Functional Groups-Driven Molecular Design for Broad-Spectrum Clusteroluminescence in Scalable Monodisperse Polymer Microspheres

Functional Groups-Driven Molecular Design for Broad-Spectrum Clusteroluminescence in Scalable Monodisperse Polymer Microspheres | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL Aggregate This is a preprint and has not been peer reviewed. Data may be preliminary. 2 September 2025 V1 Latest version Share on Functional Groups-Driven Molecular Design for Broad-Spectrum Clusteroluminescence in Scalable Monodisperse Polymer Microspheres Authors : Liang He , Xueyan Nan , Tong Wang , Zhizhou Liu , Ce Wang , Tongxu Gu , and Pengli Bai 0000-0003-1907-6825 [email protected] Authors Info & Affiliations https://doi.org/10.22541/au.175678069.97360779/v1 Published Aggregate Version of record Peer review timeline 285 views 185 downloads Contents Abstract Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract Tunable cluster luminescence (CL) in non-conjugated polymers is a longstanding challenge due to lacking efficient molecular design strategies. Herein, we report a generalizable, scalable method to fabricate monodisperse, color-tunable CL microspheres by doping cross-linked polystyrene with carboxyl-containing monomers. Aggregated chromophores in the confined network exhibit enhanced dual emission via through-space interactions (TSI) of carboxyl groups and conformational rigidity induced by hydrogen bonding/coordination, spanning 400–700 nm. Emission is precisely tuned by varying carboxyl monomer types/ratios, sulfonation time, and pH. The optimized SPSMMAs-30 (30% MMA) features uniform size and strong fluorescence across 13 flow cytometry channels (coefficient of variation <3%), meeting calibration requirements. Crucially, this enables the first kilogram-scale synthesis of CL calibration microspheres with consistent optical properties. Beyond calibration, it shows high sensitivity in tetracycline detection and promise for multicolor anti-counterfeiting. This simple, cost-effective approach expands CL’s material design space and application potential in biomedicine, diagnostics, and materials science. Functional Groups-Driven Molecular Design for Broad-Spectrum Clusteroluminescence in Scalable Monodisperse Polymer Microspheres Liang He a # , Xueyan Nan a # , Tong Wang a , Zhizhou Liu a , Ce Wang a , Tongxu Gu a *, Pengli Bai a * a Suzhou Institute of Biomedical Engineering and Technology, Chinese Academy of Sciences, Suzhou, Jiangsu 215163, PR China. # These authors contributed equally to this work. *Corresponding author: Pengli Bai, [email protected] Tongxu Gu, [email protected] Abstract Tunable cluster luminescence (CL) in non-conjugated polymer systems remains a longstanding challenge due to the absence of effective molecular design strategies for achieving efficient and broad-spectrum emission. Herein, a generalizable and scalable strategy to fabricate monodisperse, color-tunable CL microspheres by doping cross-linked polystyrene networks with carboxyl-containing monomers was reported. Chromophores were aggregated into a confined polymer network. Dual emission was strengthened by through-space interactions (TSI) of the carboxyl group and conformational rigidity induced via hydrogen bonding and coordination, spanning the visible spectrum (400 - 700 nm). The emission characteristics were precisely tuned by varying the types and ratios of carboxyl monomers, sulfonation duration, and pH conditions. Carboxyl monomers were employed to optimize emission wavelengths and improve luminescence efficiency by balancing polymer chain rigidity and flexibility, modulating intramolecular hydrogen bonds, and controlling TSI. The resulting sulfonated poly(divinylbenzene-styrene-methyl methacrylate) microspheres with 30% MMA content (SPSMMAs-30) exhibited uniform particle size and strong fluorescence across 13 standard flow cytometry channels, with fluorescence coefficient of variation values consistently below 3%, fulfilling requirements for routine flow cytometer calibration. Importantly, this study demonstrated the first successful kilogram-scale synthesis of CL calibration microspheres with consistent optical properties, highlighting the robustness and scalability of the developed method. Beyond calibration, SPSMMAs-30 also showed high sensitivity in tetracycline detection and exhibited promising performance in multicolor fluorescence-based anti-counterfeiting applications. Therefore, this work presents a simple, cost-effective, and broadly applicable approach for the fabrication of non-conjugated CL microspheres, significantly expanding the material design space and application potential of cluster luminescence in biomedicine, diagnostics, and materials science. Keywords: Spectrum-Tunable, Clusteroluminescent, Monodisperse Polymer Microspheres, Flow Optical Calibration. Introduction Multicolor organic luminescent materials have been extensively utilized in various fields, including biological imaging [1-3] , sensing [4, 5] , LED lighting [6, 7] , and anti-counterfeiting [8, 9] . Their luminescence performance, particularly the ability to achieve color-tunable emission, has been recognized as a key determinant of functionality. To achieve tunable emission across the visible spectrum (400 - 700 nm), various molecular design strategies have been developed. Traditionally, electron donor-acceptor groups were incorporated into π-conjugated systems to modulate the emission color [10-12] . However, π-conjugated materials often suffer from inherent drawbacks, including poor solubility, cytotoxicity, limited processability, and environmental risks [13, 14] . Moreover, their synthesis typically requires complex, multi-step procedures and expensive precursors, which severely restricts their scalability for practical applications. Recently, many non-conjugated fluorescent materials have been discovered, such as proteins [15] , cellulose [16] , polyacrylonitrile [17] , polyester [18] , and phenolic resin [19] . Despite the absence of π-conjugated structures, visible fluorescence was observed in the aggregated state of nonconventional chromophores (NCCs), a phenomenon known as cluster luminescence (CL) [20, 21] . Nonconjugated materials were identified as promising sustainable luminophores, demonstrating broad application potential due to their low cost, scalability, processability, and biocompatibility. A novel mechanism termed through-space interactions (TSI) was recently proposed to explain CL [22, 23] . According to TSI theory, the aggregation of electron-rich groups induces intermolecular electron delocalization, reducing band gaps and enabling visible emission. While TSI offers valuable insight into CL, the underlying photophysical principles and emissive structure-property relationships remain unclear. Consequently, the rational design of CL materials remains an open challenge. To date, three core strategies for regulating CL behavior have been identified [24, 25] : (1) Tuning NCCs type/concentration [9, 26, 27] : increasing the density of chromophore or incorporating diverse NCCs modulates electron cloud distribution, and enhances intermolecular interactions. These adjustments amplified spatial conjugation effects, leading to emission wavelength shifts. (2) Enhancing TSI-driven aggregation: introducing strong molecular interactions [28-30] , such as coordination bonds, hydrogen bonds, and ionic bonds, effectively promotes TSI, thereby improving emission efficiency. (3) Balancing structural rigidity-flexibility [31] : optimizing polymer chain mobility facilitates uniform cluster formation and enhances luminescence. Crosslinking increases chain rigidity [32-34] , suppresses non-radiative decay, strengthens TSI effects, and induces red-shift emission. However, lacking a unifying CL theory to guide luminophore design, most materials still exhibit blue-to green emissions (400 - 500 nm), with only a few cases achieving red or white light under specific conditions [6, 35-38] . The sulfonation degree of cross-linked sulfonated polystyrene microspheres has been reported to influence quantum yield (QY) and emission wavelength [39] . However, this strategy yields relatively weak emission intensities above 550 nm. In particular, flow cytometry assays revealed diminished fluorescence signals in long-wavelength detection channels (e.g., BV605, BV650, and BV786), which limited their practical application in flow calibration. Moreover, the single sulfonated CL structure displayed poor stability and accelerated decay within the red spectral region, further restricting its applicability. These limitations underscore the need to develop non-conjugated polymers with enhanced stability, broadened spectral coverage, and tunable emission. Nevertheless, achieving precise luminescence control through mechanism-driven molecular design remained a critical challenge in this research field. Polymethyl methacrylate (PMMA) has been widely used as a low-cost, non-conjugated polymer material [40, 41] . Accidentally, our group discovered that incorporating PMMA into cross-linked sulfonated polystyrene microspheres significantly enhanced emission at 580 nm and enabled broader wavelength tunability. To achieve multicolor CL, we systematically investigated the effects of carboxylic acid groups with varying rigidity, sulfonation duration, and protonation levels, enabling tunable emissions spanning blue, yellow-green, white, and orange-red. The resulting sulfonated poly(divinylbenzene-styrene-methyl methacrylate) microspheres (SPSMMAs) demonstrated remarkable monodispersity in particle size distribution and exhibited a broad dual-mode emission spectrum spanning from 400 to 700 nm. Flow cytometry analysis confirmed strong fluorescence signals across all 13 detection channels. Notably, SPSMMAs with 30% methyl methacrylate (SPSMMAs-30) exhibited comparable performance to commercial calibration beads in flow analysis, validating their applicability for routine instrument calibration. Importantly, large-scale preparation was achieved with a single-batch output exceeding 1 kg. Compared to traditional flow calibration beads, the non-conjugated polymer microspheres based on CL offer key advantages, including low production cost, scalable synthesis, and efficient large-scale fabrication. Furthermore, the SPSMMAs-30 demonstrated high sensitivity and specificity in tetracycline detection, along with anti-counterfeiting capabilities. This work established a generalizable strategy for the fabrication of monodisperse, carboxyl-doped, color-tunable CL polymer microspheres, leveraging advanced insights into CL mechanisms. Our findings pioneered the application of CL-based materials in fluidic optical calibration particles, significantly expanding the functional landscape of non-conjugated luminescent polymers. This study lays a solid technical foundation for the scalable integration of multicolor CL microspheres into biomedical research, diagnostics, and material science. Results and Discussion Synthesis of PSMMAs-30 and SPSMMAs-30 Figure 1. Design strategies and structure characterization of monodisperse CL microspheres. (A) Preparation of monodisperse CL microspheres and simplified copolymer molecular structure. SEM images of the PSMMAs-30 (B, C) and SPSMMAs-30 (D, E) microspheres. (F) Size distribution of PSMMAs-30 (CV = 1.84%) and SPSMMAs-30 (CV = 2.12%). EDS elemental mapping images of PSMMAs-30 (G) and SPSMMAs-30 (H), respectively. (I) FTIR spectra of PSMMAs-30 and SPSMMAs-30. (J) N 2 adsorption-desorption isotherms of the PSMMAs-30 and SPSMMAs-30. (K) Pore size distribution of the PSMMAs-30 and SPSMMAs-30. (L) XPS survey of the PSMMAs-30 and SPSMMAs-30. Uniform microspheres with micrometer-scale dimensions were typically difficult to obtain via direct polymerization of hydrophilic monomers such as sodium styrene sulfonate and methacrylic acid. To address this challenge, a formation-first, CL-activation strategy was proposed. In this approach, poly(divinylbenzene-styrene-methyl methacrylate) microspheres (PSMMAs) were first prepared via seed polymerization and subsequently treated with sulfuric acid to activate CL. The fabrication process of monodisperse CL microspheres was illustrated in Figure 1A. Specifically, polystyrene seed microspheres were fabricated using a previously established two-step dispersion polymerization method (Figure S1) [42] . These seeds were then uniformly swollen through a multi-step seeded swelling process, involving the addition of a swelling agent (dibutyl phthalate), initiator (benzoylperoxide), monomer (methacrylate (MMA) or styrene), and cross-linker (divinylbenzene). Following polymerization, monodisperse microspheres were obtained. The microspheres were immersed in sulfuric acid, where the formation of sulfonic acid and carboxyl groups triggers the emission of interesting CL. As shown in Figure 1B and C, the diameter of PSMMAs with 30% MMA (PSMMAs-30) was approximately 6.1 μM, and the microspheres formed a tightly packed array under scanning electron microscopy (SEM) observation, indicating excellent uniformity. After treatment with sulfuric acid, many cracks appeared on the surface of SPSMMAs-30 (Figure 1D and E), possibly due to the dissolution of uncross-linked polymers in sulfuric acid. Figure 1F presented the particle size distribution of PSMMAs-30 before and after sulfonation. The results showed that the microspheres maintained excellent monodispersity after being treated with sulfuric acid, with a CV of 2.12%. Figure 1G and H showed the elemental distribution of carbon, sulfur, and oxygen in PSMMAs-30 and SPSMMAs-30. A significant increase in sulfur content confirmed the successful sulfonation of PSMMAs-30. As shown in Figure 1I, the Fourier transform infrared spectroscopy (FTIR) spectra confirmed that -SO 3 H and -COOH groups were grafted onto PSMMAs-30. The FTIR spectrum of PSMMAs-30 exhibited clear absorption bands at 698, 757, 1455, and 1496 cm⁻¹, which were attributed to the benzene ring absorption of polystyrene. The absorption band at 1036 cm⁻¹ was due to the symmetric stretching of S═O, while the absorption bands at 614 and 1172 cm⁻¹ were associated with the asymmetric stretching of S═O. The broad peak at 3426 cm⁻¹ showed a significant increase, which was attributed to the vibration of hydroxyl of sulfonic acid groups and carboxyl groups. Additionally, the absorption band at 1750 cm⁻¹ was related to the carbonyl group of the ester group. Compared to PSMMAs-30, the absorption band of SPSMMAs-30 shifted to 1680 cm⁻¹ with a notable decrease in intensity, indicating that the ester group was successfully hydrolyzed into the carboxyl group. Further elemental analysis by X-ray photoelectron spectroscopy (XPS) (Figure 1L) revealed a new peak of S element (167.29 eV) in SPSMMA-30, confirming sulfur incorporation. The Brunauer-Emmett-Teller (BET) (Figure 1J and K) specific surface areas of PSMMAs-30 and SPSMMAs-30 were 8.27 and 31.93 m²·g⁻¹, respectively. This increase could be due to the dissolution of the uncross-linked polymer during sulfonation. The average pore diameter increased from 6.86 nm (PSMMAs-30) to 17.89 nm (SPSMMAs-30), likely attributed to the sulfonation process. These increases in specific surface areas and pore diameter were consistent with SEM results. When the polymer chains of PSMMAs-30 were sulfonated in sulfuric acid, they might become hydrophilic and fragile, facilitating the leakage of non-cross-linked polymer. The fragile organics swelled and stretched, further expanding the pore volume and specific surface area and resulting in an enlarged pore volume and specific surface area. Spectral Modulation Figure 2. Normalized UV–vis absorption, excitation, and PL spectra (1 mg/mL) of (A) SPSMMAs-0, (B) SPSVBs-30, (C) SPSMAs-30 and (D) SPSMMAs-30 in H 2 O. Inset: chemical structures of SPSMMAs-0, SPSVBs-30, SPSMAs-30 and SPSMMAs-30, respectively. Fluorescence spectra of (E) SPSMMAs-0, (F) SPSVBs-30, (G) SPSMAs-30, and (H) SPSMMAs-30 in H 2 O (1 mg/mL) under different excitation wavelengths. (I) CIE coordinates of SPSMMAs-30. (J) Concentration-dependent PL spectra of SPSMMAs-30 in H 2 O. (K) Plots of PL intensity versus concentration for SPSMMAs-30 in H 2 O. (L) Luminescent decay curve of SPSMMAs-30 in aqueous solution at 450 and 570 nm, respectively (λ ex = 365 nm). To elucidate the CL mechanism of carboxyl and sulfonic acid groups in polystyrene microspheres, four types of CL microspheres (sulfonated poly(divinylbenzene-styrene) microspheres (SPSMMAs-0), SPSMMAs-30, sulfonated poly(divinylbenzene-styrene-vinyl benzoate) microspheres (SPSVBs-30), sulfonated poly(divinylbenzene-styrene-methyl acrylate) microspheres (SPSMAs-30), and were synthesized in this study. Previous studies have shown that the sulfonic acid group in benzenesulfonic acid molecules could draw electrons from oxygen atoms through hybridization, forming lone electron pairs [43-45] . In aggregated chromophores (sulfonic acid and carboxyl groups), multiple interactions occurred, including C=O···C=O (n-π), S···C=O (n-π), O=C···C=O (π-π), and O···O short contacts. The spatial electron interactions, combined with the structural rigidity of the polymer matrix, result in strong fluorescence emission under UV irradiation. Notably, a novel π-π interaction between S=O and C=O groups emerged, which further strengthened electron transfer and structural stability. The photophysical properties of each sample were analyzed using UV-visible spectroscopy, excitation spectroscopy, and photoluminescence spectroscopy (Figure 2A-D). Carboxyl, benzoic acid, and benzenesulfonic acid groups generally absorbed ultraviolet light within 220-300 nm [46, 47] . Previous works have shown that bands above 300 nm come from n-π transitions in carbonyl and sulfur groups (S, S=O, or O=S=O), often appearing as weak peaks [44, 48, 49] . However, SPSMMAs-0 showed a strong absorption peak at 280 nm, likely coming from isolated benzenesulfonic acid groups. The broad absorption between 300-500 nm suggested that the sulfonic acid group’s aggregation boosted the n-π transition signal. Both SPSVBs-30 and SPSMAs-30 exhibited characteristic absorption peaks at 280 nm and 340 nm, likely arising from TSI-(n, π) between sulfonic acid and carbonyl groups. Of these, SPSMAs-30 showed markedly higher absorption at 340 nm compared to SPSVBs-30, indicating that the TSI interaction of SPSMAs-30 was stronger. Notably, the absorption peaks of SPSMAs-30 at 340 nm and 480 nm aligned closely with excitation peaks, indicating that sulfonic acid and carboxyl groups established dual luminescent centers via TSI-(n, π) interactions. This configuration enabled dual chemiluminescence emissions at 420 nm and 520 nm in aqueous media. It was worth noting that the 280 nm absorption peak of SPSMMAs-30 vanished and was replaced by strong peaks at 340 nm and 500 nm, aligning well with its excitation spectrum. This spectral shift implied further enhanced TSI-(n, π) interactions between sulfonic acid and carboxyl groups, leading to dual CL emissions at 450 nm and 580 nm in aqueous solution. The relationship between chain segment mobility and luminescence properties was established by measuring glass transition temperatures (Tg) and quantum yields (QY) (Table S2). For SPSMMAs-0, SPSMAs-30, SPSVBs-30, and SPSMMAs-30, Tg values were 98.05°C, 99.54°C, 81.21°C, and 85.36°C, respectively, while QYs were 3%, 2.36%, 3.34%, and 2.6%. The results revealed that excessive structural rigidity hindered the formation of large clusters, instead promoting small clusters or intrinsic luminescent systems, which led to short wavelength emission. In contrast, overly flexible structures improved QY but restricted cluster stability, size growth, and long-wavelength emission. Compared to polyacrylic acid, polymethacrylic acid exhibited higher rigidity, facilitating stable large cluster formation via TSI-(n, π) interactions and enabling long-wavelength emission. However, poly-4-vinylbenzoic acid was too rigid to form large clusters, severely limiting its long-wavelength emission. Nevertheless, excessive structural flexibility enhanced molecular chain mobility, reducing radiative transition efficiency, restricting cluster size and stability, and ultimately suppressing long-wavelength emissions. Most CL materials exhibited excitation-dependent emission (EDE) effect [50-51] . Due to the varying sizes and structures of clusters formed in CL systems, each cluster emitter had a different spatial environment and interacted differently with others. This meant that almost every cluster emitter was an independent emitting species within the system, having distinct excitation and emission wavelengths. To investigate this behavior, fluorescence spectra were recorded under different excitations (345 nm - 425 nm). As shown in Figure 2E and F, the optimal excitation wavelengths of SPSMMAs-0 and SPSVBs-30 were both around 345 nm. When the excitation wavelength gradually increased from 345 nm to 425 nm, the main emission peak showed a regular red shift, and with the increase of the excitation wavelength, its luminescence intensity gradually decayed, with an obvious EDE effect. Notably, within the 345-365 nm excitation range (Figure 2G), the secondary emission peak (420 nm) exhibited a clear red shift with decreasing intensity as excitation wavelength increased. Although the main emission peak remained relatively stable in position, its luminescence intensity increased during this phase. When the excitation wavelength reached 385 nm, the secondary peak persisted in shifting toward longer wavelengths but with substantially reduced intensity, whereas the main peak’s intensity started to decline. When the excitation wavelength reached 405-425 nm, the spectral features underwent significant changes. The secondary emission peak near 420 nm diminished progressively, while the main emission peak at 520 nm rose. This phenomenon resulted from enhanced material flexibility, which promoted more uniform cluster formation and enabled efficient CL with reduced excitation dependence. Figure 2H showed that the material exhibited a broad emission spectrum ranging from 400 to 700 nm. As the excitation wavelength increased from 345 nm to 365 nm, the primary emission peak demonstrated a red shift accompanied by enhanced intensity. Further increasing the excitation wavelength from 365 nm to 385 nm resulted in minimal spectral shift, while its intensity gradually decreased. Notably, when the excitation wavelength was extended from 405 nm to 425 nm, the dominant peak (initially at 570 nm) shifted to 600 nm; however, this red-shifted peak exhibited lower intensity compared to the secondary peak observed at 470 nm. These observations confirmed that all four materials (SPSMMAs-0, SPSMAs-30, SPSVBs-30, SPSMMAs-30) displayed typical EDE luminescence, consistent with previous reports on CL. The results strongly supported the coexistence of multiple emission centers in these materials. As shown in Figure 2I, the CIE coordinates of SPSMAAs-30 were (0.3274; 0.352), (0.3401; 0.3607), (0.3403; 0.3611), (0.323; 0.3497) and (0.3385; 0.3891), further illustrating the multicolor emission characteristics. The fluorescence lifetimes of SPSMMAs-30 at 450 nm and 570 nm were 1.92 ns and 1.45 ns, respectively (Figure 2J). This difference could be attributed to the long-distance electronic transition occurring at 570 nm. Typically, CL polymers exhibit concentration-dependent fluorescence behavior [52] , where higher polymer concentrations lead to more clusters and stronger fluorescence intensity. However, this change is positively nonlinear (in contrast to the linear relationship of traditional dyes). In this work, CL polymers were pre-crosslinked into microspheres. Due to the presence of crosslinkers, these microspheres were insoluble and retained their single-particle state even in organic solvents without dissociation or dissolution. When dispersed in solution, the relatively large spatial distance between microspheres made it difficult for them to approach each other as in conventional solutions and form effective cluster emission effects. Under such circumstances, the cluster luminescence effect was strictly confined within each polymer microsphere. In other words, polymer molecules could only aggregate into clusters and produce corresponding fluorescence emission within the local range of a single microsphere. As expected, as shown in Figure 2K and L, the fluorescence spectra of SPSMMAs-30 under different concentrations in aqueous solution were recorded, and their fluorescence emission intensities showed a good linear correlation with concentration. This result indicated that SPSMMAs-30 had good stability and were more reliable in practical applications. Figure 3. Fluorescence photos of SPSMMAs (0-80) with different MMA contents under UV light (A). Normalized emission spectra of SPSMMAs-0, SPSMMAs-10, SPSMMAs-20, and SPSMMAs-30 (B). Normalized emission spectra of SPSMMAs-30, SPSMMAs-40, SPSMMAs-50, SPSMMAs-60, SPSMMAs-70, and SPSMMAs-80 (C). Quantum yield (%) and Tg (°C) of SPSMMAs with different MMA contents (D). Fluorescence images (E) and spectra (F) of PSMMAs-30 in sulfuric acid at different reaction times. Fluorescence images (G) and spectra (H) of SPSMMAs-30 under varying pH conditions. Fluorescence image (I) and spectra (J) for SPSMAs-30 across different pH buffers. Fluorescence images (K) and spectra (L) of selected multicolor CL microspheres. The fluorescence images (left to right) correspond to: SPSMMAs-30 (5 min), SPSMAs-30 (Na + ), SPSMNAs-30 (Na + ), SPSMAs-30, SPSMMAs-30 (60 min), SPSMMAs-30 (180 min), and SPSMMAs-30. After PSMMAs was treated with sulfuric acid, a large number of carboxyl and sulfonic acid groups (non-conventional chromophores, NCCs) were generated inside the microspheres. The aggregation of NCCs and the formation of TSI led to CL. Both the type and interactions of NCCs affected the CL performance. To investigate the influence of carboxyl and sulfonic acid groups on CL, a series of polymer microspheres with different MMA contents were synthesized and treated with sulfuric acid. As expected, the fluorescence of SPSMMAs gradually changed with the MMA content. As shown in Figure 3A, when the MMA content was 0, the fluorescence of SPSMMAs-0 appeared light yellow. As the MMA content gradually increased, the color of the SPSMMAs aqueous solution gradually deepened. When the MMA content was 30%, the SPSMMAs-30 aqueous solution turned orange-red. Upon further increase in MMA content, the color of the SPSMMAs aqueous solution gradually became darker until almost no fluorescence was observed. The fluorescence characteristics of SPSMMAs with varying MMA compositions were investigated using emission spectra (Figure 3B and C). Excitation at 365 nm yielded an emission peak at 425 nm for SPSMMAs-0. As the MMA content incrementally increased from 0% to 30%, a red-shift in the blue region’s emission peak was observed, progressing from 425 nm to 470 nm. Notably, there was a concomitant enhancement in the orange region’s emission peak during this transition. At 30% MMA content, the intensity of the orange region’s emission peak surpassed that of the blue, transforming the primary blue emission into a shoulder peak and elevating the orange shoulder peak to prominence (Figure 3B). Beyond 30% MMA, the orange emission peak diminished, restoring the blue emission as the dominant emission peak (Figure 3C). The observed phenomenon was likely due to enhanced polymer chain flexibility and augmented TSI, which promoted cluster formation and consequently induced red-shifted emission. The optimal ratio of NCCs can influence both the quantity and dimensions of clusters formed. Furthermore, NCCs impact the rigidity and flexibility of polymer chains, where a balanced level of rigidity and flexibility is favorable for enhancing CL efficiency. [31] Fluorescence spectra of SPSMMAs (0-80) were measured across excitation wavelengths ranging from 345 to 425 nm. As presented in Figure S2, all samples consistently demonstrated prominent EDE behavior. Figure 3D illustrated the relationship between the QY and Tg of SPSMMAs with varying MMA contents. For SPSMMAs-0, lacking MMA, the QY was 3%. At 10% MMA content, the QY decreased to 0.67%, but it rose again to 2.6% when MMA content reached 30%. Further increments in MMA content led to a gradual reduction in QY. This behavior could be associated with spectral red-shifts and chain rigidity/flexibility. Spectral red shift indicated reduced emission energy level differences (ΔE↓), significantly lowering electron transition probabilities and suppressing radiative pathways. Insufficient cluster efficiency resulting from limited flexibility changes failed to compensate for energy losses associated with spectral red shift. Consequently, QY decreased as MMA content increased from 0% to 10%. However, enhanced flexibility promoted stronger TSI, supporting formation of larger clusters and improved QY. Notably, excessive flexibility inhibited large cluster formation while restricting molecular motion and non-radiative processes, ultimately limiting both quantum yields and long-wavelength emissions. With the increasement of content of MMA, the Tg of SPSMMAs decreased from 98.05 °C to 68.64 °C gradually (Figure 3D), indicating that the chain segment mobility from SPSMMAs-0 to SPSMMAs-80 increased continuously at room temperature, and the rigidity gradually decreased. Interestingly, in contrast to the conventional aggregation-induced emission (AIE) mechanism, the luminescence efficiency of SPSMMAs did not exhibit a direct positive correlation with its chain rigidity. Moderate molecular chain mobility played a pivotal role in enhancing the luminescence efficiency of SPSMMAs. In contrast, both excessively rigid and overly flexible molecular structures proved detrimental to the formation and stability of clusters within these materials. Such unbalanced rigidity or flexibility led to diminished luminescence efficiency and a shift towards shorter emission wavelengths, thereby underscoring the importance of achieving optimal molecular chain dynamics for superior optical performance. The fluorescence behavior of SPSMAAs-30 was systematically investigated under varying durations of sulfuric acid treatment. As shown in the emission spectra (Figure 3E) and fluorescence photographs (Figure 3F), the fluorescence characteristics of SPSMAAs-30 underwent gradual evolution with increasing sulfonation time. Blue Fluorescence emerged in SPSMAAs-30 after 5 minutes of sulfonation, with an emission peak at 420 nm under 365 nm excitation. As sulfonation time increased, the main emission peak at 420 nm decreased in intensity, whereas the shoulder peak at 570 nm exhibited enhanced fluorescence, resulting in white-light emission. With further prolongation of sulfonation, the fluorescence shifted toward the yellow region, as evidenced by the continued enhancement of the 570 nm peak and attenuation of the 420 nm shoulder peak. Upon reaching a sulfonation duration of 720 min, orange-red fluorescence emerged, with the 570 nm emission peak becoming dominant and reaching its maximum intensity. This phenomenon was associated with the number of chromophores. With increasing sulfonation time, the proliferation of chromophores led to cluster emission. As the number of emissive clusters grew, so did their size, resulting in long-wavelength emission. Due to the random distribution and varying sizes of clusters within the polymer, dual emission occurred (small clusters emitting at 420 nm and large clusters emitting at 570 nm). At shorter sulfonation times, small clusters predominated, whereas prolonged sulfonation favored the formation of larger, more stable clusters, thereby shifting the emission profile toward the red region. SPSMMAs-30 and SPSMAs-30 exhibited pH-responsive luminescence behavior. Upon addition of NaOH, the solutions emitted blue-green fluorescence, with reversibility demonstrated upon subsequent HCl introduction. Fluorescence spectra of these polymer solutions at varying pH levels were recorded (Figure 3G-J). Under alkaline conditions, both SPSMMAs-30 and SPSSMAs-30 displayed blue-shifted emission spectra accompanied by narrowed peaks. This phenomenon was attributed to the deprotonation of carboxyl (-COOH) and sulfonic acid (-SO 3 H) groups into their sodium salt forms (-COO - Na + and -SO - Na + ), which increased counterion radius and steric hindrance. [30] The resultant inhibition of sulfonic acid group aggregation led to spectral blue-shifting. Concurrently, reduced hydrogen bonding density diminished cluster aggregation and TSI, further shortening the emission wavelength. This study employed a multi-dimensional synergistic regulation strategy. By systematically varying the carboxyl content, molecular chain flexibility, sulfonation time, and protonation level, the study achieved precise multi-color tuning of cluster luminescent microspheres in the visible range. Results indicated that carboxyl group incorporation effectively modulated the TSI effect and optimized the balance of molecular chain rigidity and flexibility, significantly enhancing CL performance. Meanwhile, accurate control of sulfonation time adjusted sulfonate/carboxylate group ratios, enabling color tuning from white to orange-red. Coupled with protonation treatment to dynamically modulate surface charge states, this induced NCC protonation/deprotonation processes, achieving reversible orange-red/green switching. The synergistic multi-dimensional regulation extended emission coverage from 420 nm (blue) to 570 nm (orange-red), establishing a seven-color luminescence system. Fluorescence images and spectra in Figure 3K and L confirmed the material’s excellent optical stability. This multi-dimensional synergistic approach effectively realized efficient multicolor CL luminogens, advancing the understanding of CL mechanisms and providing a versatile platform for future multicolor optical applications. 2.3 Theoretical calculation and mechanism explanation Figure 4. Simplified molecular structures of polymers with different groups (A). Theoretical calculation of the excited state geometry of the aggregate was conducted using the DFT method at the B3LYP/6-31G (d, p) level for the frontier molecular orbital energy levels (B) and atomic distances (C). The green dashed line shows the hydrogen bond distance. The purple solid line indicates the distance between oxygen atoms (d O…O ). A schematic diagram of the mechanism by which rigidity and flexibility affect fluorescence properties was shown (D). A schematic diagram of the potential energy surface and energy gap under the aggregation state of clusters of different sizes was also presented (E). To gain a deeper understanding of the long-wavelength emission mechanism of these polymers, the frontier molecular orbital energy levels in the aggregates and the interactions between different simplified functional clusters (Figure 4A) within the molecules were studied by first-principles calculations based on density functional theory (DFT). As shown in Figure 4B and C, P1, P2, P3, and P4 configurations displayed distinct hydrogen-bonding motifs. P1 (S=O∙∙∙HO-S: -8.23 to -4.81 kcal/mol) exhibited stronger intramolecular interactions compared to P2 (S-OH∙∙∙O=C: -5.48 to -2.17 kcal/mol), which enhanced π-electron delocalization and resulted in a lower energy gap (ΔE gap ). Although both structures contained similar phenyl polymer frameworks, the -SO₃H substituent displayed a stronger electron-withdrawing effect than -COOH, causing further ΔE reduction. This resulted in P1 having a lower ΔE gap than P2. In P3, substituting three styrene sulfonic acid units with acrylic acid groups led to reduced steric hindrance and elevated hydrogen-bonding energy (-10.21 to -5.77 kcal/mol). The reduction in steric hindrance fostered a substantial number of hydrogen bonds. This, in turn, enhanced conformational flexibility and facilitated short-distance interactions between NCCs, thereby improving TSI and promoting luminescence efficiency. The introduction of a methyl group into P4 elevated its HOMO orbital energy level via electron-donating effects, consequently narrowing the energy gap. On the other hand, Methyl group incorporation was demonstrated to balance polymer rigidity and flexibility, with optimized spatial interaction correlating to enhanced TSI. [53] Previous studies indicated that larger NCC aggregates generate stronger TSI effects, enabling longer-wavelength emissions. [54] Compared to P4, P3 exhibited reduced steric hindrance and enhanced chain mobility, which hindered large aggregate formation. Methyl group incorporation increased molecular rigidity and steric bulk while preserving strong hydrogen bonding. Moderate rigidity effectively restricted molecular motion, suppressed non-radiative transitions, and improved luminescence efficiency. We hypothesize that optimal rigidity enhances electron delocalization and orbital overlap, strengthens TSI interactions, narrows the energy gap, and facilitates electron transitions for enhanced luminescence. Therefore, P4 can achieve longer wavelength emission than P3. Subsequently, to investigate the TSI in CL microspheres, excited-state chain interatomic distances incorporating both intermolecular and intramolecular interactions were systematically analyzed. The evaluation of the van der Waals radii (r B ) between atoms, provided a metric for assessing the TSI [55-56] The distance between sulfur and sulfur atoms with the participation of the carbonyl group (d S···S ) was in the range of 4.167 - 4.75 Å beyond the sum of their double r S (2•r S = 3.27 Å), [57] suggesting that the TSI was too weak to stabilize the formed clusters and further confirmed that P1 may displayed intrinsic emission of benzenesulfonic acid upon cluster formation. It was worth noting that the r O of oxygen atoms was 1.52 Å. [58] In all cluster luminescent bodies, the short-range distance between oxygen atoms ( 2.624 -2.911 Å) did not exceed twice the r O (3.04 Å). This meant that the space interactions mainly occurred between oxygen atoms, which indicated that TSI mainly occurred between the oxygen atoms of the carboxyl and sulfonic acid groups. Incorporation of MMA and MA monomers shortened interoxygen bond distances and enhanced TSI effects, whereas rigid monomer addition increased short-range interoxygen bond distances, leading to diminished TSI effects. The molecular electrostatic potential (MEP) indicated the molecular size and electrostatic potential area by color grading. As shown in Figure S3, the electron clouds of P4 and P3 were more concentrated inside the model, which further supported the existence of stronger Through-Space Interactions (TSI) in the cluster luminescent microspheres. These results indicated that the flexible carboxylation structure led to more electron cloud overlap and stronger hydrogen bonding. This intramolecular interaction also explained the reasons for the longer emission intensity and emission wavelength. To sum up, carboxyl groups were observed to form extensive hydrogen bonds, increasing molecular rigidity while enabling close oxygen atom proximity. This structural arrangement enhanced spatial conjugation and promoted luminescent properties. In addition, to clarify the effect of MMA content on fluorescence intensity, the oscillator strength ( f ) of the molecule in the excited state was further calculated using the TD-DFT method with Gaussian16 software. With the increase of MMA degree, the f values were P5 = 0.02827, P4 = 0.03276, and P6 = 0.00667 (Figure S4), respectively. It was well known that increasing the oscillator strength could promote smoother transitions and enhance luminescence, [59] which indicated that suitable carboxyl groups played a vital role in improving the CL luminescence efficiency. This was consistent with the phenomenon that rigidity and flexibility affected cluster luminescence. In summary, the molecular energy gap should be synergistically regulated by the molecular configuration (steric hindrance) and the push-pull electron effect of the functional group. When the molecular geometric configurations were not much different (P1-P2, P3-P4), the push-pull electron effect of the functional group played a major role; when the molecular configurations were far apart, the molecular configuration (steric hindrance affected electron delocalization) played a dominant role (P1-P3). At the same time, the long-wavelength emission of PL required the cluster to have sufficient conformational rigidity to suppress non-radiative deactivation and promote efficient excitation/emission processes. Carboxyl and sulfonic acid groups were commonly used NCCs, which could produce hydrogen bonds and other interactions to cause NCC aggregation, which was the basic condition for the formation of cluster luminescence. Cross-linking was an effective strategy to strengthen interchain interactions. The cross-linking points could increase the cluster size and make the chains stack more tightly, thereby obtaining stronger TSI. Since NCCs were aggregated in a limited space, the vibration and rotational freedom of the fluorophore were restricted, which might be conducive to radiative transitions and increase fluorescence emission. Figure 4D explains the mechanism of CL. It was evident that under cross-linking conditions, as the flexibility of the molecular chain increased, NCC was more likely to aggregate into larger clusters, and suitable rigidity and flexibility could lead to longer wavelength emission. Clusters with increased size exhibited lower energy gaps (ΔE 1 > ΔE 2 > ΔE 3 ) and longer emission wavelengths (Figure 4E), a phenomenon known as the cluster size distribution effect [20, 30, 60] . Given the multi-level structural characteristics of SPSMMAs, such as disordered repeating units, wide molecular weights, and linear or cross-linked structures, TSI had different intensities and multicolor emission spectra. 2.4 Multi-channel fluorescence and stability testing Figure 5. Fluorescence images of SPSMMAs-30 with different fluorescence channels: DAPI (A), FITC (B), RB (C), and CY5 (D), scale bar is 20 µm. (E) Schematic diagram of CytoFLEX three-laser flow cytometry. Histograms of SPSMMAs-30 in the same flow cytometer channel: FSC-A (F), FITC (G), PE (H), ECD (I), PE-CY7 (J), APC (K), APC-CY7 (L), BV421 (M), BV510 (N), BV605 (O), BV650 (P), BV786 (Q), AF700 (R), and PerCP (S). Performance parameters of SPSMMAs-30 compared with daily QC fluorospheres of CytoFLEX (CytoFLEX-QC) in flow cytometry testing (T). Flow cytometry (FCM), emerged in the 1960s, is widely used in cell analysis and immunodiagnostics [62] . As a complex instrument combining electronic, optical, and mechanical components, even minor device performance differences could cause result variations. Routine calibration is crucial to ensure data reliability and reproducibility, relying on specialized beads from suppliers such as Spherotech Corporation and Bangs Laboratories [63] . Conventional calibration beads were made by encapsulating multiple fluorescent dyes within a polymer matrix, enabling simultaneous detection across various excitation wavelength. However, this method required multiple dyes and strict uniformity (coefficient of variation < 3%), making the fabrication process time-consuming and technically challenging [64] . Additionally, beads prepared by swelling methods often suffer from dye leakage, reducing their long-term stability [65] . These limitations highlight the urgent need for easily manufactured, photostable, and scalable multicolor calibration beads. SPSMMAs CL microspheres, with uniform particle size and broad spectral emission, presented innovative potential. Their unique multicolor cluster-emission properties, achieved through precise spatial regulation of emissive clusters, enabled robust color coding while maintaining spectral stability. These characteristics positioned SPSMMAs CL microspheres as ideal candidates for next-generation FCM calibration standards, addressing both technical challenges and scalability limitations of traditional approaches. Multicolor emission calibration microspheres demanded stringent control over both particle size and fluorescence uniformity, with CV values typically required to remain below 3%. The effect of varying MMA contents on the size CV of SPSMMAs was investigated (Figures S5-S8). When the MMA content was below 40%, the size CV of SPSMAAs remained below 5%. Specifically, at an MMA content of 30%, SPSMMAs-30 achieved an optimal CV value of 2.12%. However, when the MMA content exceeded 50%, the size CV of SPSMMAs became significantly poor. Moreover, when the MMA content exceeded 70%, fragmentation of the microspheres was observed. These small-sized polymer particles and microsphere debris severely compromised the CV value. As a polar monomer, MMA, in contrast to the nonpolar styrene monomer, could regulate the hydrophilic-lipophilic balance of the polymer when present in lower quantities. This facilitated better dispersibility and stability of the particles in the aqueous medium, thereby enhancing the stability of the seed swelling polymerization process. Nevertheless, excessively high MMA contents hindered the swelling of all monomers into the PS seeds, leading to the emulsion polymerization of monomers outside the PS seeds and the formation of small particulate polymers. The Tg of crosslinked polystyrene microspheres was reduced by PMMA, which enhanced flexibility and enabled superior handling of internal stresses during sulfonation, thereby precluding rupture or deformation. However, after sulfuric acid treatment, SPSMMAs with superfluous MMA displayed exorbitant chain hydrophilicity and phase separation, culminating in fragmentation. In addition, when the concentration of methyl acrylate and 4-vinyl benzoate was 30%, the CV values were much higher than those of SPSMMAs-30 (Figure S9). These results demonstrated that SPSMMAs-30 exhibited the optimal broad emission and particle size uniformity. Then, the fluorescence properties of SPSMMAs-30 were verified. As shown in Figure 5A-D, the fluorescence images of SPSMMAs-30 were obtained in four different channels (DAPI, FITC, RB, and CY5) of the fluorescence microscope. Figure 5E showed the basic principle of CytoFLEX three-laser flow cytometry (Beckman Coulter, USA), which was equipped with 405 nm, 488 nm, and 638 nm lasers, along with 13 detection channels. Correspondingly, following testing of SPSMMAs-30 via a 13-channel flow cytometer, strong fluorescence signals were detected across all channels (FITC, PE, ECD, PerCP, PE-CY7, APC, AF700, APC-H7, BV421, BV510, BV605, BV650, BV786) shown in Figure 5F-S. Notably, both fluorescence intensity distribution and particle size distribution remained consistently below 3% CV across all detection channels. To further validate the performance of SPSMMAs-30, CytoFLEX-QC were analyzed under identical testing conditions (Figure S10). Despite some minor differences in Figure 5T, the performance of SPSMMAs-30 was generally similar to that of commercial flow cytometry calibration microspheres in flow cytometry assays, indicating that the performance of SPSMMAs-30 was usually consistent with that of commercial products. In addition, the distribution of sulfur elements in different microspheres was measured by EDS 20 times, and the CV value of the S content was calculated to be 3.37% (Figure S11), which was close to its fluorescence CV. The results showed that the fluorescence CV can be easily adjusted by controlling the uniformity of the sulfonation degree. Due to their monodispersity and broad excitation/emission characteristics derived from CL, SPSMMAs-30 represented an ideal substitute for multi-dye coded flow fluorescence calibration microspheres. Figure 6. (A) PL spectra and image at the single-particle level. (B) The excitation wavelength is 365nm. The time trace of emission intensity from a single SPSMMAs-30 and CytoFLEX-QC under continuous laser illumination for more than 30 min. The bin time for each data point is 10 ms. (C) Fluorescence intensity of SPSMMAs-30 at different temperatures. (D) The fluorescence intensity of SPSMMAs-30 across 13 channels under natural light over 180 days. Each measurement was referenced to the CytoFLEX-QC stored in the dark at 4°C. (E) The fluorescence intensity of CytoFLEX-QC across 13 channels under natural light over 180 days, with each measurement referenced to the CytoFLEX-QC maintained at 4°C in the dark. Similarly, the fluorescence intensity of SPSMMAs-30 over 13 channels when stored at 4°C in the dark for 180 days (F), with each measurement based on the CytoFLEX-QC stored under the same conditions. Flow cytometry recorded the optical signal of a single particle during the examination process. Therefore, to comprehensively and accurately test the single-particle luminescence properties of SPSMMAs-30, a customized single-particle fluorescence microscope (Olympus IXplore 73) was used. As shown in Figure 6A, individual SPSMMAs-30 particle exhibited orange fluorescence under 365 nm excitation. The spectrum of this single SPSMMAs-30 was also recorded under the same excitation condition. An obvious double peak emission phenomenon was observed at 420 nm and 580 nm, with the spectrum covering a wide range from 400 nm to 800 nm. This result was consistent with the previous findings obtained via fluorescence spectroscopy, proving that the spectrum could fully and completely cover all available channels during flow cytometry single particle testing. Figure 6B showed the fluorescence intensity versus time traces of single SPSMMAs-30 and CytoFLEX-QC under continuous wave excitation with a power density of 5 × 10² W/cm². The results indicated that SPSMMAs-30 maintained better emission more effectively after long-term laser irradiation compared to CytoFLEX-QC. This outstanding photostability highlights the superior anti-photobleaching performance of SPSMMAs-30, making them well-suited for long-term fluorescence applications. Considering temperature fluctuations might affect molecular motion and cluster integrity [31, 61] , the CL properties of SPSMMAs-30 at different temperatures were examined (Figure 6C). Fluorescence intensity was demonstrated to remain stable between 4°C and 37°C, with only a slight decrease observed at 50°C, indicating robust thermal stability across the tested temperature range. To evaluate ionic strength effects, samples were incubated in NaCl solutions with different concentrations (Figure S12). Remarkably, SPSMMAs-30 preserved over 97% of its initial fluorescence even at 0.2 M NaCl, highlighting exceptional tolerance to high ionic environments. Besides anti-photobleaching properties, the environmental stability of calibration microspheres serves as a critical evaluation parameter. SPSMMAs-30 and CytoFLEX-QC were subjected to stability tests in three organic solvents (ethanol, acetone, and DMSO) (Figure S13). Significant differences were observed between the two materials. CytoFLEX-QC exhibited massive non-fluorescent particles after 12-hour ethanol treatment, suggesting dye leakage caused by solvent dissolution. Prolonged immersion in acetone and DMSO led to near-complete dissolution of CytoFLEX-QC microspheres, likely due to their low-crosslinked polymer network. Conversely, SPSMMAs-30 retained fluorescence in all tested solvents, which was attributed to its stable CL mechanism instead of passive dye encapsulation. The highly crosslinked 3D polymer network of SPSMMAs-30 effectively prevented solvent penetration, providing excellent resistance to swelling and dissolution. Stability tests under extreme conditions, including exposure to organic solvents, temperature variations, and high ionic strength, were conducted and demonstrated robust stability of SPSMMAs-30. This superior performance was attributed to the combined effects of its unique CL mechanism and highly cross-linked structure, which provided reliable material support for flow cytometry calibration applications. The stability of fluorescence was critical for calibration particles, as it directly influences the accuracy and reliability of measurement results. Each measurement was performed using the CytoFLEX-QC stored at 4°C in the dark as a baseline to evaluate the time-dependent changes in the fluorescence intensity of 13 channels of SPSMMAs-30 and CytoFLEX-QC under natural light. As shown in Figure 6D and E, when CytoFLEX was exposed to natural light, the fluorescence signal of its long-wavelength channels (specifically including PE-CY7, APC-CY7, AF700, and BV-786) began to decay rapidly after only 15 days. In contrast, SPSMMAs-30 exhibited a slower decay rate in the same channels under identical conditions. After 60 days of light exposure, CytoFLEX-QC’s long-wavelength fluorescence signals had decreased by approximately 50%, while SPSMMAs-30 showed a smaller decline of about 30%. This performance gap widened over time. After more than 180 days, the fluorescence signal of the long-wavelength channel of CytoFLEX-QC decreased to about 20%, indicating that most of the fluorescence signal had been lost, which significantly impacted the measurement results. At the same time, the fluorescence signal of the long-wavelength channel of SPSMMAs-30 maintained approximately 30%, demonstrating better photostability. Figure 6F illustrated the changes in fluorescence intensity of 13 channels of SPSMMAs-30 stored at 4°C in the dark for 180 days. Data analysis indicated that SPSMMAs-30 preserved over 90% of its original fluorescence intensity. These results showed that SPSMMAs-30 had excellent resistance to photobleaching and fluorescence stability. In summary, SPSMMAs-30 demonstrated significant anti-photobleaching properties and environmental stability. The underlying stability mechanisms were identified as follows: First, the material’s robust cross-linked architecture was shown to suppress swelling in aqueous environments, thereby enhancing cluster structural integrity through steric hindrance effects. Second, a gradient distribution of surface hydrophilicity and internal hydrophobic regions was observed, wherein compact cross-linked networks and benzene ring-derived hydrophobic barriers effectively restricted water molecule penetration into cluster cores. This synergistic system, resulting from high cross-linking density and optimized hydrophobicity, ultimately conferred superior photostability and environmental robustness on the luminescent microspheres. 2.5 Large-scale preparation of SPSMMAs-30 Figure 7. Large-scale preparation of PSMMAs-30 using a 10 L reactor (A). Weighing Photo of Single-Batch-Scale Preparation of PSMMAs-30 (B). Photograph of a single batch of SPSMMAs-30 solution, wt = 10% (C). Photograph of a single batch of SPSMMAs-30 solution under UV light (D). SEM images of PSMMAs-30 (E) and SPSMMAs-30 (F) prepared at scale. (G) Histogram of SPSMMAs-30 prepared at scale in channel 13 of the flow cytometer. Large-scale preparation can minimize the adverse effects of batch differences and ensure the accuracy and reliability of test data. This study presented a successful case of large-scale preparation of cluster luminescent calibration microspheres. Specifically, a single batch weighing up to 1384.4 g of PSMMAs-30 was successfully prepared using an advanced 10 L reactor (Figure 7A and B). Subsequently, 1 kg of PSMMAs-30 was treated with 2 L of sulfuric acid. After a sufficient reaction and a series of washing steps to remove residual sulfuric acid and other impurities, 10 L (weight percentage, 10%) of SPSMMAs-30 dispersion solution was finally obtained (as shown in Figure 7C). When irradiated with ultraviolet light, the 10 L dispersed solution was observed to emit bright orange fluorescence (Figure 7D). This visually demonstrated that the microspheres possess desirable fluorescence properties similar to those of SPSMMAs-30 prepared in small batches. Under close observation with SEM, it was found that the size of both the scaled-up SPSMMAs-30 was 5.5 μm, showing excellent uniformity throughout the sample (Figure 7E and F). To further verify the performance of the scaled-up SPSMMAs-30, it was placed on an advanced 13-channel flow cytometer (Cytoflex, Beckman Coulter, USA) for detection. Strong fluorescence signals were detected in all fluorescence channels (Figure 7G), including FITC, PE, ECD, PerCP, PE-CY7, APC, AF700, APC-H7, BV421, BV510, BV605, BV650, and BV786. Importantly, the fluorescence CV across all channels remained below 3%, confirming high uniformity and signal consistency. This achievement represented an important breakthrough in the large-scale production of high-quality fluorescent calibration microspheres based on CL. 2.6 Tetracycline Detection and Anti-Counterfeiting Figure 8 . (A) The fluorescent spectra of SPSMMAs-30 (λ ex = 356 nm, 1 mg) with different TC concentrations (3 mL) after 180 min interaction. (B) Quantitative change of I 570 /I 450 with TC concentrations. (C) Plot of the relationship between B/G values and TC concentrations under UV light irradiation. (D) Plot of B/G values against TC concentrations under natural light. (E) The anti-interference capability of TC was assessed under two detection modes: visible light and 365 nm UV light colorimetric. Both TC and nine common metal ions (Na⁺, Cl⁻, Zn²⁺, Mg²⁺, Cu²⁺, Fe²⁺, Fe³⁺, Ba²⁺, and Mn²⁺) were prepared at a concentration of 5 μM each. (F) A smartphone device was employed for scanning and monitoring TC. (G) Bland-Altman plots comparing predicted vs. actual concentrations for colorimetry-only, fluorescence-only, and combined colorimetry-fluorescence methods; (H) ANN performance evaluation. (I) ANN confusion matrix assessing TC classification accuracy by concentration category. Tetracycline antibiotics (TC) have been extensively used in veterinary practice to enhance livestock growth and treat infections, resulting in substantial residue accumulation in animal-derived products. Long-term intake of TC-contaminated food could induce antibiotic resistance in pathogens, thereby posing significant risks to public health [66, 67] . Consequently, reliable quantification of TC residues became imperative. To evaluate the feasibility of SPSMMAs-30 for TC detection, SPSMMAs-30 solutions at varying concentrations of TC (0 - 15 μM) were first analyzed under 356 nm excitation following 180 min incubation. As illustrated in Figure 8A, following exposure to TC, fluorescence intensity of SPSMMAs-30 decreased, with the primary peak at 570 nm declining faster than the secondary peak at 450 nm. At a TC concentration of 10 μM, the primary emission peak shifted to 450 nm. The ratio of primary-to-secondary emission peak intensities decreased as TC concentration increased, demonstrating a linear relationship within the 1-5 μM range (Figure 8B). The linear fit yielded a quantitative equation for TC determination: y = 2.183 - 0.109x, R 2 = 0.992. The detection limit is based on a 3δ/k (δ is the standard deviation of the blank signal, and k is the slope of the linear calibration plot). The limit of detection (LOD, S/N = 3) was calculated as 0.334 μM. Due to SPSMMAs-30’s wide excitation/emission range, its solution color shifted from orange-red to yellow-green under both UV and sunlight as TC concentration increased. Figure 8C and D revealed that SPDMMAs-30 solutions underwent distinct color changes upon TC addition under both sunlight and 365 nm UV light. Naked-eye visual identification of 2.5 μM TC was achievable, while lower concentrations exhibited no detectable changes. TC quantification at diluted concentrations was achieved via image analysis employing the blue and green (B/G) ratio. Within the 1-10 μM range, linear correlations between B/G ratios and TC concentrations were observed under both lighting conditions, yielding quantitative equations: y = 1.071 - 0.09x, R 2 = 0.998, y = 0.936 - 0.09x, R 2 = 0.996. The detection limit (LOD, S/N = 3) was determined as 0.173 μM and 0.337 μM. To evaluate the probe’s anti-interference performance, the B/G ratio of SPDMMAs-30 in aqueous solution was measured in the presence of nine common metal ions (Na + , Cl⁻, Zn 2+ , Mg 2+ , Cu 2+ , Fe 2+ , Fe 3+ , Ba 2+ , and Mn 2+ , 5 μM). As shown in Figure 8E, SPSMMAs-30 demonstrated strong anti-interference capability at 5 μM. A smartphone-compatible TC scanning device was developed to enable faster and more sensitive detection, using images of SPSMMAs-30 captured under both sunlight and 365 nm UV irradiation during TC testing (Figure 8F). Fluorescence changes in SPSMMAs-30 enabled real-time, on-site antibiotic detection via a smartphone paired with a commercial colorimetric scanning application. Two RGB datasets derived from colorimetric and fluorescence images were analyzed via machine learning-based ANN regression, which accurately predicted TC concentrations. Figure 8G shows that the combined colorimetry-fluorescence approach achieved excellent agreement with true concentrations (R 2 ≈ 1, MSE = 0.0108), highlighting the ANN’s robust predictive capacity. This dual-mode method outperformed single-mode detection. The Bland-Altman analysis (Figure 8H) revealed that 99% of data points lay within the 98% confidence interval, validating the model’s precision. Furthermore, the ANN confusion matrix (Figure 8I) confirmed reliable differentiation of RGB signals across TC concentration categories. This demonstrates that SPSMMAs-30 holds significant potential as a fluorescent probe for trace chemical analysis in aqueous media. Figure 9. (A) pH-responsive lotus patterns were fabricated using SPDMMAs-30. (B) pH-responsive orchid patterns were prepared with SPDMAs-30. (C) SPDMMAs-30 and SPDMAs-30 in PVA solutions (5 wt%, pH-responsive) were loaded into a 384-well plate for red filter (610 – 1100 nm) information encryption/decryption. (D) Cu 2+ /EDTA responsive SPDMMAs-30 and SPDMAs-30PVA solutions (5 wt%) were employed for red filter (610 - 1100 nm) information decoding. Upon dispersion of SPSMAs-30 and SPSMMAs-30 in polyvinyl alcohol (PVA) solutions at pH = 10 and 12, respectively, their fluorescence exhibited a consistent blue-green. Fluorescence emission peaks were observed at 450 nm - 460 nm under both conditions (Figure S14). This phenomenon enhanced the design flexibility for fluorescent anti-counterfeiting labels. The pH-responsive property of SPSMMAs-30 was exploited to create a single-pigment pH-color-changing painting. Lotus petals and leaves were stained using SPSMMAs-30 solution (pH = 10, excess NaOH), resulting in uniform green coloration. Post-treatment with HCl solution (pH = 1) induced selective color changes: petals transitioned to orange-red, whereas leaves retained green. Orchid petals and leaves were treated with SPSMAs-30 solution (pH = 10, excess NaOH), rendering the entire orchids green. Subsequent exposure to HCl solution (pH = 1) induced yellow-green petals and green leaves, thereby confirming the capacity of pH-responsive materials for spatially selective color modulation. Multi-layer anti-counterfeiting security labels were developed as an effective and straightforward approach for product authentication and information concealment [68, 69] . A composite label was fabricated on the microarray, incorporating SPDMMAs-30 gel (pH = 10) and SPDMAs-30 gel (pH = 10) to form the numeral ”888”. Under ultraviolet excitation, all microarrays exhibited uniform green fluorescence, enabling primary-level information encryption. Subsequent treatment with specific reagent volumes (0.1 M HCl, 10 µL) and optical filters (610–1100 nm) revealed the secondary encoded information within ”999”, demonstrating spatially resolved decoding capability. Additionally, SPDMMAs-30 and SPDMAs-30 were incorporated into a customized (quick response) QR code. Fluorescence signals were initially quenched via Cu²⁺ solution treatment. As shown in Figure 9D, fluorescence quenching was observed in all microarrays following Cu²⁺ addition. Subsequent introduction of EDTA restored fluorescence, revealing dual fluorescent QR codes. However, interference between the two fluorophores under UV exposure was difficult to resolve. A 610 - 1100 nm filter was subsequently applied to selectively decode the concealed ”red QR code” layer. This strategy thus enables multi-layered information encryption: UV illumination alone yields scrambled data, while filtered analysis ensures accurate content retrieval, enhancing both security and cryptic functionality. Conclusion In summary, we have successfully developed a scalable fabrication strategy for CL polymer microspheres for the first time and systematically investigated the luminescence mechanisms. The resulting SPSMMAs microspheres exhibited high monodispersity and an emission spectrum spanning the full visible range (400-700 nm). To elucidate the structure–property relationships governing CL behavior, we employed DFT calculations to explore the effects of polymer chain rigidity, flexibility, and carboxyl functionalization. MMA was shown to provide appropriate rigidity and facilitate intermolecular hydrogen bonding, thereby enhancing TSI, red-shifting emission wavelengths, and improving overall luminescence efficiency. Notably, SPSMMAs-30 microspheres demonstrated strong fluorescence across 13 commonly used flow cytometry detection channels, with fluorescence coefficient of variation (CV) values below 3%, effectively meeting the requirements for daily cytometer calibration. They exhibited excellent photostability and resistance to photobleaching compared to conventional dye-based calibration beads, enabling their long-term storage and reuse. The emission properties of SPSMMAs-30 were well maintained across both gram- and kilogram-scale syntheses, confirming the reliability and scalability of the synthetic method and paving the way for industrial-level production. Besides, these microspheres also displayed high sensitivity and specificity in tetracycline detection and exhibited promising potential for multicolor fluorescence-based anti-counterfeiting applications. Therefore, this work offers a simple, cost-effective, and robust strategy for fabricating CL-based microspheres. It not only addresses critical challenges in spectral coverage and stability but also significantly reduces production costs and broadens the application landscape of CL materials in various fields. Experimental Section All experiment details are provided in the supporting information. Acknowledgments This work was supported by Ministry of Science and Technology of the People’s Republic of China (Grant No. 2022YFC2406600), the Youth Innovation Promotion Association of the Chinese Academy of Sciences (No. Y2023088), and the Innovative Key Project of Suzhou Institute of Biomedical Engineering and Technology, the Chinese Academy of Sciences (Grant NO. CX202501001). We thank the technical support of Public Technology Center in Suzhou Institute of Biomedical Engineering and Technology (SIBET). Author Contributions L. He and X. Nan contributed equally to this work. L. He, X. Nan, and P. Bai performed conceptualization. L. He, T. Wang, and Z. Liu performed the methodology. C. Wang performed visualization. L. He wrote the original draft. L. He, X. Nan, T. Gu, and P. Bai wrote, reviewed, and edited the original draft. Conflict of Interest The authors declare no conflict of interest. References [1] X. Yang, R. Hu, A. Qin, B. Z. Tang, Eur. Polym. J. 2024 , 204 , 112704.[2] K. 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Chen, Adv. Mater. 2022 , 34 , 1. Information & Authors Information Version history V1 Version 1 02 September 2025 Peer review timeline Published Aggregate Version of Record 13 Oct 2025 Published Copyright This work is licensed under a Non Exclusive No Reuse License. Collection Aggregate Keywords clusteroluminescent flow optical calibration monodisperse polymer microspheres spectrum-tunable Authors Affiliations Liang He University of Science and Technology of China School of Biomedical Engineering View all articles by this author Xueyan Nan University of Science and Technology of China School of Biomedical Engineering View all articles by this author Tong Wang University of Science and Technology of China School of Biomedical Engineering View all articles by this author Zhizhou Liu University of Science and Technology of China School of Biomedical Engineering View all articles by this author Ce Wang University of Science and Technology of China School of Biomedical Engineering View all articles by this author Tongxu Gu University of Science and Technology of China School of Biomedical Engineering View all articles by this author Pengli Bai 0000-0003-1907-6825 [email protected] University of Science and Technology of China School of Biomedical Engineering View all articles by this author Metrics & Citations Metrics Article Usage 285 views 185 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Liang He, Xueyan Nan, Tong Wang, et al. 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