal moiety with a tunable and strong fluorescence quenching property. [23]The quenched emissions of tetrazine-chromophore conjugates are fully recovered after the tetrazine quencher is controllably decomposed through photoconversion, [24] inverse electron demand Diels-Alder (iEDDA) reaction with strained alkene [25][26][27] and strained-alkyne, [28] or [4+1] cycloaddition with isonitriles. [29,30][33][34][35] Consequently, tetrazine-based fluorogenic dyes have been extensively developed, with significant efforts aiming at enhancing their emission enhancement ratio (I AC /I BC , where I AC and I BC are the photoluminescence intensities after and before click reactions) of the post-click product.To achieve this, amplifying the emission-quenching efficiency of the tetrazine unit is of utmost importance.For example, initial studies focused on Förster resonance energy transfer (FRET) [36,37] or through-bond energy transfer (TBET) [38,39] to direct n-π* emission quenching.Consequently, ultrahigh I AC /I BC (>1000-fold) was achieved in blue to green probes via connecting fluorophore and tetrazine in proximity distance. [38]Later on, red to near-infrared (NIR) turn-on emissions, which are challenging to attain through FRET or TBET-type quenching, [40] were realized by employing Dexter energy transfer (DET), [41] monochromic molecular designs or internal conversion (IC), [42][43][44][45][46] and photo-induced electron transfer (PeT) directed by sophisticated control of molecular conformations. [47]As a unique approach, molecular disaggregation upon iEDDA reaction was also applied to enhance the I AC /I BC of NIR cyanine dyes. [48]These tetrazine-based fluorogenic dyes have been successfully employed in bioorthogonal imaging of various cellular targets such as lipids, [49] sugars, [50] proteins, [51] and nucleic acids. [52]However, each design strategy requires tailored molecular designs, which have their limitations in generalizability.
Therefore, we are motivated to develop a general and simple strategy for enhancing tetrazine's quenching efficiency and achieving ultrahigh fluorogenicity.In this regard, we consider aggregation, a spontaneous phenomenon of hydrophobic molecules in aqueous systems, will be an effective solution to increase tetrazine-chromophore proximity and interactions, enhancing tetrazine quenching effects. [23,53]owever, most conventional planar fluorophores suffer from aggregation-caused quenching (ACQ) effect, [54][55][56] which diminishes post-click turn-on emission in aggregates, making it difficult to apply this aggregation strategy for fluorogenic probe designs.[63] However, they have not explicitly addressed the mechanism and relation between the aggregation and the high I AC /I BC .Supported by these results, we were motivated to investigate the effect of aggregations on the fluorogenicity of tetrazine-AIEgen complexes.
In this study, we demonstrated that by linking tetrazine to various AIEgens, the weakly emissive tetrazine-AIEgen complex further suppresses their emission upon aggregation.Following an iEDDA reaction, the resulting emissive pyridazine compounds enhance their emission upon aggregation, affording ultrahigh I AC /I BC .Further investigation concludes that this effect arises from the synergistic interplay between the intermolecular quenching effect of the tetrazine moiety and the AIE property of fluorogens.We also showcased that this ultra-fluorogenic system is applicable for site-selective fluorogenic imaging of multiple organelles in living cells, highlighting its potential for advanced imaging applications in biological systems.Drawing inspiration from the sociological term "Matthew effect" [64] -describing the phenomenon that the rich become richer, and the poor become poorer-we coined this unique fluorogenicity as the "Matthew effect" in aggregate emission, highlighting intensified darkness of quenched species and amplified brightness of emissive species in the aggregate state.This "Matthew effect" potentially offers a universal strategy for achieving ultra-fluorogenic nanoaggregate systems, which are simply achieved by AIEgen-quencher conjugations.

Initial design and synthesis
To investigate how aggregation and AIEgens affect the fluorogenicity of tetrazine probes, we designed and synthesized a series of tetrazine-dye conjugates.Tetrazine units were synthesized under Pinner synthesis with a thiol catalyst, [65] and sequential Pd-catalyzed coupling reactions connected tetrazine to chromophores.The obtained compounds were recrystallized whenever possible, and purity was confirmed by high-performance liquid chromatography to avoid any impurity effects (shown in Figures S81-S90).Their molecular structures were thoroughly characterized by nuclear magnetic resonance spectroscopy and high-resolution mass spectrometry (shown in Figures S25-S56).Single crystals were obtained except for MTPA-Tz, and X-ray diffraction (XRD) analysis supports the structure [66] (Figures S20-S23).We prepared four AIEgen-tetrazine conjugates (AIE-Tz) and one normal planar fluorophore: pyrene-tetrazine conjugate (PR-Tz) as a negative control for the entire experiment.AIEgens were designed by including well-known AIE-triggering units-tetraphenyl ethylene (TPE) or triaryl amines (TPA)in their chromophores. [67]To obtain a general perspective of fluorogenicity with diverse emission wavelengths, TPAbased chromophores were connected with electron donors: para-substituted methoxy groups (M), and with electron acceptors: pyridine and benzo[c][1,2,5]thiadiazole (B), expecting bathochromic shifted emissions from donoracceptor charge transfer effect. [68]The obtained AIE-Tzs are named TPE-Tz, TPA-Tz, MTPA-Tz, and MBTPA-Tz, respectively.All molecules reacted quantitatively with the strained alkyne compound (1R,8S,9s)-bicyclo[6.1.0]non-4yn-9-ylmethanol(BCN-OH), providing emissive pyridazine compounds named PR-Pz, TPA-Pz, TPE-Pz, MTPA-Pz, and MBTPA-Pz (Figure 1A).The obtained AIEgen-pyridazine conjugates (AIE-Pz) covered a broad emission range from violet to NIR (380-780 nm) in water (Figure 1B) with an emission maximum at 419, 481, 538, and 636 nm for TPA-Pz, TPE-Pz, MTPA-Pz, and MBTPA-Pz, respectively.Trans-cyclooctene (TCO) is another well-utilized click reaction partner of tetrazine, known for its excellent selectivity and reactivity.However, the products of the TCO-tetrazine click reaction are a mixture of dihydropyridazine tautomers, one of which retains emission quenching properties. [46,69,70]oreover, these dihydropyridazines spontaneously undergo a rapid, yet not instant, oxidation process to form pyridazine groups. [45,69]Due to the difficulty in obtaining uniform mixtures and maintaining consistent fluorogenicity after TCO-tetrazine reactions, we have decided to focus solely on BCN-OH as the reaction partner for the tetrazine group.

Photoluminescence measurements of AIE-Tz
After synthesizing AIE-Tz, we first investigated their emission properties in dilute solutions in dimethyl sulfoxide (DMSO).Not surprisingly, both AIE-Tzs and PR-Tz only exhibited very weak emissions in dilute solutions (quantum yield < 0.002, shown in Table S1).This could be due to two reasons: first, AIEgens do not show strong emission in solution because of their molecular motions, [71] and second, the tetrazine unit acts as a quenching unit.However, we still observed non-negligible emissions from AIEgens or pyrene moieties that could cause background emission problems during fluorescence imaging.
Next, we investigated their emission properties in the aggregate state in water.The aggregate formation upon water addition was confirmed with dynamic laser scattering measurements (Figure S5).Interestingly, not only PR-Tz, whose fluorophore is impacted by the ACQ effect, but also AIE-Tzs demonstrated further suppressed emission upon aggregation (Figure 2D), apart from the negligible emissions from the tetrazine moiety peaked at around 600 nm (Figure S2).Specifically, TPE-Tz solution exhibited very weak emission at 470 nm coming from the TPE core, [72] and this emission's intensity decreased upon aggregation (Figure 2A).TPA-based tetrazine molecules displayed slightly stronger emission than TPE-Tz in dilute solution, and the emission was also well-quenched upon water addition (Figure 2B and Figure S2).This more notable emission of TPA tetrazine adducts over TPE-Tz could be a consequence of the less flexible structure of TPA units compared to TPE units.Indeed, PR-Tz, which has the most rigid structure, showed the strongest emission among all tetrazine compounds in DMSO, and a significant emission decrease was observed upon water addition (Figure 2C).This phenomenon of AIE-Tzs suppressing their emissions upon aggregation effectively highlights tetrazine's potential to reduce the background signals from fluorogenic dyes, thereby contributing to ultrahigh I AC /I BC in the aggregate state.This intriguing observation further spurred our interest to explore this quenching effect more thoroughly.

Intermolecular quenching effect of AIE-Tz
To understand why AIE-Tz exhibits emission quenching in the aggregate state despite the incorporation of AIEgens, we further investigated the interaction between the tetrazine moiety and AIEgens in the aggregate state.Single crystal XRD analysis and molecular dynamics (MD) simulations provided aggregate models of MBTPA-Tz, and intermolecular centerto-center distances between the tetrazine and surrounding aryl rings were calculated.As expected, we observed proximal molecular interactions in the aggregate state: 3.6-4.7 Å from the XRD result and 3.9-4.9Å from the MD calculation (Figures S6-S10 and S24).The single crystal structure of MBTPA-Tz revealed a tetrazine-chromophore distance as close as 3.668 Å (Figure 3A).This distance is comparable to or even shorter than that of reported single molecular tetrazine fluorogenic dyes known to efficiently quench emissions through DET mechanism, suggesting the existence of molecular orbital interactions. [41]Therefore, this result indicates that tetrazine enhances its emission quenching efficiency upon aggregation via intermolecular interactions with AIEgens, while keeping its intramolecular quenching pathways as well.This intermolecular quenching effect of the tetrazine unit was experimentally confirmed using a commercially available AIEgen: 4-(1,2,2-triphenylvinyl)benzonitrile (TPE-CN) as a fluorescence indicator (Figure 3B).We measured the emission intensity of 10 µM TPE-CN, co-aggregated with TPE-Tz at different concentrations.Consequently, we observed that the emission of the aggregate was apparently quenched by 50.3% with 0.1 µM TPE-Tz addition; the emission intensity dropped 94.6% and even 99.7% with 1 and 10 µM TPE-Tz addition, respectively (Figure 3B).Considering there is no direct through-bond connection between TPE-CN and TPE-Tz, this quenching effect only arises from intermolecular interactions between TPE-Tz and TPE-CN directed by aggregation, highlighting tetrazine's enhanced quenching efficiency upon aggregation.

Photoluminescence measurements of AIE-Pz
Tetrazine quenchers were easily converted to pyridazine moieties by BCN-OH addition, and the aggregate emission of the obtained AIE-Pz was next investigated.Similar to AIE-Tz, AIE-Pz formed aggregates upon increasing water fraction (Figure S5).Because the tetrazine quencher was removed, AIE-Pz exhibited stronger emission both in dilute solutions and aggregates (Table S1).Most importantly, AIE-Pzs demonstrated enhanced emission upon aggregation, unlike AIE-Tzs (Figure 4D).Specifically, TPE-Pz and MBTPA-Pz displayed the most typical AIE properties (Figure 4A and Figure S3), with significantly increased emission upon water addition.TPA-Pz and MTPA-Pz exhibited twistedintramolecular charge transfer (TICT) [73] type AIEgen-like behavior: upon water addition, increased polarity caused charge separation in the excited state, initially quenching the emission.However, as the water fraction further increased, the emission turned on again, induced by AIE (Figure 4B and Figure S3).The PR-Pz negative control suffered from the ACQ effect and quenched its emission upon aggregation as expected (Figure 4C).In short, we achieved an emission enhancement system in AIE-Pzs aggregates.

Turn-on emissions of AIE-Tz upon "click" reaction
We successfully achieved a unique system in which nonemissive tetrazine molecules become even darker, and emissive pyridazine molecules become even brighter in aggregate states.Encouraged by this result, we calculated the I AC /I BC of the post-click product in the aggregate state to verify the impact of this aggregation-formation strategy on the I AC /I BC .The PR-Tz negative control demonstrated a very weak I AC /I BC around 20 in an aqueous environment due to the strong ACQ effect in water (Figure 5B).In contrast, TPE-Tz, possessing the most canonical AIEgen unit, displayed a significantly high I AC /I BC up to 4833 (Figure 5A,C).Even TPA derivatives, which act as less active AIE units, resulted in a high I AC /I BC (Figure S4  calculation proved that this was because TPAPz was affected by n-π* type quenching from the pyridazine unit and only showed weak emission (quantum yield < 0.02, Table S1 and Figure S16) even after the click reaction.
Multiple reports suggest a negative correlation between emission wavelength and I AC /I BC for tetrazine fluorogenic probes. [37,40,70]We plotted I AC /I BC values of previously reported tetrazine dyes against their emission wavelength to confirm this claim.As a result, the plotted data revealed a distinct and consistent trend, illustrating that as the emission wavelength increased, the I AC /I BC decreased.This visualization successfully substantiated the negative correlation between these two parameters.Most importantly, we observed that AIE-Tz demonstrated a very strong turnon property compared to previously reported molecules in each emission wavelength.Notably, the 4833-fold emission enhancement of TPE-Tz is one of the best I AC /I BC values among previous reports (Figure 5C and Table S2).In conclusion, we discovered that connecting AIEgens to tetrazine units is an effective and general strategy for achieving strong fluorogenic signals in aqueous systems.

Theoretical investigation of tetrazine quenching mechanism
To better understand the quenching mechanism and turn-on mechanism, DFT and time-dependent DFT calculations were carried out on MBTPA-Tz and MBTPA-Pz in single molecule and aggregate states.We focused on these molecules considering MBTPA-Pz's long emission wavelength with high I AC /I BC , which is ideal for biological applications.A comprehensive explanation of the assignment process for individual excited states can be found in Section 3 of Supporting Information.
Tetrazine moieties cause significant emission quenching through various mechanisms.In general, the primary quenching mechanism involves accessing the dark n-π* transition state in the tetrazine moiety.When the tetrazine moiety is conjugated with a chromophore, this access comes from the IC process, termed internal conversion to dark state (ICDS). [43]Alternatively, when access comes from energy transfer processes such as FRET [70] or DET [41] between tetrazine and fluorophore, the quenching process is summarized as energy transfer to dark state (ETDS). [37]dditionally, when tetrazine is located close to a fluorophore, the electron-deficient tetrazine unit can act as a strong acceptor, causing a non-radiative electron-transfer (ET) state through the PeT mechanism. [47]At the same time, AIEgens with highly twisted and rotational structures are well known to quench their emission through molecular motion. [71]As one of the molecular flexibility-triggering quenching pathways, we focused on the access to TICT [73,74] state, in which molecules are twisted to intramolecularly separate charge between electron-rich and electron-poor moieties that cause weaker emission.
When we investigated our system (Figure 6), not surprisingly, we observed two major non-radiative states under the emissive π-π* state in MBTPA-Tz molecular state: (1) the dark n-π* state caused by the tetrazine unit and (2) the TICT state caused by the AIEgen.Those states were confirmed as dark states by their negligible oscillator strength values (f < 0.01).For MBTPA-Tz, tetrazine is conjugated with AIEgen; therefore, the access to the dark n-π* state is from IC.We determined that emission quenching is due to both ICDS and TICT processes.Once MBTPA-Tz is aggregated, the dark state characteristics change.Restricted molecular configuration prohibits access to the TICT quenching state.Instead, we found a newly formed dark ET state whose hole and electron are separated in two molecules, accompanied by low-lying dark n-π* states induced by the tetrazine moieties.
This result matches the intermolecular quenching effect observed in AIE-Tz molecules: close interaction between tetrazine and AIEgen induces PeT quenching and provides intermolecular access to the n-π* dark state through energy transfer as well as maintaining IC pathways.In summary, MBTPA-Tz aggregate quenches emission based on ICDS, ETDS, and PeT processes.These multiple quenching pathways amplify the quenching effect of tetrazine and explain the experimental result of the aggregation-enhanced quenching effect of MBTPA-Tz (Figure 2).
Upon iEDDA reaction, the tetrazine moiety is damaged, and the tetrazine-triggered quenching states are henceforward eliminated.As a result, the pyridazine molecule exhibits different behavior.In the single-molecule state of MBTPA-Pz, the n-π* dark state is no longer observed, but the TICT state persists, resulting in weak fluorescence in dilute solution.However, when aggregation occurs, the TICT formation is restricted due to the limited free volume and hydrophobic environment in the solid state.Consequently, no quenching state exists beneath the emissive π-π* state, allowing for strong emission in the MBTPA-Pz aggregate.As a result, the emission of MBTPA-Pz is switched on as the molecular flexibility-triggering quenching pathways are restricted in the aggregate state.

"Matthew effect" in aggregate emission
In short, we successfully developed a system in which nonemissive tetrazine-containing molecules become even less emissive, and emissive pyridazine molecules become even more emissive in the aggregate state, resulting in ultrahigh I AC /I BC .We refer to this dark-bright states amplification effect in aggregates as the "Matthew effect" in aggregate emission (Figure 7), drawing from the sociological term that describes the phenomenon in which the poor become poorer and the rich become richer in academic society. [64]here exist a number of emission quenchers applied in fluorogenic probe designs other than tetrazine.[77] Considering that both energy and electron transfer quenching are stimulated by molecular proximity, and given the close molecular interactions observed in the aggregate environment of this study, this "Matthew effect" is expected to be demonstrated by a variety of AIEgen-quencher complexes, not limited to AIE-Tz.Therefore, we believe this report will establish a general design principle of highly fluorogenic nanoprobes, highlighting the advantage of utilizing aggregate systems in probe designs.

Bioorthogonal fluorogenic live cell imaging
Lastly, we applied the fluorogenic nanoaggregate for bioorthogonal fluorogenic imaging in living cells.First, MBTPA-Tz was added to cells, and confocal laser microscopy was used to observe the emission signals inside the cells.As expected, no emission was observed even after a 60 min incubation (Figure 8B).After washing the cells thoroughly, BCN-OH was added and incubated for 30 min, resulting in the observation of turn-on emission inside the cell without the washing procedure (Figure S18).This result confirms that the iEDDA reaction between MBTPA-Tz and BCN-OH can bioorthogonaly take place in congested living systems.
To utilize this bioorthogonal turn-on emission for more specific purposes, the hydroxy group of BCN-OH was functionalized with several side chains targeting specific organelles: a triphenylphosphine group (TPP) to target mitochondria, a 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE) unit to target the cell membrane, and a halotag ligand (Halo) to localize to the hydrophobic environment inside the cell.These compounds were designated as BCNTPP, BCNDPPE, and BCNHalo, correspondingly (Figure 8A).The biocompatibility of MBTPA-Tz and these bicyclononyne (BCN) compounds was confirmed by 3-(4,5-dimethylthiazole-2-yl)-2,5diphenyltetrazolium bromide (MTT) assay (Figure S17).By adding BCNTPP or BCNHalo to cells incubated with MBTPA-Tz, strong turn-on emission was observed at the mitochondria and lipid droplets, respectively.Here, excellent colocalization signals alongside commercially available organelle-staining dyes (MitoTracker Deep Red FM and BODIPY 493/503) were observed, with Pearson correlation coefficients up to 0.95 and 0.84 (Figure 8C,E), respectively.However, when BCNDPPE was added, no turn-on emission was observed at the cell membrane.This could likely be attributed to BCNDPPE being initially internalized into the cell and taking some time to be expressed on the cell surface.To address this problem, BCNDPPE was added first and allowed to incubate for 24 h, followed by a thorough washing of the cells and the subsequent addition of MBTPA-Tz.As a result, strong turn-on emission was observed at the cellular membrane (Figure 8D), which was further confirmed by colocalized signals using a commercially available membrane tracker (CellMask Plasma Membrane Stains) with a Pearson correlation coefficient of up to 0.93.
Consequently, MBTPA-Tz aggregates were proven to selectively target and visualize biological targets labeled with a strained alkyne group.To our best knowledge, this is the first example of tetrazine-functionalized AIEgen for imaging multiple cellular environments.[63] Therefore, this result highlights the flexible localization property of AIEgen-based nanoprobe, which expands the applications of this bioorthogonally activatable fluorogenic system.Furthermore, it is noteworthy that turn-on signals from MBTPA-Tz aggregates show better photo-bleaching stability than any commercially available bioprobes used in this study (Figure S19).These compelling findings provide substantial evidence that the MBTPA-Tz nanoprobe can effectively serve as a versatile biological imaging tool, as demonstrated by its exceptional multi-organelle targeting capabilities.
F I G U R E 7 Proposed working mechanism for "Matthew effect" in aggregate emission: upon aggregation, the "dark" AIE-Tz become even darker, while the "bright" AIE-Pz with their tetrazine moiety removed by click reactions become more emissive, leading to the observed high emission enhancement ratio of the post-click product (I AC /I BC up to 4833).α AIE , emission enhancement ratio upon aggregation; AIE, aggregation-induced emission; AIEgen, AIE luminogens; AIE-Pz, AIEgen-pyridazine conjugates; AIE-Tz, AIEgen-tetrazine conjugates.

CONCLUSION
In this study, we have demonstrated a general strategy to develop ultra-highly fluorogenic tetrazine probes.By connecting a tetrazine quencher to an AIEgen, we observed a unique feature where non-emissive tetrazine compounds further quench their emission upon aggregation, and emissive pyridazine compounds enhance their emission upon aggrega-tion.We termed this dark-bright states amplification effect in aggregates, which significantly enhances the I AC /I BC , as the "Matthew effect" in aggregate emission.
Based on the experimental and theoretical investigation, we concluded that this "Matthew effect" arises from the synergetic contributions of intermolecular quenching effects of the tetrazine moiety and AIE properties of AIEgens.
Lastly, we demonstrated that this highly fluorogenic aggregate can be applied for bioorthogonal fluorogenic imaging in living cells: by controlling the BCN location, we can observe turn-on signals in specific cellular environments such as mitochondria, cellular membranes, and lipid droplets.80] This report highlights the advantage of considering aggregate systems in probe designs. [17]Based on its mechanism, the "Matthew effect" is expected to be realized not only by tetrazine but also by other emission quenchers when they are connected to AIEgen.Therefore, this simple molecular design will stand as a general and powerful strategy for achieving ultrahigh emission enhancement in diverse fluorogenic systems, transcending single molecular properties through aggregation.

A C K N O W L E D G M E N T S
We would like to express our deepest gratitude to Prof. Terence Wong at The Hong Kong University of Science and Technology, Prof. Motonari Uesugi at Kyoto University, Dr. Hiromichi V. Miyagishi at Hokkaido University, Prof. Neal K. Devaraj at the University of California, San Diego, and Prof. K. Barry Sharpless and Dr. John Cappiello at Scripps Research for their invaluable guidance, comments, and insightful suggestions.The authors acknowledge the funding support from the Hong Kong PhD Fellowship Scheme (PF18-15484), the National Natural Science Foundation of China (21788102 and 22274106), the Research Grants Council of Hong Kong (16306620, 16303221, N_HKUST609/19, and C6014-20W), the Innovation and Technology Commission (ITC-CNERC14SC01), and the Shenzhen Science and Technology Innovation Committee (JCYJ20180507183832744).This work was partly supported by the JSPS KAKENHI (JP23H01977, JP23H04631) and the JST, the establishment of university fellowships toward the creation of science technology innovation (JPMJFS2132).

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 they have no conflicts of interest.

D ATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available in the Supporting Information of this article.

F I G U R E 2
Aggregation-enhanced quenching of chromophore-tetrazine (Ch-Tz) adducts.Photoluminescence (PL) spectra of (A) TPE-Tz, (B) MTPA-Tz, and (C) PR-Tz in the dimethyl sulfoxide/water mixtures with different fractions of water (f w ).(D) Plots of peak emission intensities (I) of Ch-Tz adducts against water fractions in the aqueous mixtures.It needs to be noted that since the compounds are all dark tetrazine species, the emission intensities here are significantly low.Therefore, some inevitable noise was also captured to obtain these minimum signals with this magnified view.Nevertheless, a clear trend of enhanced quenching upon aggregation was observed.
), as evidenced by MTPA-Tz (I AC /I BC = 2439) and MBTPA-Tz (I AC /I BC = 209).Only TPA-Tz did not exhibit strong turn-on emission (I AC /I BC = 93) among AIE-Tz.A density functional theory (DFT) F I G U R E 4 Aggregation-induced emission (AIE) of pyridazine adducts with AIE luminogens (AIEgens) and aggregation-caused quenching (ACQ) of a pyridazine adduct with an ACQ luminophore (ACQphore).Photoluminescence (PL) spectra of (A) TPE-Pz, (B) MTPA-Pz, and (C) PR-Pz in the dimethyl sulfoxide/water mixtures with different fractions of water (f w ).(D) Plots of peak emission intensities (I) of the pyridazine adducts with AIEgens or ACQphore against water fractions in the aqueous mixtures.

F I G U R E 5
Emission enhancement of post-inverse electron-demand Diels-Alder (iEDDA) click reaction products of AIEgen-tetrazine adducts in the aggregate state.(A) Photoluminescence (PL) spectra of TPE-Tz and TPE-Pz, with an emission enhancement ratio (I AC /I BC ) of the post-click product, where I AC and I BC are the PL intensities after and before click reactions.(B) I AC /I BC of chromophore-tetrazine adducts in the aggregate state after iEDDA click reaction.(C) Comparison of I AC /I BC between this and previous works.AIEgen, aggregation-induced emission luminogens.

F I G U R E 6
Schematic illustration of representative (de)excitation processes of MBTPA-Tz and MBTPA-Pz at molecular and aggregate states with hole/electron distributions obtained from quantum chemical calculations.The electron and hole are labeled in cyan and pink colors, respectively.d CT , charge transfer distance; ET, electron transfer state; f, oscillator strength; FC, Franck-Condon state; ICT, intramolecular charge transfer state; TICT, twisted intramolecular charge transfer state.