s2fieldsofstudy:
Chemistry
Materials Science

text:
Flexible Alkylene Bridges as a Tool To Engineer Crystal Distyrylbenzene Structures Enabling Highly Fluorescent Monomeric Emission

Abstract To design ultrabright fluorescent solid dyes, a crystal engineering strategy that enables monomeric emission by blocking intermolecular electronic interactions is required. We introduced propylene moieties to distyrylbenzene (DSB) as bridges between the phenyl rings either side of its C=C bonds. The bridged DSB derivatives formed compact crystals that emit colors similar to those of the same molecules in dilute solution, with high quantum yields. The introduction of flexible seven‐membered rings to the DSB core produced moderate distortion and steric hindrance in the DSB π‐plane. However, owing to this strategy, it was possible to control the molecular arrangement with almost no decrease in the crystal density, and intermolecular electronic interactions were suppressed. The bridged DSB crystal structure differs from other DSB derivative structures; thus, bridging affords access to novel crystalline systems. This design strategy has important implications in many fields and is more effective than the conventional photofunctional molecular crystal design strategies.

Introduction
Recently, ultrabright solid-state fluorescent materials containing organic dyes have been applied to the fabrication of organic light-emitting devices (OLEDs) [1] and nanoparticles, [2] as well as for circularly polarized luminescence experiments, [3] in bioimaging, [4] and for lasers. [5] The wide applicability of these materials is a function of their versatility, in terms of molecular design, and excellent processability. Optimization of the luminous efficiencies, emission colors, crystallinities, and molecular orientations of solid-state fluorescent dyes are required for the design of advanced OLEDs. At present, the challenges associated with these devices are as follows. 1) Developing fluorescent dyes that exhibit high luminescence efficiencies without concentration quenching in their solid states is essential. [6] J-aggregates are fluorescent dyes that exhibit strong solid-state luminescence; however, the fluorescence wavelengths of their aggregated forms are different with respect to the monomer emissions. [7] These simple systems function based on external shielding by bulky substituents (site isolation effect), [8] or by preventing association through hydrogen bonding or ion-pair formation. [9] Another type of systemaggregation-induced emission (AIE) luminogens (AIEgens) has been investigated. [10] However, in these systems, the dye molecules are large. Small dye molecules and crystals with small free volumes are desired for electronic device applications. [11] 2) Predicting solid-state fluorescence spectra from dilute-solution or distributed-matrix fluorescence spectra is challenging. Numerous organic dyes form aggregates of � 2 molecules in the excited state, [12] and the fluorescence wavelengths often change due to intermolecular electronic interactions. Therefore, monomeric emissive dyes with high quantum yields possess advantages in the field of materials design. 3) To obtain monomeric emission, controlling the dye arrangement is essential. Methodologies based on arranging molecular πskeletons appropriately such that dye molecules without bulky substituents (without increased free volume) exhibit monomeric emission remain underdeveloped. Therefore, novel functional groups must be attached to the π-electron framework to control the crystalline system. In addition, introducing a new functional group into an existing π-electronic compound may afford a different crystalline structure. Increased diversity in crystal engineering is important for the development of material design strategies.
In this study, we prepared highly fluorescent monomeric emissive dyes, di-bridged distyrylbenzenes (DSBs), DBDB[7]s, with highly dense crystalline structures, by introducing flexible cyclic structures into π-conjugated DSB systems. We propose a new crystal engineering method to control luminescence and electronic properties in which simple alkylene bridges are introduced to π-electron compounds. We designed DBDB[7]s with reference to a well-known fluorescent dye, viz., DSB. [13,14] The DBDB[7] structures include two seven-membered rings with C=C bonds bridged loosely by propylene chains, which are much smaller functional groups than those studied in previous reports ( Figure 1). This structure design suppressed intermolecular electronic interactions without distancing the chromophores -a typical result of steric-hindrance-based intermolecular-interaction-suppression strategies. This result is in stark contrast with DSB-bearing H-aggregates [15] -the DBDB[7]s reported herein underwent only very minor emission-wavelength shifts. Moreover, the DBDB[7]s exhibit AIE properties, controllable fluorescence emission, and are not mechanochromic.

Results and Discussion
In this study, we classified DSB analogs into two categories dependent on their photophysical properties: Category A is represented by DBDB[7] and structural homologues, whereas Category B includes not only DSB but also other related dyes that exhibit similar photophysical properties. DSB, PPB, and DSDMB have been previously reported. [15][16][17] We classified the novel compound DBDB[6] as a Category B DSB owing to its similarity to DSBs in terms of their photophysical properties. We synthesized DSBs and bridged DSBs (DBDB[6] and DBDB[7]s); detailed synthetic methods [18] are described in the Supporting Information. Previously, we proposed the concept of bridged stilbenes for AIEgen design. [19] In that case, a flexible ring structure mechanically controlled the accessibility of the configuration taken during the process of quenching the excited-state stilbene. Compared to the melting point of 4phenylstilbene, that of bridged 4-phenylstilbene (8-([1,1'-biphenyl]-4-yl)-6,7-dihydro-5H-benzo[7]annulene) [19] was decreased by 120°C, and there was not a significant increase in the free volume in the crystal. This result inspired the design of the new dyes presented herein. Similar results were obtained for curved nanographene [20] and carbon nanotubes, [21] whose solubility was improved by introducing a seven-membered ring.

Photophysical properties
The photophysical properties, that is, the molar absorption coefficient (ɛ), the maximum absorption wavelength (λ abs ), maximum fluorescence emission wavelength (λ fl ), and quantum yield Φ fl , in both tetrahydrofuran (THF) and the solid-state, are listed in Table 1. Although DSBs have been reported previously, [15][16][17] a systematic study of the photophysical properties of DSBs, in both dilute solution and the solid state, has not been reported. Therefore, we synthesized DSBs to perform a study of this type. For example, for DSB, λ abs , λ fl , and Φ were measured in dilute dichloromethane and in a single crystal; for PPB, λ abs and λ fl were measured in dilute THF and in the aggregated form; and for DSDMB, λ abs was measured in dilute chloroform only.
Dilute solutions of DSB, DSDMB, and DBDB[6] in THF exhibit similar λ abs of~350 nm and Φ THF of~0.90. Meanwhile, PPB, which contains methyl groups at the C=C bonds, exhibits ã 40 nm blue-shifted λ abs of 314 nm and nearly quenched fluorescence with a Φ THF of 0.01. DBDB[7], with a flexible propylene bridge, also exhibits a blue-shifted λ abs (320 nm) and a low Φ THF (0.11). Moreover, introducing methyl groups at the central benzene or bridging C=C bonds results in further blueshifting of λ abs (DBDMDB[7]: λ abs = 273 nm, DBαMDB[7]: λ abs = 284 nm), with Φ THF values of < 1 %. [22] The value of ɛ depends on the planarity of the molecular structure and decreases in the following order: structures without cyclic moieties > six-membered ring > seven-membered ring > methyl-substituted C=C; this order correlates with increasing π-conjugation length and destabilization of the Franck-Condon (FC) energy at the excited state. All the compounds, except DBαMDB[7], 5DBαMDB[7], and tBuDBαMDB[7] (these had no emissions; we refer to these as the DBαMDB[7]s compounds in this report), exhibited similar Figure 1. Previously reported organic dyes that exhibit solid-state fluorescence [8a,b] and the solid-state-fluorescing organic dye designed in this work. Functional groups are shown in red. fluorescence maxima λ fl,THF (392-426 nm). This also implies that their lowest singlet excited states have similar geometries (S1 min structures), regardless of the existence of a bridged structure or of the introduction of methyl groups at the C=C bonds.
We calculated the radiative and non-radiative transition rates (k r and k nr , respectively) of DSB, DSDMB, DBDB[6], and DBDB [7] in THF solutions. In the case of DSB, fluorescence lifetimes (τ) of both the solution and solid have two components. However, since the contribution of one of the lifetime components was far more significant, that value was used to approximate the rate constant. All the k r values were similar (5.2-7.1 × 10 8 s À 1 ), but for DBDB [7], k nr is more than 50fold larger than those of the other compounds (Table 1). The fluorescence quenching of the DBDB[7]s in solution occurs through pyramidal structures due to cleavage of π-conjugation of the C=C bonds, similar to the situation in stilbene derivatives. [19,23] In addition, the conjugation lengths shorten, and the FC energies are destabilized.
This fact explains the low ɛ, λ abs , and Φ THF values of the DBDB[7]s and PPB. The photophysical properties of DSDMB and DBDB[6] are similar to those of DSB, as the planarity of the DSB structure is preserved in these molecules.
The  [7]. We analyzed the differences between the fluorescence properties in solution and the solid state for the two categories shown in Figure 2.

Chemistry-A European Journal
Research Article doi.org/10.1002/chem.202201884 the quantum yield is > 0.8 higher in the solid state than in solution. Although new band structures were observed for these dyes, the changes were small; therefore, the fluorescence properties are comparable to those of monomeric luminescence. DBDB[7], DBDMDB[7], and DBαMDB[7] agglomeration experiments were performed to better understand the photophysical characteristics of these molecules and to determine whether their photophysical properties in the agglomerated state and the solid state differ. The agglomeration study of DBDB[7], DBDMDB[7],and DBαMDB[7] in THF/water reveals that λ abs , λ fl , and ɛ in a 90 % water suspension were similar to those in dilute THF solutions. However, for the aggregates in 70 or 80 % water suspensions, large blue-shifts in λ fl (particularly for DBαMDB [7]) and decreases in ɛ were observed, possibly owing to differences in the molecular conformations taken compared to those in dilute THF solution or the solid state (Figures S11-13, Tables S1-3).
Photophysical measurement of powdered DBαMDB[7] was performed ( Figure S6). λ fl and Φ solid were similar before and after grinding, revealing that DBαMDB[7] is not a mechanochromic dye. [25] 5DBαMDB[7] exhibits emission characteristics similar to those of DBαMDB[7], whereas tBuDBαMDB [7], which has a bulky substituent, exhibits a high Φ solid value (0.84), although λ fl,solid for this molecule is approximately 40 nm shorter than those for the other molecules due to conformational change. tBuDBαMDB [7] in the solid state and DBαMDB[7] in a 70-80 % water suspension exhibit identical conformations, as their λ fl values are similar. Steric hindrance caused by bulky substituents at the axial positions in tBuDBαMDB [7] results in a larger distance between the π-electron systems in the core than in the other DBDB[7]s. The introduction of substituents along the πelectron framework axis enables control over the crystal structure and fluorescence wavelength ( Figure 4).
Although the conjugation lengths were shortened by the introduction of flexible bridges, it is apparent that this design strategy is advantageous for the development of AIEgens because DBDB[7]s exhibited almost no emission in THF but strong fluorescence in the solid state.

Category B
In a previous study, [15,16] DSB and PPB were found to form Haggregates in the solid state. Compared to PPB in solution, solid-state PPB exhibits a longer λ fl,solid ( Figure 3b) and a higher Φ solid value.
The fact that the λ fl and Φ fl values of DSDMB and DBDB[6] are comparable to those of the DSBs implies that these molecules are likely to form H-aggregates, and the crystal structures of DSDMB and DBDB [6] are similar to that of DSB ( Figures S21, S22, and S24). In DSB with methyl groups introduced at the lateral positions (DSDMB) or DSB with a

Chemistry-A European Journal
Research Article doi.org/10.1002/chem.202201884 highly planar six-membered-ring core (DBDB[6]), monomeric emission is unfavorable in the solid state, and in solution.

Single-crystal X-ray structure analysis
We analyzed the relationships between the photophysical properties and crystal structure among the investigated dyes.

Category A
The torsion angles between the C=C bonds and the central and outer benzene rings are ϕ i and ϕ o , respectively, as shown in Figure 5.
In DSB, the central and outer benzene rings occupy almost parallel planes.  DBαMDB[7] was dissolved in a hexane/dichloromethane mixture, and then the solution was gradually evaporated under atmospheric conditions to yield two types of single crystals: a cross type, with twisted central and outer benzene rings, and a parallel type, with almost no twisting ( Figure 5). In both structures, the magnitudes of ϕ o and ϕ i are similar, and the central and outer benzene rings are twisted equally with respect to the bridging double bond. The λ fl and Φ values of the two DBαMDB[7]-cross/parallel single crystals are shown in Figure 6.
In DBDB[7], DBDMDB[7], and DBαMDB[7], the benzene rings of adjacent overlapping molecules are almost orthogonal; thus, the electronic interactions are negligible. These three molecules exhibit solid-state monomeric emission. The molecular structure is probably similar to that in dilute THF solution, as their fluorescence properties in dilute THF solution and the solid state are similar.
In the case of DBαMDB[7], the molecular conformation can be controlled by recrystallization, and the conjugation length and luminescence properties of the chromophore can therefore be manipulated. Moreover, the λ fl values of the DBαMDB[7] aggregate in a 70 % water suspension and the DBαMDB[7]parallel crystal were shown to be similar, indicating that these conformations are the same.
Finally, in terms of the molecular densities and occupancies, no difference was observed between the DSBs and DBDB[7]s (Table S6). Introducing a flexible bridging structure into the πframework results in a different crystal, but the free volume is not increased.
The molecular structure of 5DBαMDB [7] was similar to that of DBαMDB[7]-cross, and that of tBuDBαMDB [7] was identical to that of DBαMDB[7]-parallel ( Figure S20). For tBuDBαMDB [7], the distance between the benzene rings in adjacent molecules that overlapped in-phase was approximately 1.1 Å longer than that for DBαMDB[7]-parallel. We were not able to analyze the fluorescence properties of the DBαMDB[7]s in detail because

Chemistry-A European Journal
Research Article doi.org/10.1002/chem.202201884 they exhibited no fluorescence in dilute THF solutions (AIE). However, these molecules are expected to exhibit monomeric fluorescence in the solid state, based on their Φ solid and τ solid values and crystal structures. [26] Category B Each molecule is highly planar, and DSB, PPB, and DBDB[6] adopt edge-to-face herringbone structures. The intermolecular distances between the chromophores (d chr , Figure 7c) of DSB and DBDB[6] are 3.6 Å, whereas that of PPB is 3.7 Å. We define the tilt angle between the central and outer benzenes in adjacent molecules as α and β, respectively (Figure 7a). The magnitudes of α follow the order DBDB[6] > PPB > DSB, whereas those of β follow PPB > DSB > DBDB[6] (Figure 7b). In PPB, the electronic interactions are weak because the molecule exhibits a d chr value larger than those of the other compounds, and α and β are > 70°, suggesting large dihedral angles between adjacent molecules. Thus, the quantum yield of PPB is higher than those of the other DSBs. Conversely, DSDMB adopts a face-to-face zigzag column structure. In DSDMB, the d chr value is 3.7 Å, which is the same as that of PPB, but stronger electronic interactions exist compared to those of PPB owing to the faceto-face structure, which might explain its low Φ solid value. DBDB[7] has unique crystal structures differing from those of any DSB derivatives, and exhibits many excellent characteristics. The introduction of flexible cyclic structures into π-cores contributes to diversity in crystal chemistry.

Theoretical study
We calculated the transition from the ground to the excited states of a single molecule with the configuration it takes in a single crystal using time-dependent self-consistent field theory at the ωB97XD/6-311G (d,p) level of theory. The calculated absorption wavelengths in the solid state are similar to the experimental values (Table S10). It is challenging to precisely assess the absorption wavelengths and molecular packing structures in the solid state using low-resolution diffuse-reflection spectra or these calculations. Therefore, herein we present a qualitative discussion of the photophysical property trends. Each of the compounds exhibited an S o !S 1 transition in which the highest occupied molecular orbital (HOMO)-lowest unoccupied molecular orbital (LUMO) transition was dominant (Table S10). The HOMO and LUMO electron cloud distributions and energy levels are shown in Figures S31 and S32 as well as Table S11. In DBDMDB[7], π-conjugation is localized and not extended. In contrast, in the other compounds, π-conjugation is extended over the entire molecule. Because ϕ i of DBDMDB[7] is > 90°, the π-conjugation is localized within the outer rings. Considering the HOMO and LUMO energies of all the compounds with respect to those of DSB, the HOMO energies are within � 0.4 eV but the LUMO energies differ by up to 1.5 eV. The HOMO energy levels of DBαMDB[7]-cross and DBαMDB[7]parallel, in particular, are similar, although their molecular structures differ considerably.
We calculated the overlap between the natural bond orbitals (NBOs) of adjacent molecules for the DSBs, DBDB[7], DBDMDB[7], and DBαMDB[7]-cross/parallel in single crystals at the ωB97X/6-311G(2d,2p) level of theory ( Figures S33-S39). We analyzed only the overlap between occupied and unoccupied C=C bonding orbital of adjacent molecules because our focus was the molecular light-emitting behaviors in the solid state. Overlap between the NBOs in DSDMB was observed, but in DBDMDB [7], with the introduction of the bridging structure, NBO overlap was not observed. The NBO overlap of DBαMDB[7]-parallel is 0.49 kcal mol À 1 less than that of PPB; conversely, that of DBDB[7] is 0.79 kcal mol À 1 greater than that of DSB, and that of DBαMDB[7]-cross is 0.63 kcal mol À 1 greater than that of PPB.
These results are not in accordance with the results of our photophysical study, which indicate that there is little electronic interaction between individual DBDB[7] molecules. However, NBO analyses evaluate the states of bonding orbitals isolated from the stabilizing structure of the π-conjugated system. Therefore, it is reasonable to suggest that the DBDB[7] molecules exist in unique crystal structures with few intramolecular electronic interactions, despite the close proximity of their adjacent π-conjugated planes.

Conclusion
We have successfully synthesized bridged DSBs (DBDB[7]s), and crystal structures with compact packing, and solid-state monomeric fluorescence spectra similar to the dilute-solution fluorescence spectra were obtained. Unlike conventional design methods based on the use of bulky functional groups or capsules to isolate dyes, introducing small functional groups (flexible propylene bridges) to a π-conjugated system yielded monomeric emission in the solid state. The π-distortion and appropriate degree of steric hindrance generated by the flexible propylene bridges enabled the DBDB[7]s to form compact crystal structures with few intermolecular electronic interactions. The DBDB[7]s also possess the following valuable characteristics: 1) AIE character (Φ solid > 0.80); 2) low melting Figure 7. a) Arrangements of adjacent DSB, [14] PPB, [15] and DBDB[6] molecules in the solid state. The angles between the central (top row, α) and outer (bottom row, β) benzene rings are defined. b) Values of α and β for each compound. c) Definition of d chr .
points (Table S7) and good processability because they can adopt many conformations owing to their flexible cyclic structures; and 3) they are not mechanochromic. The fact that they are not mechanochromic means that these dyes have no limitations in terms of the processing methods that can be used in applications, and hence they can be incorporated into materials and devices. In other words, their application performance is, to a certain extent, independent of the environment. These unique properties suggest that the DBDB[7] molecules will contribute to future material design strategies. For example, a desired luminescence color can be generated by arranging multiple dyes with different luminescence wavelengths without them interacting with each other. The crystal structure obtained by introducing flexible cyclic structures to π-cores will depend on the selected π-electron molecule. This strategy dramatically expands the diversity of crystal structure fabrication approaches.
We plan to introduce alkylene bridges to various skeletons and further study the crystal chemistry of this class of molecules in the future. Development of the flexible-alkylene-bridge concept should reveal novel functions for materials based on hardly soluble rigid-rod π-conjugated molecules and polymers. [27] For these rod-shaped molecules, there is a tradeoff between improving the processability and molecular alignment; this is an obstacle to improving device performance that we are currently attempting to resolve.

Experimental Section
Measurement methods of photophysical properties: UV-Vis spectra were recorded on a JASCO V-670 UV-vis spectrophotometer, and fluorescence spectra were recorded on a JASCO FP-6500 spectrofluorometer. The wavelengths obtained by the fluorescence spectrometer were converted to wavenumber using the equation I(ṽ) = λ 2 I(λ) 4 . Absolute quantum yields were measured by a Hamamatsu Photonics Quantaurus QY apparatus.
All photophysical measurements were performed using dilute solutions with optical densities (ODs) around 0.1 at the maximum absorption wavelength in 1 cm path length quartz cells at room temperature (298 K). In addition, all sample solutions were deaerated by bubbling with argon gas for 15 min prior to the quantum yield.
Emission lifetimes were obtained using a Horiba FluoroCube timecorrelated single-photon counting system. The excitation light sources were LED pulse lamps (NanoLED, 269 and 379 nm). The solution samples were dissolved in THF and purged with argon for 20 min before lifetime measurements. In all samples, the time-toamplitude converter ranges were 50 ns, and the amounts of counts were 10000. Reptation rates were 1 MHz in solution samples and 100 kHz in solid samples.
Diffuse-reflectance spectra were recorded on a JASCO FP-6500 spectrofluorometer equipped with an integration sphere detector. Thus, the experimental error arising from the fluorescence, typically encountered with diffuse-reflectance spectrometers producing polychromatic outgoing light, was avoided. Samples and references were charged in a JASCO powder sample cell to obtain a sufficiently thick powder layer. Just before each measurement, the synchronous spectrum of the NaBr powder was measured as a reflectance spectrum of a standard reference r standard (λ). Then, a reflectance spectrum of each sample r sample (λ) was obtained following the same procedure. In addition to neat powder samples, the spectra of samples adsorbed on the NaBr powder were measured at a concentration of 1.0 × 10 À 3 M. The obtained reflectance spectra r sample (λ) and r standard (λ) were converted to Kubelka-Munk functions f(r ∞ ) by using the following equation:

Keywords: aggregation-induced emission
· crystal engineering · distyrylbenzene · organic light-emitting devices (OLEDs) · solid state fluorescence peak wavelength was apparently measured, it should be recalled that a commercially available fluorescence spectrometer possesses a measurement error of 3-5 nm, and hence the observed difference should be neglected.
[26] Determining whether organic dyes strictly exhibit monomeric emission is difficult; however, luminescence spectrum changes caused by an intermolecular electronic interaction should be observable. The photophysical properties of PMMA films doped with a DBDB[7] (0.1 wt %) were measured (Figures S15-S19 and Table S5), and λ abs and λ fl of almost all these films were similar to those in THF and the solid state, respectively.


A Rapid Fluorescence Sensor for the Direct Quantification of Rongalite in Foodstuffs

Rongalite was reported illegally used as a food additive for bleaching purposes and improving the tenderness of foodstuffs, which may endanger public health. At present, rongalite was mostly detected by indirect methods via derivatization or determining its decomposition products. In this study, we developed a new fluorescence sensor for the direct quantification of rongalite based on the principles: (1) dopamine reacts with resorcinol and generates strong fluorophore (azamonardine); (2) rongalite could inhibit the production of fluorophores and then result in lower fluorescence intensity. Hence, the rongalite concentration was inversely proportional to fluorescence intensity of fluorophore. Several crucial reaction conditions of fluorescence sensor were further optimized, such as dopamine and resorcinol concentration, pH values, and reaction time. Under the optimal conditions, the limit of detection of fluorescence sensor was 0.28–0.38 μg/g in vermicelli, wheat and rice powder samples, exhibiting almost 3.5-fold improvement compared to that of lateral flow immunoassay. Moreover, the detection time was substantially decreased to 20 min. The recoveries in spiked samples were 80.7–102.1% with a coefficient of variation of less than 12.6%. In summary, we developed a direct, high throughput, selective and accurate fluorescence sensor that poses a promising application for the rapid detection of rongalite in foodstuffs.

Introduction
Rongalite, also known as sodium formaldehyde sulfoxylate, is a versatile and commercially available reagent, which is typically used as an industrial bleaching agent for vat dyeing as well as a green reagent in organic synthesis [1][2][3]. Although rongalite possesses diverse applications, its addition to food as a decolourant is prohibited. This is because the unstable rongalite in acidic solution and the heated condition is decomposed in equimolar amounts into formaldehyde and sodium bisulfite [4,5], both of which are serious threats to human health. As one of the decomposition products, formaldehyde plays a key role in food preservation but is associated with pain, nausea, dermatitis, central nervous system injury, and carcinogenic effects when the human body ingests formaldehyde in excessive quantities [6,7]. Sodium bisulfite, another decomposition product, is used as a color stabilizer and antioxidant in the food industry, however, it may generate harmful gas such as H 2 S and SO 2 under certain conditions [8]. Rongalite possesses similar physical and chemical properties to both formaldehyde and sodium bisulfite, resulting in the motive for the abusive and substitutional use of rongalite in the food processing industry. As reported, the rongalite was illegally used as a food additive for bleaching purposes and to improve the tenderness of wheat powder, rice powder, tofu, vermicelli, and other foodstuffs, which caused adverse effects on human health in different countries [9,10]. Thus, it is urgent and necessary to develop a reliable, rapid, and sensitive detection method for rongalite analysis in food samples.
Until now, the analysis method for rongalite includes indirect and direct methods. The indirect methods generally defined the decomposition products (formaldehyde and sodium bisulfite) as detection makers rather than rongalite directly, maybe leading to ambiguous and inaccurate detection results [11,12]. In addition, the indirect methods mainly consisted of instrumental methods such as capillary electrophoresis [13] and high-performance liquid chromatography [14], which suffer from sophisticated instrumentation and tedious sample preparation. Hence, the indirect methods were not a promising method for accurate and convenient analysis of rongalite. Recently, several direct methods that adopted rongalite itself as the detection maker were reported as alternative methods for the rapid detection of rongalite. For instance, based on a rongalite-specific aptamer, Li et al. established a sandwich lateral flow strip (LFIA) assay with the limit of detection (LOD) of 1.0 µg/mL [15]; Jing et al. developed a sandwich-type enzyme-linked immunosorbent assay (ELISA) with the LOD of 0.57 ng/mL (Table S1) [16]. Although the aptamer-participated methods were rapid and sensitive, the key reagent aptamer with high specificity and affinity was hard to be prepared. Recently, Abubakar et al. reported a fast but low-flux electrochemiluminescence (ECL) method for the detection of rongalite in foodstuffs with a LOD of 0.07 µg/mL (Table S1) [9]. Nowadays, with the increasing demand for quicker and higher throughput screening of rongalite, ongoing effort should be focused on developing new and generic detection methods, and the rongalite residue in food should be timely assessed.
Fluorescence-based sensing platforms were a promising alternative system for achieving rapid implementation, sensitive and high-throughput detection of chemical contaminants [17,18]. In fluorescent analysis, fluorophores serve as an important signal reporter and achieve the goal of quantitative analysis through "turn on" or "turn off" responses. Recently, the fluorophores that were prepared through a one-step bimolecular reaction attracted lots of attention and were used in the analysis field [19,20]. For example, Acuna et al. described a strong yield fluorophore (azamonardine product) that was prepared by a simple reaction of resorcinol and catecholamines, and then the fluorophore could be used for analytical and imaging techniques [21]. Based on the above study, some researchers employed dopamine (or resorcinol) as the catalytic product of tyrosinase (or alkaline phosphatase), and then added the incorporating substrate to induce a fluorescence response [22][23][24][25].
Inspired by the above research, we propose that the introduction of a strong reductant (rongalite) could inhibit the introduction of fluorophores, achieving the quantification of rongalite in foods. Herein, we explored the fluorescence intensity response between fluorophore and diversity concentrations of rongalite and construct a simple on-site detection system for the rapid detection of rongalite in vermicelli, wheat and rice powder. This research provides a new strategy for the determination of rongalite in foods quality control.

Materials and Apparatus
Rongalite, dopamine hydrochloride and resorcinol were purchased from Macklin Company (Shanghai, China). Formaldehyde solution (37-40%) was obtained from J &K Scientific (Beijing, China). Glucose, sucrose, glutamate, prolamin and Ca 2+ were supplied by Aladdin Industrial Corporation (Shanghai, China). Sodium carbonate, sodium hydroxide and hydrochloric acid were purchased from Sinopharm Chemical Reagent Co., Ltd. (Beijing, China). All the reagents were analytical grade.
Sterile syringe filters (0.45 µm) were obtained from Merck (Merck Millipore, Burlington, MA, USA). White opaque microplates were supplied by Corning Ltd. (Corning Life Science, Hartford, NY, USA). Ultra-pure water was used in all aqueous solution and was produced by Millipore purification system (MilliQ gradient A10, Millipore, Bedford, MA, USA). Fluorescence excitation and emission spectra, absorption spectra, optical density, and fluorescence intensity were measured using a microplate reader (SpectraMax M2e, Molecular Devices, San Jose, CA, USA). The pH values were measured by a pH meter

Characterization of the Fluorescence Sensing Platform
Dopamine and resorcinol were dissolved in ultra-pure water at the concentrations of 50 and 20 µM, respectively. The dopamine (30 µL) and resorcinol (30 µL) solution were injected into the microplates, followed by the addition of 200 µL NaOH solution (pH 9.5) or rongalite standard solution (5 µg/mL, in NaOH solution, pH 9.5). The mixture solution was incubated in a metal bath at 45 • C for 20 min before detection. The absorption spectrum, fluorescence spectrum, optical density (OD), and fluorescence intensity of four solutions (dopamine, resorcinol, mixture solution of dopamine and resorcinol, mixture solution of dopamine, resorcinol and rongalite, 260 µL) were measured using a microplate reader. Note that dopamine hydrochloride was abbreviated to dopamine in this paper.

Dopamine and Resorcinol Concentration-Response
The checkerboard assay and two-point (0 and 0.25 µg/mL rongalite) competitive format was adopted to study the optimal concentrations of dopamine and resorcinol solution. Briefly, the concentrations of dopamine solution were 5, 10 and 50 µM with the resorcinol solution ranging from 1.25 to 40 µM. Then, the fluorescence intensity (λ ex , 420 nm, λ em , 465 nm) was measured, and the inhibition ratio (Equation (1)) at different concentrations was recorded to evaluate the response tendency.
where F 0 and F 1 are the fluorescence intensity of the wells in the absence of rongalite and in the presence of rongalite, respectively.

pH Values Optimization
The NaOH solution at different pH values (from 6 to 12, with a concentration gradient of 1) was prepared to explore the relationship between fluorescence intensity and pH values. The microplates were incubated at 45 • C for 20 min, and then the fluorescence spectrum (λ ex , 420 nm) and fluorescence intensity (λ ex , 420 nm, λ em , 465 nm) were measured, separately.

Carbonate Concentration Optimization
The carbonate solution was more stable than the NaOH solution, thus we further optimized the carbonate concentration at 0, 5, 7.5, 10, 12.5, 15, 17.5 and 20 mM. The detailed experiment procedure was similar to the "Dopamine and resorcinol concentration-response" section, except for replacing the NaOH solution with carbonate solution.

Reaction Time Optimization
The reaction time of the fluorescence sensing system was optimized to evaluate whether the reactions reached equilibrium. In total, six time points from the reaction process were selected (5, 10, 15, 20, 30 and 40 min), and the corresponding fluorescence spectrum and intensity were measured.

Fluorescence Sensing Platform Development
Under the optimal conditions, various rongalite solutions (sample extracts or other related compounds, 200 µL) at consecutive concentrations were added to the mixture reaction system of dopamine (30 µL) and resorcinol (30 µL) and incubated for 20 min. The fluorescence intensity (λ ex , 420 nm, λ em , 465 nm) was measured and the sigmoidal curve was fitted using a four-parameter logistic equation (Equation (2)) with the assistance of OriginPro 8.0 (Northampton, MA, USA). Some agents were evaluated to explore the where A and B represent the responses at high and low asymptotes of the curve, C stands for the concentration of the analytes resulting in 50% inhibition, D represents the slop factor, and X is the calibration concentration.

Sample Preparation
The sample preparations of vermicelli, wheat and rice powder were operated according to the previous study with some modifications [16]. 1.0 g wheat or rice powder (vermicelli was ground into powder) was dispersed in 5 mL carbonate solution (2 mM, pH 10), and the mixture was maintained for 10 min under ultra-sonication (40 • C). After 15 min of centrifugation at 10,000 rpm, the supernatant solution (1.0 mL) was collected and filtered with sterile syringe filters (0.45 µm), diluted with carbonate solution 4 times, and then subsequently submitted to the fluorescence sensing platform for the rongalite detection.

Limit of Detection
Negative samples were supplied and verified by Anhui Public Inspection Research Institute Co., Ltd. (Hefei, China). The LOD was defined as the concentration corresponding to the average fluorescence intensity of 20 negative samples plus 3 times the standard deviation (three independent experiments) [26,27].

Method Validation
Three samples, including vermicelli, wheat and rice powder were spiked with rongalite at different concentrations. After sample pretreatment, the samples were detected using the fluorescence sensing platform. Subsequently, the recovery ratios and variations in intraand inter-assays were calculated.

Assay Principle for the Fluorescence Sensing Platform
According to the previous reports, under the alkaline condition, the dopamine could react with resorcinol within several minutes, producing in situ azamonardine fluorophores via a nucleophilic attack (shown in Scheme 1) [25]. In this research, we introduced a rongalite solution in the reaction system, and a surprising decreased optical density and fluorescence intensity were observed in Scheme 1. In detail, the absorption peaks of dopamine and resorcinol were both 351 nm (Figure 1a). A new absorption peak for the reaction product of dopamine and resorcinol was 415 nm with an optical density of 1.06, and its optical density was weakened with the addition of rongalite. Subsequently, the fluorescence signal of the above solutions was further characterized. The maximum excitation and emission wavelengths of the product solution were centered around 420 nm and 465 nm ( Figure 1b). It is obvious that the fluorescence intensity (1,3125) decreased to 625 since the addition of rongalite. Thus, the in situ system could be used for the quantification of rongalite in samples. We finally selected the fluorescence system for the subsequent research rather than optical signals because the fluorescence system has higher sensitivity owing to its high signal-to-noise (S/N) ratio. rongalite in samples. We finally selected the fluorescence system for the subsequent research rather than optical signals because the fluorescence system has higher sensitivity owing to its high signal-to-noise (S/N) ratio. Scheme 1. Reaction mechanisms of fluorescence sensor for rongalite detection. Resorcinol reacts with dopamine and produces blue fluorophore (azamonardine) under alkaline conditions. The fluorescence intensity of azamonardine sharply decreased with the addition of rongalite, and the standard curve is constructed based on fluorescence intensity. Subsequently, the rongalite concentration in foodstuffs is measured via microplate reader. We hypothesize that there existed two possibilities for the addition of rongalite to reduce the fluorescence intensity: (1) the rongalite acts as a protective agent and prevents the oxidizing of dopamine or reduce the reaction product, resulting in the decreased generation of azamonardine fluorophores; (2) the rongalite undergoes a reduction reaction with dopamine or resorcinol, which reduce the concentration of reaction substrate and inhibited the production of fluorophores. To illustrate the principle of the in situ fluorescence assays, the chemicals in the reaction system were characterized using HPLC-MS. rongalite in samples. We finally selected the fluorescence system for the subsequent research rather than optical signals because the fluorescence system has higher sensitivity owing to its high signal-to-noise (S/N) ratio. Scheme 1. Reaction mechanisms of fluorescence sensor for rongalite detection. Resorcinol reacts with dopamine and produces blue fluorophore (azamonardine) under alkaline conditions. The fluorescence intensity of azamonardine sharply decreased with the addition of rongalite, and the standard curve is constructed based on fluorescence intensity. Subsequently, the rongalite concentration in foodstuffs is measured via microplate reader. We hypothesize that there existed two possibilities for the addition of rongalite to reduce the fluorescence intensity: (1) the rongalite acts as a protective agent and prevents the oxidizing of dopamine or reduce the reaction product, resulting in the decreased generation of azamonardine fluorophores; (2) the rongalite undergoes a reduction reaction with dopamine or resorcinol, which reduce the concentration of reaction substrate and inhibited the production of fluorophores. To illustrate the principle of the in situ fluorescence assays, the chemicals in the reaction system were characterized using HPLC-MS.  We hypothesize that there existed two possibilities for the addition of rongalite to reduce the fluorescence intensity: (1) the rongalite acts as a protective agent and prevents the oxidizing of dopamine or reduce the reaction product, resulting in the decreased generation of azamonardine fluorophores; (2) the rongalite undergoes a reduction reaction with dopamine or resorcinol, which reduce the concentration of reaction substrate and inhibited the production of fluorophores. To illustrate the principle of the in situ fluorescence assays, the chemicals in the reaction system were characterized using HPLC-MS. The dopamine hydrochloride and resorcinol exhibited ion peaks at 188.0 and 109.0 (m/z, [M-H] − ) ( Figure S1a,b). The ion peaks of rongalite couldn't be found at 117 (m/z, [M-H] − ), because of the high-temperature vaporization of rongalite. As shown in Figure S1c, the intense ion peak at 258.1 (m/z, [M-H] − ) was attributed to azamondardine (reaction product of dopamine and resorcinol, without purification), which was consistent with the previous research [25]. The intensity of azamondardine sharply decreased from 8.9 × 10 7 to 4.0 × 10 3 , when rongalite was presented in the reaction system ( Figure S1d). Moreover, we observed that the mixture of dopamine and rongalite, or resorcinol and rongalite wouldn't produce a new chemical reaction. This was because the ion peaks intensity of dopamine and resorcinol remained almost unchanged, and moreover there didn't exist new ion peaks ( Figure S2a,b). Herein, our first supposition, whereby rongalite served as a protective agent and prevented the yielding of azamonardine fluorophores, was true and proved to be a new strategy for the detection of rongalite.

Dopamine and Resorcinol Concentration-Response
Dopamine and resorcinol concentration-response is closely related to the sensitivity of the fluorescence sensing platform. In theory, the higher concentration of dopamine and resorcinol would generate bright fluorescence, resulting in faint competitive affection of rongalite and lower sensitivity. On contrary, rongalite at low concentrations could inhibit the yield of fluorescence when a lower concentration of reactant was used. In this case, the fluorescence sensor possesses excellent sensitivity but is unstable due to the obvious variation coefficient of lower fluorescent intensity. Here, we explored the dopamine and resorcinol concentration-response by analyzing the variation of fluorescent intensity via a two-point competitive format.
The fluorescent intensity heightened evidently with the concentration of dopamine varying from 5 to 50 µM and resorcinol changing from 1.25 to 40 µM, due to the reaction product (azamonardine fluorophores) increased. However, the corresponding fluorescent intensity also increased in the presence of rongalite (Figure 1c). It is hard to select the superior point of dopamine and resorcinol concentration-response. Subsequently, the inhibition ratio was evaluated and described in Figure 1d. We found that dopamine concentration has an obvious effect on the inhibition ratio at the same resorcinol concentration. The lower inhibition ratio (6.7-29.7%) was observed when dopamine concentration was 50 µM. There was no significant difference in terms of inhibition ratio (44.9-52.2%) when resorcinol concentration ranged from 2.5 to 20 µM and dopamine concentrations were 5 and 10 µM. Finally, the 5.0 µM and 10.0 µM were separately selected as the optimal response concentrations for dopamine and resorcinol, considering the appropriate fluorescence intensity (388) and the highest inhibition ratio values (52.2%).

Optimization of Reaction pH, Carbonate Concentration and Response Time
Three experiment parameters, including reaction pH, carbonate concentration and response time, were further optimized for improving the stability, sensitivity and saving the reaction time of the fluorescence sensor.
The pH values were a vital parameter for the reaction system because the formation of azamonardine relies on a rigid alkaline condition. Here, we investigated the effect of pH values on fluorescence intensity under the optimal dopamine and resorcinol concentrationresponse. The fluorescence intensity raised following the increase of pH values (Figure 2a), and its corresponding values at 465 nm were exhibited in Figure 2b. Briefly, the fluorescence intensity was close to 0, when the pH values were 6 and 7. This result indicated the chemical reaction between dopamine and resorcinol was hardly to occur under acidity or neutral conditions, which was consistent with previous research [20]. Meanwhile, the inhibition ratio was low (−2.2% to 28.9%, Figure 2c). In the absence of rongalite, the fluorescence intensity increased from 117 to 488 when the pH values ranged from 8 to 12; however, the fluorescence intensity in the presence of rongalite (0.25 µg/mL) also increased at the same time ( Figure S3). Thus, the inhibition ratios increased followed by a decrease. A higher inhibition ratio usually means higher sensitivity. Hence, 10 was selected as the optical reaction pH value for the fluorescence sensor with an inhibition ratio of 54.3%. Generally, the buffer solution plays a vital role in fluorescence analysis, which furnishes stable and accurate detection conditions by sustaining the pH and ionic strength at constant values [28]. In this research, the carbonate buffer solution served as the system buffer solution, considering it was the frequently used alkaline buffer solution. We hypothesize the ionic strength at different concentrations might interfere with the stability of the fluorescence signal. Here, we explored the affection of ionic strength concentration of carbonate buffer (pH 10) on fluorescence intensity and inhibition ratio. The discrete degree of fluorescence signal was low at different ionic strength concentrations, illustrating the ionic strength did not affect the stability of the fluorescent signal (Figures 2d,e, and  S4). However, a slight variation in fluorescence intensity and inhibition ratio was observed, which confused us. Finally, the ionic strength at 2 mM contributed to the higher inhibition ratio (54.9%) and was selected as the excellent concentration (Figure 2f).
The fluorescence signal of the product will continue to rise or be unstable if the reaction has not reached equilibrium. Consequently, we evaluated the response time to develop an accurate and constant fluorescence sensor. As shown in Figure 3a,b, the fluorescence signal sharply increased from 136 to 438 between 5 min and 20 min and tended to be balanced at 20 min. Aiming to improve detection efficiency, we choose 20 min as the optical response time. Generally, a rapid detection method could provide a report in <25 min [29,30]. Therefore, our fluorescence sensor has obvious advantages in terms of saving time. Generally, the buffer solution plays a vital role in fluorescence analysis, which furnishes stable and accurate detection conditions by sustaining the pH and ionic strength at constant values [28]. In this research, the carbonate buffer solution served as the system buffer solution, considering it was the frequently used alkaline buffer solution. We hypothesize the ionic strength at different concentrations might interfere with the stability of the fluorescence signal. Here, we explored the affection of ionic strength concentration of carbonate buffer (pH 10) on fluorescence intensity and inhibition ratio. The discrete degree of fluorescence signal was low at different ionic strength concentrations, illustrating the ionic strength did not affect the stability of the fluorescent signal (Figures 2d,e and S4). However, a slight variation in fluorescence intensity and inhibition ratio was observed, which confused us. Finally, the ionic strength at 2 mM contributed to the higher inhibition ratio (54.9%) and was selected as the excellent concentration (Figure 2f).
The fluorescence signal of the product will continue to rise or be unstable if the reaction has not reached equilibrium. Consequently, we evaluated the response time to develop an accurate and constant fluorescence sensor. As shown in Figure 3a,b, the fluorescence signal sharply increased from 136 to 438 between 5 min and 20 min and tended to be balanced at 20 min. Aiming to improve detection efficiency, we choose 20 min as the optical response time. Generally, a rapid detection method could provide a report in <25 min [29,30]. Therefore, our fluorescence sensor has obvious advantages in terms of saving time.

Development of Fluorescence Sensor
Under the optimal conditions, a series of rongalite concentrations were prepared for construct a standard curve, i. e., 0, 0.01, 0.03, 0.09, 0.27, 0.81, 1.30 and 2.43 μg/mL. The fluorescence intensity faded with the increase of the rongalite concentration in the buffer

Development of Fluorescence Sensor
Under the optimal conditions, a series of rongalite concentrations were prepared for construct a standard curve, i.e., 0, 0.01, 0.03, 0.09, 0.27, 0.81, 1.30 and 2.43 µg/mL. The fluorescence intensity faded with the increase of the rongalite concentration in the buffer solution (Figure 4a,b). Subsequently, a standard curve was fitted using a four-parameter logistic equation (Figure 4c) with the half inhibitory concentration of 0.22 µg/mL (R 2 , 0.993), and its linear range was 0.05 µg/mL to 0.90 µg/mL (marked with the blue outline).

Development of Fluorescence Sensor
Under the optimal conditions, a series of rongalite concentrations were prepared for construct a standard curve, i.e., 0, 0.01, 0.03, 0.09, 0.27, 0.81, 1.30 and 2.43 μg/mL. The fluorescence intensity faded with the increase of the rongalite concentration in the buffer solution (Figure 4a,b). Subsequently, a standard curve was fitted using a four-parameter logistic equation (Figure 4c) with the half inhibitory concentration of 0.22 μg/mL (R 2 , 0.993), and its linear range was 0.05 μg/mL to 0.90 μg/mL (marked with the blue outline) We explored the selectivity of fluorescence sensor by analyzing some reducing agents (formaldehyde, sodium bisulfite), and matrix factors (glucose, sucrose, glutamate prolamin, Ca 2+ ). As shown in Figure 3c, although formaldehyde and sodium bisulfite displayed a response to the fluorescence intensity, the response concentration that made the signal decrease in half was higher than 10 μg/mL. Hence, these interfering factors could be recognized as the negligible response. Other interfering factors would not generate the signal response, which indicated the fluorescence sensor was selective enough for the rongalite detection.
Compared with the instrumental analysis, a simple and convenient sample preparation method was a crucial section for rapid detection [31,32]. Generally, for combined forms of analytes and samples, the analytes in samples were extracted using organic solvent followed by dilution; otherwise, they were extracted with buffer solution for dissociated forms of analytes and samples or analytes with higher water solubility. In this research, the carbonate buffer solution (pH 10, 2 mM) was selected to extract the rongalite in samples. The matrix effect of the extract was expected to be removed by dilution and was explored in two steps. Firstly, the extraction solution of samples was diluted with carbonate solution for 2, 4 and 8-fold, and then its fluorescence intensity was measured using the fluorescence sensing platform. Owning to the matrix factors could interfere with the trigger of fluorescence signal, hence, the lowest dilution corresponding to the fluorescence intensity similar to that in buffer solution was preliminarily selected as the point We explored the selectivity of fluorescence sensor by analyzing some reducing agents (formaldehyde, sodium bisulfite), and matrix factors (glucose, sucrose, glutamate, prolamin, Ca 2+ ). As shown in Figure 3c, although formaldehyde and sodium bisulfite displayed a response to the fluorescence intensity, the response concentration that made the signal decrease in half was higher than 10 µg/mL. Hence, these interfering factors could be recognized as the negligible response. Other interfering factors would not generate the signal response, which indicated the fluorescence sensor was selective enough for the rongalite detection.
Compared with the instrumental analysis, a simple and convenient sample preparation method was a crucial section for rapid detection [31,32]. Generally, for combined forms of analytes and samples, the analytes in samples were extracted using organic solvent followed by dilution; otherwise, they were extracted with buffer solution for dissociated forms of analytes and samples or analytes with higher water solubility. In this research, the carbonate buffer solution (pH 10, 2 mM) was selected to extract the rongalite in samples. The matrix effect of the extract was expected to be removed by dilution and was explored in two steps. Firstly, the extraction solution of samples was diluted with carbonate solution for 2, 4 and 8-fold, and then its fluorescence intensity was measured using the fluorescence sensing platform. Owning to the matrix factors could interfere with the trigger of fluorescence signal, hence, the lowest dilution corresponding to the fluorescence intensity similar to that in buffer solution was preliminarily selected as the point eliminating the matrix effect of samples. Compared with the fluorescence intensity in buffer solution (in the absence of rongalite), 2-fold dilution of extraction solution exhibited obvious matrix effect along with the decreased signal ( Figure S5). Nevertheless, 4-and 8-fold dilution could avoid the matrix effect. Obviously, a 4-fold dilution appeared to avoid the matrix effect. Secondly, the matrix calibration curve was constructed via spiking rongalite at different concentrations in the 4-fold extraction solution. As shown in Figure S6, all the extraction calibration curves were in good agreement with the standard curve in buffer solution, which indicated the 4-fold dilution eliminated the matrix effect. Eventually, a 4-fold dilution of extraction solution was selected as the preferred preparation method for samples considering the higher sensitivity of the fluorescence sensor.
Detailed parameters about average fluorescence intensity of 20 negative samples and standard deviation were displayed in Table S2. Subsequently, the calculation results were submitted to the standard curve and obtained the LOD. Thus, the LOD was measured after Foods 2022, 11, 2650 9 of 12 sample preparation, and was calculated as 0.38, 0.28 and 0.32 µg/g for vermicelli, wheat and rice powder, and the linear range was about 1-18 µg/g.
Despite possessing sensitive properties, the reported indirect assays generally quantified rongalite via derivatization or determining its decomposition products [11]. In contrast, our research could achieve the goal of direct and rapid detection of rongalite. Here, we summarized several direct and rapid detection methods for rongalite in foodstuffs and compared them with our fluorescent assay (Table S1). The proposed fluorescence sensor displayed an excellent performance in LOD (0.28-0.38 µg/g) compared with that of the lateral flow immunoassay (1 µg/mL) but exhibited higher LOD compared with other assays. Actually, LOD is not the only criterion for evaluating assay performance; therefore, other parameters should be taken into accounts, such as the required materials and linear range for the assay and the pretreatment procedure for samples. Among the assays, the ECL method has the widest linear range (4.7-118 µg/mL) [9]. It was also observed that the dilution factor was 40, which means the ECL method was susceptible to the matrix effect of samples and the stability of the detection result might be interfered. Comparatively, our proposed fluorescent sensor possessed higher matrix tolerance with a dilution factor of 20. The reported ELISA displayed an excellent LOD of 0.57 ng/mL. However, the experimental procedure was complex and the operation time was 110 min. Compared with ELISA, our fluorescent assay possessed the advantage in terms of less time (20 min), and saved 5.5-fold time. Actually, ELISA and LFIA both required aptamer as the recognition material. As reported, aptamer was usually acquired through multi-round exponential enrichment, its screening procedure being full of considerable workload. Preparing two aptamers to recognize different sites of the small molecule was especially a challenge. Thus, the ELISA and LFIA for quantification of rongalite based on aptamer both need a particular material, which might restrict its wide application in foodstuffs safety testing. In contrast, the fluorescent assay in this study adopted the traditional chemicals (dopamine, resorcinol) as the substrate, and exhibited a satisfactory detection time, high throughput, sensitivity as well as accuracy, possessing irreplaceable properties.

Accuracy of the Fluorescence Sensor
As previously reported, there are two main approaches to verify the accuracy of the rapid detection method: (1) detecting real samples using both instrumental analysis methods and rapid detection methods simultaneously, and comparing the consistency or correlation of detection results; (2) spiking the analyte in negative samples at different concentrations for preparing positive samples and measuring the recovery ratio using rapid detection method [33,34]. In this research, the accuracy of the proposed fluorescence sensor was analyzed by recovery ratios and the variations in intra-and inter-assays. The rongalite-contaminated samples were prepared by spiking 1, 2, and 4 µg/g in vermicelli, wheat and rice powder samples, respectively. The recovery ratio was 80.7-102.1% with a coefficient of variation of less than 8.6% (Table 1), which illustrates the fluorescence sensor was a promising method for rapid detection of rongalite in samples.

Conclusions
In conclusion, we developed a fluorescence sensor for the detection of rongalite within 20 min. This assay was constructed by introducing the chemical reaction system of dopamine, resorcinol and rongalite, the higher concentration of rongalite could inhibit the production of azocine fluorophores and then result in lower fluorescence intensity. Hence, the rongalite concentration was inversely proportional to fluorescence intensity of fluorophore, this detection mechanism was firstly proposed by our team. The fluorescence sensor possessed the advantages of high throughput, easy operation and rapid, exhibited a LOD of 0.28-0.38 µg/g in vermicelli, wheat and rice powder samples, respectively. The recovery ratio was higher than 80.7% with a coefficient of variation of less than 12.6%, which could satisfy the accuracy of the rapid detection. The novelty of this research was that we constructed a new fluorescence sensor, which contributed to the superiority of higher sensitivity, easy operation, time-saving and low cost for the detection of rongalite. This fluorescence sensor could be a potential tool for the rapid measurement of rongalite in foodstuffs.

Supplementary Materials:
The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/foods11172650/s1, Figure S1: HPLC-MS of dopamine hydrochloride, resorcinol and mixture solution; Figure S2: HPLC-MS of the mixture of dopamine hydrochloride and rongalite, the mixture of resorcinol and rongalite; Figure S3: fluorescence intensity at different pH values; Figure S4: fluorescence intensity at different carbonate solution concentrations; Figure S5: matrix effect of vermicelli, wheat and rice powder for the fluorescence sensor; Figure S6: matrix calibration curve of vermicelli, wheat and rice powder; Table S1: an overview of the reported method for the rapid detection of rongalite; Table S2: limit of detection of three samples using fluorescence sensor.