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Inhibitory Mechanism of Baicalein on Acetylcholinesterase: Inhibitory Interaction, Conformational Change, and Computational Simulation

Alzheimer’s disease (AD) is the most prevalent chronic neurodegenerative disease in elderly individuals, causing dementia. Acetylcholinesterase (AChE) is regarded as one of the most popular drug targets for AD. Herbal secondary metabolites are frequently cited as a major source of AChE inhibitors. In the current study, baicalein, a typical bioactive flavonoid, was found to inhibit AChE competitively, with an associated IC50 value of 6.42 ± 0.07 µM, through a monophasic kinetic process. The AChE fluorescence quenching by baicalein was a static process. The binding constant between baicalein and AChE was an order of magnitude of 104 L mol−1, and hydrogen bonding and hydrophobic interaction were the major forces for forming the baicalein−AChE complex. Circular dichroism analysis revealed that baicalein caused the AChE structure to shrink and increased its surface hydrophobicity by increasing the α-helix and β-turn contents and decreasing the β-sheet and random coil structure content. Molecular docking revealed that baicalein predominated at the active site of AChE, likely tightening the gorge entrance and preventing the substrate from entering and binding with the enzyme, resulting in AChE inhibition. The preceding findings were confirmed by molecular dynamics simulation. The current study provides an insight into the molecular-level mechanism of baicalein interaction with AChE, which may offer new ideas for the research and development of anti-AD functional foods and drugs.

Introduction
Alzheimer's disease (AD), a progressive neurodegenerative disease featuring hidden onset, is clinically the most prevalent dementia form, which manifests as cognitive impairment, such as memory loss, out-of-control behaviour, and emotional disorder [1,2]. According to Alzheimer's Disease International's data in September 2021, the number of AD patients worldwide is 55 million. With the increase in the ageing population, the number of AD patients is estimated to reach 78 million by 2030 and 139 million by 2050, with a new AD case appearing every 3 s [3]. AD has become a major challenge impacting the health of older adults and has become a public health concern [4].
The aetiology of AD is currently unknown [5]. Scholars both at home and abroad have proposed multiple hypotheses about its pathogenesis, among which the "cholinergic hypothesis" is the most developed and well-known [6][7][8]. Degeneration of basal cholinergic neurons in the forebrain and loss of associated cholinergic neurotransmitters in the cerebral cortex and other regions lead to the degradation of cognitive function in AD patients [9]. Acetylcholinesterase (AChE, Acetylcholinesterase, E.C.3.1.1.7) is a serine protease that catalyses the hydrolysis of acetylcholine (ACh), an essential neurotransmitter in vivo, and decomposes it into acetic acid and choline, thus blocking nerve impulse transmission [10,11]. Foods high in flavonoids have been found to reduce the neurodegenerative pathological process of AD animal models and improve cognitive function [18]. Epidemiological studies have also demonstrated that eating a substantial amount of foods rich in flavonoids can improve normal cognitive function and reduce the incidence of dementia in the local population [19]. Flavonoids are highly effective in alleviating reversible neurodegenerative pathological processes and improving age-related cognitive decline [20] and thus have become a hot spot in the research and development of anti-AD drugs and functional food factors both at home and abroad.
Baicalein (5,6,7-trihydroxyflavone, as shown in Figure 1A) is an effective active component monomer of flavonoids extracted from the root of Scutellaria baicalensis Georgi [21], and it exerts numerous pharmacological effects, including antioxidant [22], scavenging oxygen free radicals [23], anti-inflammatory [24], antimicrobial [25], anti-carcinogenic [26] and neuroprotective effects [27]. The effects of baicalein on alleviating AD and improving cognition and brain protection have been reported [28][29][30]. Baicalein can activate the PI3K pathway, inhibit the levels of GSK3β and BACE1, reduce the content of total Aβ, and play a neuroprotective role in mice, thereby enhancing memory [31]. Wei [32] confirmed that baicalein can effectively alleviate cognitive impairment induced by Aβ  in rats and change the protein expression level in the cerebral cortex and hippocampus. Studies by Han [33] and other scholars have shown that baicalein is a selective and specific inhibitor of BACE and AChE that can be utilised to prevent and treat AD. Xie [34] investigated the Foods high in flavonoids have been found to reduce the neurodegenerative pathological process of AD animal models and improve cognitive function [18]. Epidemiological studies have also demonstrated that eating a substantial amount of foods rich in flavonoids can improve normal cognitive function and reduce the incidence of dementia in the local population [19]. Flavonoids are highly effective in alleviating reversible neurodegenerative pathological processes and improving age-related cognitive decline [20] and thus have become a hot spot in the research and development of anti-AD drugs and functional food factors both at home and abroad.
Baicalein (5,6,7-trihydroxyflavone, as shown in Figure 1A) is an effective active component monomer of flavonoids extracted from the root of Scutellaria baicalensis Georgi [21], and it exerts numerous pharmacological effects, including antioxidant [22], scavenging oxygen free radicals [23], anti-inflammatory [24], antimicrobial [25], anti-carcinogenic [26] and neuroprotective effects [27]. The effects of baicalein on alleviating AD and improving cognition and brain protection have been reported [28][29][30]. Baicalein can activate the PI3K pathway, inhibit the levels of GSK3β and BACE1, reduce the content of total Aβ, and play a neuroprotective role in mice, thereby enhancing memory [31]. Wei [32] confirmed that baicalein can effectively alleviate cognitive impairment induced by Aβ 1-40 in rats and change the protein expression level in the cerebral cortex and hippocampus. Studies by Han [33] and other scholars have shown that baicalein is a selective and specific inhibitor of BACE and AChE that can be utilised to prevent and treat AD. Xie [34] investigated the inhibitory effect of 20 flavonoids on AChE and explored their structure−activity relationship in the preliminary stages. However, the detailed molecular mechanism of baicalein against AChE is yet to be explored.
In the current study, UV-Vis absorption, fluorescence, and circular dichroism (CD) spectroscopy were used to investigate the inhibitory activity, inhibitory kinetics, binding properties, number of binding sites, type of acting force, and effect on the conformation of AChE by baicalein. A PC12 cytotoxicity test was performed to assess the safety of baicalein. For the interaction of baicalein with AChE, the molecular simulation was utilised to predict binding sites, binding conditions, and major amino acid residues. Molecular dynamics (M.D.) simulation was used to investigate the stability of protein skeleton, peptide flexibility, and free energy of amino acid residues before and after the binding of baicalein with AChE. The findings may provide novel insights into the mechanism of baicalein in AChE inhibition and serve as a reference for the research and development of flavonoids, such as baicalein, as anti-AD food functional factors and drugs.

Enzyme Activity Assay
AChE activity was evaluated per Ellman's method, albeit with a slight modification [35]. Quartz cuvettes (3 mL) were utilised for measuring the assay mixture activity. Solutions containing varying amounts of baicalein, phosphate-buffered saline (0.1 M, pH 7.6), and 50 µL of AChE (2 U/mL, 3.4 × 10 −8 M) were cultivated in 30 min at 25 • C. After the pre-incubation period, 50 µL of 5, 5-dithiobis-(2-nitrobenzoic) acid (DNTB, 5 mM) was added. The reaction was then initiated with 50 µL of acetylthiocholine iodide (ATCI, 15 mM). The absorbance at 412 nm was monitored every 5 s with the TU−1901 dual-beam UV-Vis Spectrophotometer (Persee; Beijing, China). The samples were measured thrice, and the average value was considered. Enzyme activity without baicalein was identified to be 100%. Herein, the measured half-inhibitory concentration (IC 50 ) for baicalein and the relative enzymatic activity agreed with those reported previously [36]. Galantamine hydrobromide served as the positive control.

Kinetic Analysis of the Inhibitory Type
To assess the kinetic mode of AChE inhibition displayed by baicalein, the inhibitory effect was determined with four different concentrations of baicalein (0, 4, 8, and 10 µM) when different substrate concentrations of ATCI coexisted in the same way of as the AChE activity assay. The Lineweaver-Burk plots and Michaelis-Menten enzyme kinetics were applied to analyse the inhibitory type. For competitive type inhibition, the dynamic double reciprocal equation was used as follows [37]: where (I) and (S) denote the inhibitor and substrate concentrations, respectively; ν represents the enzyme reaction rate irrespective of the presence of baicalein; ν max is the maximum catalytic reaction rate; K i , K m , and K app m stand for the inhibition constant, Michaelis−Menten constant, and apparent Michaelis constant, respectively.
The kinetic process and rate constants for enzyme inactivation were determined through the time-course test. Baicalein with concentrations of 2, 4, 8, and 16 µM was used for analysing the time-course. Enzyme activity at each concentration was measured every 3 min for 0-30 min, every 6 min for 30-60 min, and then at 70 and 80 min.

Fluorescence Spectrum Measurement
A spectrofluorometer (model F-7000; Hitachi, Tokyo, Japan) containing a thermostat bath was used to measure the fluorescence spectra. The AChE solution (2.5 mL; 0.17 µM) was introduced into a 1.0 cm path length quartz cell, followed by titration through the successive addition of 2.0 mM of diluted baicalein solution (to obtain the concentration range of 0-16 µM). The well-mixed solutions were set aside for three min for equilibration purposes. The fluorescence spectra were measured in the 290-450 nm range, and the relevant excitation wavelength set was 280 nm at 25 • C, 31 • C, and 37 • C. Excitation and emission bandwidths were 2.5 nm. In addition, synchronous fluorescence spectra for AChE with baicalein were conducted by fixing the interval of excitation and emission wavelength constants at 15 nm and 60 nm. Fluorescence data were amended according to our previous reports to erase the re-absorption possibility and the inner filter effects of UV absorption [38].

CD Spectra Measurements
Far-UV CD spectra were measured using a spectrometer (MOS 450 CD; Bio-Logic, Claix, France) with a 1.0 mm path length quartz cuvette. All the spectra were measured in the 200-240 nm range at 25 • C, and the associated scan speed was 60 nm min −1 . The AChE in the PBS was completely blended with baicalein at varying concentrations and then equilibrated for three min before CD measurements. The spectra were amended by subtracting the spectrum of the buffer with that of baicalein at the same concentration as the sample solution. Three scans were conducted, and the ellipticity in millidegrees indicated the results. The contents of the discrepant secondary structures (α-helix, β-sheet, β-turn, and random coil) from AChE were measured based on CD spectroscopic data following the online SELCON3 program (http://dichroweb.cryst.bbk.ac.uk/html/home.shtml. Accessed on: 18 August 2021) [39].

Cytotoxicity of Baicalein in PC12 Cells
PC12 cells were purchased from the NanJing KeyGen Biotech Co. Ltd. (Nanjing, China). These cells were cultivated in high-glucose DMEM supplemented with 10% FBS, 100 U/mL of penicillin and 100 µg/mL of streptomycin at 37 • C under a humidified 5% CO 2 atmosphere. Baicalein was dissolved in DMSO to prepare a 5 mM stock solution and diluted with serum-free media before use. PC12 cells were seeded into a 96-well plate at the density of 2 × 10 4 cells/well (100 µL) and cultured for 24 h. Next, the medium was changed, and the cells were treated with baicalein at 0, 1, 10, 20, 40, and 80 µM. Each concentration had six replicate wells, and the culture was continued for 24 h. To determine the effect of baicalein on cell viability, 20 µL of MTT (5 mg/mL) was added to each well. After 4 h, the resulting formazan crystals were dissolved by adding 150 µL of DMSO for 10-15 min. The absorption (O.D.) of each hole at 570 nm wavelength was measured using an enzyme-labelled instrument (Varioskan LUX; Thermo Fisher Scientific, Waltham, MA, USA). Cell viability was expressed as the percentage of the control.

Molecular Docking
Molecular docking was adopted for visualising the interaction of baicalein with AChE through the Discovery Studio 4.5 program (neotrident, Beijing, China) [40]. The crystallo- graphic structure of the donepezil-AChE complex (PDB ID: 4EY7) was acquired through the RCSB Protein Data Bank (http://www.rcsb.org/. Accessed on: 28 September 2021) [41]. According to the special tetramer structure in AChE, Chain A was selected for simulation optimisation, and hydrogenation and polarity addition were conducted. The 3D structure of baicalein was plotted using the Chem3D Ultra 8.0 (Cambridge Soft, Waltham, MA, USA), and molecular optimisation was performed to obtain the conformation with minimum energy. Then, the CHARMm force field algorithm was used, wherein AChE and baicalein served as the receptor and ligand, respectively. Therefore, to validate the accuracy of docking, the co-crystallised ligand donepezil was re-docked into the active site of AChE. The molecular docking was performed using the CDOCKER algorithm, with 100 running times, and the docking tolerance was set to 0.25 Å. The best binding pose with the lowest energy was selected for examining the interplay of baicalein with AChE.

M.D. Analysis
M.D. simulation was conducted using the GROMACS 4.5.6 software, with the AMBER 99SB-ILDN force field [42,43]. The PDB file for AChE was complied with the PDB file for molecular docking. The AChE-and AChE−baicalein complex systems were solvated using the explicit SPC solvent model [44] and then placed within a dodecahedron box full of water molecules, ensuring a minimum of the 1 nm distance between all protein atoms and the box wall. The system surface charges were neutralised by adding 18 Na + and nine Cl − , followed by energy minimisation. The minimised system was equilibrated through NVT at 300 K and one bar for 100 ps, whereas M.D. simulation was conducted by NPT ensemble. Finally, the running time of M.D. simulations was set to 60 ns [45].

Statistical Analysis
All samples were tested in triplicate, and the results are indicated as mean ± standard deviation. Data analysis was performed with a one-way analysis of variance in Origin 9.0 (Origin Lab, Northampton, MA, USA), and a p-value of <0.05 was considered statistically significant.

Inhibitory Effect of Baicalein on AChE
Baicalein and galanthamine hydrobromide ( Figure 1) significantly prohibited AChE activity in a dose-dependent manner ( Figure 2A), with IC 50 values of 6.42 ± 0.07 µM and 0.29 ± 0.02 µM, respectively. The AChE activity was almost completely inhibited by galanthamine. However, AChE activity inhibition by baicalein displayed a decreasing trend. When baicalein reached a concentration of 26.7 µM, the final inhibition rate was 90%. Although baicalein inhibited AChE less effectively than galantamine, it was recognised as a compound with potential bioactivity that might prevent both Aβ aggregation and amyloid fibril plaque formation [33]. Moreover, baicalein demonstrated a lower IC 50 value than baicalin (204.1 ± 16.5 µM), a flavone glycoside of baicalein. In vitro glycosylation of dietary flavonoids decreased plasma protein affinity [46]. Flavone glycosylation also altered the distribution and density of the electronic cloud among the rings, causing significant steric hindrance [47]. Thus, baicalein's inhibitory activity was substantially greater than its glycosylation activity. It may be deduced that glycosylation reduces AChE inhibitory activity, and the conclusion was reached in our prior study [40]. Furthermore, the hydroxyl groups in the A ring may play a key role in AChE inhibition by baicalein, thereby supporting the docking results, suggesting that the hydroxyl groups of flavonoids form hydrogen bonds containing AChE active site residues [48]. The solvent and temperature influencing factor tests were performed to optimise the experimental conditions, considering the impact of solvent and temperature on AChE activity. No evidence of AChE inactivation was available in the ethanol concentration range of 3%, as shown in Figure S1 (supplementary data). The enzyme activity revealed a prolonged inactivation trend with an increase in the solvent concentration. Thus, the final ethanol concentration in the reaction system was regulated by 3%. The activity of AChE slowly increased between 19-37 °C and was relatively stable between 37-43 °C, whereas it decreased rapidly at >43°C temperature ( Figure S2). Baicalein is a yellow, reductive component that darkens during incubation. With an increase in the incubation temperature, the colour darkens more noticeably, interfering with the UV measurement. Therefore, the incubation temperature was adjusted to room temperature (25 °C).

Inhibition Kinetics of Baicalein
The plots for ν vs. AChE concentration with varying concentrations of baicalein were drawn to assess the inhibition reversibility against AChE ( Figure 2B). When baicalein was added, the straight lines through the origin exhibited favourable linearity, and the slope progressively reduced, indicating that the baicalein-induced inhibition was reversible, and the binding mode was noncovalent interaction between baicalein and AChE. [49] Lineweaver-Burk plots were used to determine the inhibitory type and kinetic parameters of baicalein against AChE. The AChE activity is shown in Figure 2C at varying ATCI concentrations with varying baicalein levels. The lines of double reciprocal plots were intersected on the Y axis, which indicated a competitive inhibition, similar to chrysin's impact The solvent and temperature influencing factor tests were performed to optimise the experimental conditions, considering the impact of solvent and temperature on AChE activity. No evidence of AChE inactivation was available in the ethanol concentration range of 3%, as shown in Figure S1 (supplementary data). The enzyme activity revealed a prolonged inactivation trend with an increase in the solvent concentration. Thus, the final ethanol concentration in the reaction system was regulated by 3%. The activity of AChE slowly increased between 19-37 • C and was relatively stable between 37-43 • C, whereas it decreased rapidly at >43 • C temperature ( Figure S2). Baicalein is a yellow, reductive component that darkens during incubation. With an increase in the incubation temperature, the colour darkens more noticeably, interfering with the UV measurement. Therefore, the incubation temperature was adjusted to room temperature (25 • C).

Inhibition Kinetics of Baicalein
The plots for ν vs. AChE concentration with varying concentrations of baicalein were drawn to assess the inhibition reversibility against AChE ( Figure 2B). When baicalein was added, the straight lines through the origin exhibited favourable linearity, and the slope progressively reduced, indicating that the baicalein-induced inhibition was reversible, and the binding mode was noncovalent interaction between baicalein and AChE [49]. Lineweaver-Burk plots were used to determine the inhibitory type and kinetic parameters of baicalein against AChE. The AChE activity is shown in Figure 2C at varying ATCI concentrations with varying baicalein levels. The lines of double reciprocal plots were intersected on the Y axis, which indicated a competitive inhibition, similar to chrysin's impact on xanthine oxidase [50]. The secondary plot for K app m vs. (I) (insert Figure 2C) demonstrates linear fitting, assuming a single inhibition site or a single class of inhibition sites [37]. The K m and K i values obtained from Equations (1) and (2) were 1.674 and (4.32 ± 0.26) µM, respectively.

Inactivation Kinetics and Rate Constants
The time courses for AChE inhibition with varying concentrations of baicalein were examined in 80 min to acquire the inactivation kinetics and rate constants ( Figure 2D). The figure shows that the inhibition of AChE by baicalein was time dependent. The enzyme activity reduced rapidly in the first 20 min and then gradually became steady after 40 min. Thereafter, the enzyme activity did not change dramatically. Subsequent semilogarithmic plot analysis revealed that the baicalein-induced inactivation was a monophasic first-order process with no intermediates. When the baicalein concentration ranged between 2 µM and 16 µM, the rate constants (k) of inactivation increased from (1.48 ± 0.03) × 10 −4 s −1 to (5.18 ± 0.08) × 10 −4 s −1 . By using the equation ∆∆G • = −R.T. ln k, the transition free energy change (∆∆G • ) was measured as 21.85 kJ mol −1 s −1 , which reduced to 18.74 kJ mol −1 s −1 upon addition of baicalein. The variation in ∆∆G • potentially induced enzyme inactivation (

Fluorescence Quenching of AChE by Baicalein
The remarkable inhibitory effect of baicalein on AChE indicated that the inhibitor was possibly bound to the enzyme [52]. Fluorescence quenching experiments were conducted to examine the binding property, binding constant, and binding site to further investigate the interactions between baicalein and AChE [53]. As shown in Figure 3A, the maximum fluorescence emission peak of AChE occurred at 343 nm after excitation at 280 nm, and baicalein exhibited no interference with AChE fluorescence since it did not display any signal under the same conditions (Curve l). The AChE fluorescence quenching was conducted by serially adding baicalein with no visible peak shift, implying that baicalein interacts with AChE.
The fluorescence quenching data, including quenching constant (K SV ) and bimolecular quenching constant (K q ), were assessed using the known Stern−Volmer equation to elucidate the quenching mechanism for AChE fluorescence through baicalein [54]. The Stern-Volmer plots exhibited favourable linearity at 25 • C, 31 • C, and 37 • C ( Figure 3B), indicating the presence of a single quenching type alone. Additionally, the relevant K SV values (shown in Table 2) reduced with an increase in temperature, proving that the fluorescence quenching mechanism through baicalein was static. Moreover, the corresponding K q values of 10 13 L mol −1 s −1 magnitude order were substantially higher than the quenching rate constant for maximum scattering collision (2.0 × 10 10 L mol −1 s −1 ) [55].The results also indicate that the static quenching mode triggered the quenching of baicalein to form a ground-state complex rather than a dynamic collision. The fluorescence quenching data, including quenching constant (KSV) and bimol lar quenching constant (Kq), were assessed using the known Stern−Volmer equatio elucidate the quenching mechanism for AChE fluorescence through baicalein [54].  Table 2) reduced with an increase in temperature, proving that the orescence quenching mechanism through baicalein was static. Moreover, the correspo ing Kq values of 10 13 L mol −1 s −1 magnitude order were substantially higher than quenching rate constant for maximum scattering collision (2.0 × 10 10 L mol −1 s −1 ) [55] Table 2. The quenching constants (K SV ), binding constants (K a ), and relative thermodynamic parameters for baicalein−AChE interaction under three temperature conditions.

Binding Parameters and Thermodynamics
The binding constant (K a ) for the AChE-bacalein complex and the binding site number (n) were measured using Equation (3) [54]: (Q t ) and (P t ) denote the bacalein and AChE concentrations, respectively. The order of magnitude of K a values was 10 4 L mol −1 (Table 2), which indicated that bacalein bound to AChE with a moderate affinity. The n values approached 1, indicating the presence of merely one binding site for bacalein on AChE, which is congruent with the results of the Lineweaver−Burk plot analysis [56].
In general, four noncovalent interaction forces exist between small molecules and macromolecules, namely hydrogen bonds, van der Waals forces, electrostatic forces, and hydrophobic interactions [57]. To identify the binding forces between bacalein and AChE, the thermodynamic parameters were measured following the Van't Hoff equation: where K a denotes the binding constant, and R represents the gas constant (8.314 J mol −1 K −1 ). The temperatures (T) used were 25 • C, 31 • C, and 37 • C. As shown in Table 2, ∆G • and ∆H • showed negative values, which indicated that the interplay of bacalein with AChE was spontaneous, and the binding was exothermic. Furthermore, the positive ∆S • and negative ∆H • values indicated that the hydrogen bonds and hydrophobic interactions exerted main effects on the complex formation [58].

Synchronous Fluorescence Spectroscopy
For the investigation of AChE-related conformational changes, synchronous fluorescence spectroscopy is used. The fluorescence spectra between 15 and 60 nm at the wavelength interval (∆λ) indicate the fluorescence characteristics for Tyr and Trp residues and can reveal the microenvironment information of the proteases. As shown in Figure 3C,D, the increase in baicalein concentration resulted in the bathochromic shifts from 294 to 295 nm for Tyr residues and 285 to 287 nm for Trp residues of AChE. The findings indicate that the hydrophobicity of Tyr and Trp residues in the milieu was increased and that baicalein triggered a conformational change in AChE and influenced the microenvironment for amino acid residues. Tyr and Trp residues were contrasted in contribution based on their ratios in synchronous fluorescence quenching (RSFQ = 1 − F/F 0 ). With an increase in the concentration of baicalein, the RSFQ value for Trp residues reached 63.83%, which was higher than that for Tyr residues (58.48%) ( Figure 3E). The current finding implies that the Trp residue contributed more to the interplay between baicalein and AChE than the Tyr residue because Trp residue was closer to the binding site and had more substantial binding power with baicalein [59].

CD Spectra
CD may provide information on the stereochemistry of a protein-bound drug and protein conformation through secondary structure analysis, consequently revealing information about the binding process [60]. As shown in Figure 3F, two negative CD absorption bands appeared in the AChE spectra at approximately 210 and 222 nm, due to π→π* and n→π* transitions in amide groups, which have been defined as characteristic bands of the α-helix structure [55]. Moreover, the presence of baicalein increased the negative intensity of the band at 210 nm relative to the intensity of the band at 222 nm, indicating that baicalein interacted with AChE mainly by π→π* transition and altered the conformation of AChE. The findings of thermodynamics analysis show that the hydrophobic interactions and hydrogen bonds were the dominant forces during the interplay of baicalein with AChE. With the increase in the molar ratios of baicalein to AChE from 0:1 to 6:1, the α-helix and β-turn contents increased gradually, whereas the contents of the β-sheet and random coil decreased ( Table 3). The current result is consistent with that of Manavalan et al. [61]. The observed increase in the α-helix contents indicated that the AChE structure became increasingly compact, which may have prevented the substrate from accessing the active cavity, resulting in a decrease in the AChE catalytic activity.

Cytotoxicity of Baicalein in PC12 Cells
The cytotoxicity of baicalein in PC12 cells was evaluated using the MTT assay. Figure 4 demonstrates that the viability of the PC12 cells changed slightly under different concentrations of baicalein. However, the change was not statistically significant compared with that of the control group, indicating that baicalein was nontoxic to PC12 cells at the concentration range of 1-80 µM. mation about the binding process [60]. As shown in Figure 3F, two negative CD absorption bands appeared in the AChE spectra at approximately 210 and 222 nm, due to π→π* and n→π* transitions in amide groups, which have been defined as characteristic bands of the α-helix structure [55]. Moreover, the presence of baicalein increased the negative intensity of the band at 210 nm relative to the intensity of the band at 222 nm, indicating that baicalein interacted with AChE mainly by π→π* transition and altered the conformation of AChE. The findings of thermodynamics analysis show that the hydrophobic interactions and hydrogen bonds were the dominant forces during the interplay of baicalein with AChE. With the increase in the molar ratios of baicalein to AChE from 0: 1 to 6: 1, the α-helix and β-turn contents increased gradually, whereas the contents of the βsheet and random coil decreased ( Table 3). The current result is consistent with that of Manavalan et al. [61]. The observed increase in the α-helix contents indicated that the AChE structure became increasingly compact, which may have prevented the substrate from accessing the active cavity, resulting in a decrease in the AChE catalytic activity.

Cytotoxicity of Baicalein in PC12 Cells
The cytotoxicity of baicalein in PC12 cells was evaluated using the MTT assay. Figure  4 demonstrates that the viability of the PC12 cells changed slightly under different concentrations of baicalein. However, the change was not statistically significant compared with that of the control group, indicating that baicalein was nontoxic to PC12 cells at the concentration range of 1-80 µM.

Molecular Docking
The co-crystallised ligand donepezil was re-docked into the active site of AChE to validate the accuracy of the docking protocol. The outcome is depicted in Figure 5A, and the root mean square deviation (RMSD) between the docking and original co-crystallised pose was 0.844 Å.
The binding position, binding mode, and force type of the interaction between baicalein and AChE were predicted using a molecular simulation technique. According to the X-ray crystal structure analysis, the active site of AChE contains a narrow and deep gorge comprising two binding sites, namely the Ser-His-Glu catalytic site at the gorge bottom and peripheral anion site (PAS) at the gorge entrance.
As shown in Figure 5B, baicalein penetrated the active site of AChE, and the binding pattern was consistent with the competitive inhibition type of baicalein on AChE. Baicalein's lowest binding energy to AChE was −33.26 kJ mol −1 , close to the thermodynamic experimental data. Figure 5C,D demonstrate the baicalein binding area and the main amino acid residues interacting with baicalein in AChE in 3D and 2D modes. Baicalein formed three hydrogen bonds with AChE. The C6-hydroxyl group in the A ring of phenylchromen formed a hydrogen bond with the O atom of Trp86 (catalytic site) at the choline-binding site, with the bond length 2.04 Å. The C7-hydroxyl group formed two hydrogen bonds with the oxygen atom of Tyr72 and Asn87 residues, with bond lengths of 3.85 Å and 2.67 Å, respectively. The main phenylchromen had apparent π-π stacking, and hydrophobic interaction with the PAS active site residues Trp86, Tyr124, Phe338, and Tyr337 inhibited the AChE activity by suppressing aromatic inner surface induction. The Val73, Asp74, Tyr341, Pro88, Gln71, Ser125, Leu130, Gly126, Gly121, Gly120, and His447 residues surrounded the whole baicalein molecule by van der Waals forces. These residues were found in either the middle gorge area or the substrate-binding site, where they interacted with baicalein to decrease AChE activity. Furthermore, the PAS locus was linked to the allosteric control of AChE. [62]. Therefore, it is plausible to conclude that baicalein can simultaneously induce the allosteric structure of AChE, causing the gorge entrance to constrict and thereby preventing the combination of the substrate and enzyme active site and eventually inhibiting AChE activity.

M.D. Simulation
M.D., a typical computer simulation tool for studying protein stability, can visibly illustrate the dynamic changes of the protein skeleton with time and space. Understanding the link between enzyme structure and function is critical for understanding the inherent flexibility of proteins. As a result, M.D. simulations of the AChE crystal and the bacalein-AChE complex were run in 60 ns.
The findings of the RMSD were used to evaluate the dynamic changes in system stability and determine the time when the system reaches a steady state. The RMSD values for free AChE varied from 0.15 to 0.24 nm and tended to reach equilibrium at 35 ns, as shown in Figure 6A. The RMSD value of the protein skeleton fluctuated slightly in the first 35 ns, and a stable complex was formed slowly through hydrogen bonds or intermolecular forces; the RMSD value of the enzyme became stable after 35 ns. Overall, the RMSD value of the complex did not vary greatly, indicating that the binding of baicalein and AChE would only marginally alter the freedom of protein movement and that the stability of the baicalein−AChE complex was comparable to that of AChE [63].  The flexibility of amino acid residues was demonstrated by root mean square fluctuation (RMSF) during the whole simulation duration. High RMSF values of amino acids provide high flexibility during binding. The local amino acid residues of 70−100, 110−150, and 330−380 in AChE showed an obvious fluctuation, possibly due to the participation of amino acid residues at the sites in the allosteric formation of the enzyme to form stable complexes, as is consistent with the main amino acid residues during the interaction with baicalein in molecular docking. The presence of baicalein reduced the RMSF value of AChE residues (as shown in Figure 6B), indicating that baicalein limited the flexibility of the AChE structure, which might be due to the increase in the content of the rigid structure (α-helix) of AChE due to interaction [64].
The radius of gyration (Rg) is an important parameter that reflects the compactness of protein structure [65]. After 45 ns of simulation, the Rg value tended to become stable and slightly lower than that of free AChE ( Figure 6C), indicating that baicalein might induce the AChE structure to become more compact. The hydrogen bonds and other forces formed during the bonding process may have rendered the steric structure of AChE more stable, which conformed to the CD analysis results. Figure 6D depicts the variation in the number of hydrogen bonds fabricated in the complex during simulation. The hydrogen bonds varied from 0 to 3, with 1−2 being the most prevalent. The solvent-accessible surface area (SASA) of a system in interaction with solvents indicates surface changes in the system. To further verify the change in the microenvironment hydrophobicity of Trp and Tyr residues, the SASA values of the Trp and Tyr residues were compared before and after stimulation ( Figure 6E,F). The SASA of Trp residue decreased noticeably, implying that Trp residues' microenvironment hydrophobicity had improved. The overall SASA for Tyr residues, on the other hand, tended to decline slightly, which indicated a minor increase in the microenvironment hydrophobicity of Tyr residues [40]. The results corresponded with those of synchronous fluorescence research.

Conclusions
The present study investigated the inhibitory action of baicalein on AChE and the underlying mechanism at the molecular level by using several spectroscopic and computer simulation techniques. Baicalein was discovered to be a highly effective reversible competitive inhibitor of AChE. Baicalein could statically quench AChE fluorescence. The hydrogen bond and hydrophobic interaction aided the binding between baicalein and AChE. Baicalein bound to AChE at one binding site, and the order of magnitude of the binding constant was 10 4 L mol −1 . A cytotoxicity test showed that baicalein did not affect the activity of PC12 cells. The interaction of baicalein with PAS residues (Tyr72, Tyr124, Phe338, and Tyr337) caused an increase in the α-helix content AChE, making the structure of the enzyme more compact and increasing the surface hydrophobicity of AChE. Through hydrogen bonding, hydrophobic interaction, and van der Waals force, baicalein may embed into the active site of AChE to form a relatively stable complex. The binding caused a conformational shift in the enzyme and tightened the structure of the gorge entrance, preventing the substrate from entering and binding with the enzyme's active site, eventually inhibiting AChE activity. M.D. simulation analysis showed that the binding of baicalein with AChE might influence the stability of the protein skeleton. As fluctuating peptides, the 70-100, 110-150, and 330-380 residues might participate in causing conformational changes in the enzyme and forming stable complexes. Furthermore, a slight decrease in the Rg value reflects a moderate compact structure of AChE. Overall, the current study investigated the mechanism of AChE inhibition by baicalein and provided a new direction for the research and development of anti-AD food functional factors and drugs.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/foods11020168/s1, Figure S1: Effect of ethanol on AChE activity; Figure S2: Effect of temperature on AChE activity.


s2fieldsofstudy:
Chemistry
Materials Science

text:
Binder-Free Fabrication of Prussian Blue Analogues Based Electrocatalyst for Enhanced Electrocatalytic Water Oxidation

Developing a cost-effective, efficient, and stable oxygen evolution reaction (OER) catalyst is of great importance for sustainable energy conversion and storage. In this study, we report a facile one-step fabrication of cationic surfactant-assisted Prussian blue analogues (PBAs) Mx[Fe(CN)5CH3C6H4NH2]∙yC19H34NBr abbreviated as SF[Fe-Tol-M] (where SF = N-tridecyl-3-methylpyridinium bromide and M = Mn, Co and Ni) as efficient heterogeneous OER electrocatalysts. The electrocatalysts have been characterized by Fourier transform infrared (FT-IR) spectroscopy, powder X-ray diffraction (PXRD), scanning electron microscopy (SEM) coupled with energy dispersive X-ray (EDX) analysis, and X-ray photoelectron spectroscopy (XPS). In the presence of cationic surfactant (SF), PBAs-based electrodes showed enhanced redox current, high surface area and robust stability compared to the recently reported PBAs. SF[Fe-Tol-Co] hybrid catalyst shows superior electrochemical OER activity with a much lower over-potential (610 mV) to attain the current density of 10 mA cm−2 with the Tafel slope value of 103 mV·dec−1 than that for SF[Fe-Tol-Ni] and SF[Fe-Tol-Mn]. Moreover, the electrochemical impedance spectroscopy (EIS) unveiled that SF[Fe-Tol-Co] exhibits smaller charge transfer resistance, which results in a faster kinetics towards OER. Furthermore, SF[Fe-Tol-Co] offered excellent stability for continues oxygen production over extended reaction time. This work provides a surface assisted facile electrode fabrication approach for developing binder-free OER electrocatalysts for efficient water oxidation.

Introduction
The global energy demand is anticipated to be doubled due to the rapid expansion of technology and depletion of fossil fuels. To achieve "dual-carbon" goals, it is not only necessary to control carbon emissions from traditional fossil fuels, but also to find suitable alternative energy sources [1]. Hydrogen can be used as a promising clean energy source because of its high energy content and environmentally friendly nature, therefore, it has been extensively studied during the past decades. Incredible efforts have been devoted to the inexpensive and efficient production of hydrogen as well as to scrutinize some strategic techniques [2][3][4][5]. The oxygen evolution reaction (OER), a half-reaction of water electrolysis, is alleged to be the rate-limiting step that significantly impedes the overall efficiency of electrochemical hydrogen production [6]. High thermodynamic potential, as well as slow kinetics for OER leading to higher overpotential, necessitates the use of an efficient electrocatalyst to favor the reaction at lower overpotential values [7,8]. Furthermore, water electrolysis is reliant on pH and significant literature reports the use of basic and acidic conditions for oxygen evolution and hydrogen evolution reactions, respectively. However, extreme conditions of pH do not meet the green chemistry rules. So, the search for efficient and robust water oxidation catalysts (WOC) that could work under milder conditions is crucial for electrochemical energy devices [6,9]. IrO 2 and RuO 2 show excellent electrochemical performance, but their applications have been constrained due to their high cost and scarcity [10]. First-row transition metals such as manganese, cobalt, nickel, and iron-based materials, have drawn enormous considerations as appropriate electrocatalysts for the OER due to their low cost, high activity, and long-term stability [11][12][13]. Despite the high catalytic activities of metal oxides, particularly cobalt oxide [14], they have low stability and a high tendency to decompose in an acidic medium. Prussian blue (PB) and its analogues (PBAs, Na 2 M[Fe(CN) 6 ], M = Fe, Co, Mn, Ni, Cu, etc.) belong to a large class of transition-metal hexacyanoferrates with an open framework structure, numerous redox-active sites, and strong structural stability. Therefore, due to their unique structural features, PB and its analogs have been thoroughly investigated as new alternative catalysts for energy-based applications [15].
Subsequently, the investigation of Prussian blue analogues (PBAs) as WOCs by the group of Galán-Mascarós is among the targeted studies showing that metal hexacyanometalates are robust and stable in a wide pH range [15]. However, PBAs present low current densities (~1 mA·cm −2 at overpotential >600 mV) due to their high crystallinity and low mechanical resistance attributing to their poor interfacial matching with the electrode surface. In follow-up studies by the group of Ferdi, pentacyanometallate-based PBA i.e., [CoFe(CN) 5 -PVP] exhibited a current density of 1 mA cm −2 at comparatively lower overpotential η = 510 mV [16]. The study further shows that the OER reaction occurs more efficiently on the surface of amorphous material than on that of a crystalline one, owing to more surface-active sites. This work provided the room to explore and investigate the effect of N-donor ligands on the water oxidation performance of PBAs. It has also been observed that conventional electrode fabrication using a binder, can lead to the undesirable electrode interface, i.e., higher resistance amid electrocatalyst and substrate, thereby lessening the active sites and flaking off the catalyst etc. Therefore, binder-free electrocatalysts are advantageous to boost the catalytic performances of PBAs [17,18]. Moreover, the in situ growth of a conductive layer i.e., graphene, CNTs and surfactants, etc., onto an electrode and electrocatalyst interface is an effective tactic to construct an electron pathway and regulate electron transference between them [17][18][19][20].
Surfactants can act as structure-directing agents and have a considerable influence on electrochemical reactions [21,22]. A surfactant-based conductive layer between the electrode and electrocatalysts has played a significant role in the enhanced film growth, increasing stability and with faster charge transfer kinetics [17].
Herein, we have investigated the effect of cationic surfactant i.e., N-tridecyl-3-methylpyridinium (SF) on the electrocatalytic performance of PBA films towards water oxidation reaction. We have developed the strategy of preparing binder-free, surfactant-assisted PBA films on the glassy carbon electrode (GCE). The addition of this surfactant immobilizes PBAs on the bare GCE and significantly enhances the charge transfer between the analyte and the electrode surface [23,24]. Thus, the fabrication of SF[Fe-Tol-M] films on GCE has shown significant improvement in electrochemical performance towards water oxidation in terms of currents density, redox activity, and electrochemical stability.

Chemicals and Instruments
All the reagents and solvents (analytical grade) were purchased from Sigma-Aldrich (St. Louis, MO, USA) and used as received without any further purification. All salt solutions were prepared in deionized water (resistivity: 18 mΩ·cm) at 25 • C.
The electrochemical measurements were carried out on a Gamry 1010E potentiostat/galvanostat equipped with a standard three-electrode system comprising of platinum wire, Ag/AgCl (3 M KCl) and modified glassy carbon (2 mm diameter) as counter, reference, and working electrodes, respectively. FT-IR absorption spectra of compounds were recorded on a Thermo Nicolet-6700 spectrophotometer in the frequency range of 4000-400 cm −1 . The NMR spectra ( 13 C and 1 H) were recorded on a Bruker Advanced Digital 300 MHz spectrometer (Switzerland) using D 2 O as a solvent and the chemical shifts (δ) are given in 'ppm'. Shimadzu-1800 double beam spectrophotometer was used to determine the characteristic peak positions in UV-visible region (200-800 nm). PXRD analyses were carried out on a PANalytical X'Pert multipurpose X-ray diffraction system, in the 2θ range of 10-80 • . SEM images were obtained by using FEI Nova SEM-230 coupled with Bruker EDX system at an accelerating voltage of 3 kV. XPS analyses of the synthesized PBAs were performed by mounting the samples onto a holder with a conducting carbon tape to avoid surface charging during the measurements. The samples were dispersed in acetone and drop-casted onto a silicon wafer. The solvent was dried under nitrogen flow. In order to prevent charging during the photoelectron measurement, a highly doped silicon wafer with natural oxide was used as a substrate. After preparation of the sample, it was introduced to the UHV chamber. The UHV chamber operating at a vacuum pressure of~1 × 10 −9 mbar was equipped with a water-cooled X-ray gun (Mg/Al, Specs XR 50) and a hemispherical analyzer (Specs Phoebos 100). For excitation, the MgKα radiation (1253.6 eV) source was used in this experiment. Large lens mode was used for the detection of the photoelectron with pass energy of 30 eV. For data analysis the CASA-XPSTM software was used. The measured data was fitted using the CASA XPSTM software and the background was subtracted using Shirley's methods. A simplified Voigt function was used for fitting with sample width at half maximum (FWHM) for doublet and the ratio between 2p 3/2 and 2p 1/2 was found to be 0.5. Electrocatalytic performance of PBA-based modified electrodes was tested on Gamry interface 1010E on the workstation at ambient temperature.

Synthesis of the Surfactant
Pyridine-based cationic surfactant was synthesized (Scheme S1) according to our reported method with slight modification [25,26]. Briefly, surfactant was synthesized by refluxing equimolar amounts of 3-methyl pyridine (0.20 g; 2.15 mmol) and tridecyl bromide (0.57 g; 2.15 mmol) in 25 mL dried toluene as a solvent for 10 h. After cooling to room temperature, the reaction mixture was filtered off and the brownish viscous compound was obtained by rotary evaporation. The purity of SF was confirmed by FT-IR and NMR spectroscopy (see Supplementary Information).

Synthesis of the Sodium Pentacyanoammineferrate(II)
Sodium pentacyanoammineferrate(II) (Na 3 [Fe(CN) 5 [16]. Briefly, 15 g of [Fe-NO] was dissolved in 40 mL of water followed by the addition of 6 g of NaOH under constant stirring at 10 • C. Afterwards, NH 4 OH (25% v/v) was added to this solution until saturation, followed by the addition of cold methanol [16]. The resulting yellow precipitates were aged overnight at 0 • C and recrystallized using NH 4 OH/CH 3 OH solution that were finally dried under vacuum (Scheme S2). The obtained yield was calculated to be 50%.

Synthesis of Sodium Pentacyano(methylaniline)ferrate (II) [Fe-Tol]
The Prussian blue precursor was synthesized by a ligand substitution reaction of [Fe-NH 3 ] with 2-methylaniline by slight modification in a reported procedure [16,27,28]. The 2-methylanaline (0.65 mL; 6.14 mmol) was added slowly into the [Fe-NH 3 ] solution (2 g; 6.14 mmol) under constant stirring at room temperature (Scheme S3). The reaction was allowed to continue in a covered flask for 24 h. The reaction mixture containing the green-colored precipitates was then concentrated to a 5 mL solution and was washed with cold methanol followed by centrifugation (6000 rpm) to remove unreacted [Fe-NH 3 ]. Then, cold diethyl ether (250 mL) was added to the suspension (approximately 50 mL) under constant stirring. The precipitates were isolated and dried under vacuum overnight at room temperature which resulted in a green-colored powder with a yield of about 63%.

Modification of Electrode for OER
The deposition of the PBA films onto the GCE was accomplished by first placing a drop of surfactant solution (in methanol) followed by the addition of 10 µL of 10 mM [Fe-Tol] and 15 mM of desired metal salt solutions i.e., Co(NO 3 and SF[Fe-Tol-Mn] films, respectively. The adsorption of surfactant to GCE occurs through the attachment of polar head groups of the surfactant to the hydrophilic ionic groups on GCE, which provides hemimicelles of the surfactant. During hemimicelles' formation, the head groups expose themselves to the aqueous solution because of interactions between the hydrocarbon chains [29]. Cyclic voltammetry (CV) measurements were recorded in 50 mM phosphate buffer (pH 7) containing 1 M NaNO 3 as electrolyte between 0-1.5 V versus Ag/AgCl at the scan rate of 50 mVs −1 .

Characterization
The surfactant-assisted PBAs were synthesized in different steps and characterized by several techniques. Firstly, [Fe-NO] was reduced to [Fe-NH 3 ] (Scheme S1) and then -NH 3 ligand was substituted by 2-methylaniline (Scheme S2).  (Figure 1a). Furthermore, the spectrum of SF[Fe-Tol-Co] mainly exhibits a doublet with a binding energy of 722.8 and 709.7 eV for Fe 2p 1/2 and 2p 3/2 , which is attributed to the 2+ oxidation state of iron in SF[Fe-Tol-Co] and confirms that the Fe 2+ is retained after PBA formation [30]. Similarly, the spectrum of SF[Fe-Tol-Co] is shown in Figure 1b which exhibits a doublet for Co with a binding energy of 797.1 and 781.4 eV for Co 2p 1/2 and 2p 3/2 , respectively corresponding to the 2+ oxidation state of cobalt [31,32]. In addition to the main photoelectron peak, shake-up satellite peaks are also visible at the higher binding energies in the spectrum. The XPS overview spectra of SF[Fe-Tol-M] (M = Co, Ni and Mn) are shown in Figure S2, where corresponding photoelectron peaks and Auger peaks are indicated. The substitution of 2-methylaniline moiety has further been confirmed by UV-visible and FT-IR spectroscopy. In the UV-visible spectra (Figure 1c), broad absorption at 645 nm, corresponding to the n→π* transition for [Fe-Tol] shows the presence of 2-methylaniline in [Fe-Tol] complex [33]. Similarly, the band observed at 395 nm is accredited to the metal to ligand charge transfer (MLCT) transition i.e., Fe II → π* (L), which is typical for the coordination of Fe II (CN) 5 to organic ligand [34] that confirms the successful replacement of -NH 3 by 2-methylanilie. In the FT-IR spectrum of [Fe-Tol] (Figure 1d PXRD analysis was performed to understand the morphological features of the synthesized materials. The PXRD patterns of the as-synthesized PBAs and their lattice structures are given in Figure 1e. All PBAs demonstrated identical XRD patterns, identified as a face-centered cubic lattice. The crystallinity of the synthesized PBAs is lower than that of precursors and hexacyanoferrates, which might be attributed to the presence of organic ligand resulting in lower crystallinity [37]. 1360 cm −1 for aromatic ring and CH bending endorse the replacement of the NH3 group [35]. In case of SF[Fe-Tol-M], the (CN ) has been shifted to 2067-2063 cm −1 due to the interaction of Fe 2+ − CN − M 2+ [36]. The broadband in the range of 3400-3200 cm −1 and 1610-1595 cm −1 are attributed to stretching and bending modes of the -OH group of water molecules present in the interstitial spaces of SF[Fe-Tol-M]. PXRD analysis was performed to understand the morphological features of the synthesized materials. The PXRD patterns of the as-synthesized PBAs and their lattice structures are given in Figure 1e. All PBAs demonstrated identical XRD patterns, identified as a face-centered cubic lattice. The crystallinity of the synthesized PBAs is lower than that of precursors and hexacyanoferrates, which might be attributed to the presence of organic ligand resulting in lower crystallinity [37].
The SEM images of PBAs shown in Figure Table S1. During the EDX measurement, different areas were focused to identify the corresponding metal (M) peaks as can be seen in the EDX spectra shown in Figure S3.   Table S1. During the EDX measurement, different areas were focused to identify the corresponding metal (M) peaks as can be seen in the EDX spectra shown in Figure S3. Considering the percentage content of Na, Mn, Co, Ni and Fe for each catalyst, the molecular formulae are approximated as Co 1. 28

Electrocatalytic Performance
The transition metal-based (especially the ones based on iron, cobalt, and nickel etc.) OER electrocatalysts undergo a pre-oxidation of the low-valence metal before the OER catalysis and form the catalytically active high-valence species. The high-valence active species formed in situ accumulates and hence leads to the substantial increment in the OER activity [39,40]. The group of Galán Mascarós put forward a phase conversion approach to transform Co(OH)1.0(CO3)0.5•nH2O into CoFe PBA thin film, resulting in an

Electrocatalytic Performance
The transition metal-based (especially the ones based on iron, cobalt, and nickel etc.) OER electrocatalysts undergo a pre-oxidation of the low-valence metal before the OER Molecules 2022, 27, 6396 6 of 10 catalysis and form the catalytically active high-valence species. The high-valence active species formed in situ accumulates and hence leads to the substantial increment in the OER activity [39,40]. The group of Galán Mascarós put forward a phase conversion approach to transform Co(OH) 1.0 (CO 3 ) 0.5 ·nH 2 O into CoFe PBA thin film, resulting in an improved OER activity [41]. Up untill now, along with the rapid and scalable synthesis approach of the PBAs-based catalysts, related elucidation of the pre-oxidation process before OER catalysis is also needed [39].  Table S2). The favorable reaction kinetics during OER catalysis and superior activity of SF[Fe-Tol-Co], compared to other two PBAs, is further supported by the Tafel slopes values (Figure 2b Table 1 presents the C dl , ECSA, and RF, for all the prepared electrodes [42,44]. The high current density, significantly lower over-potential value, and high ECSA of SF[Fe-Tol-Co] compared to the recently reported ones, can be attributed to the binder-free fabrication approach. A conductive layer of the surfactant also favors the charge transfer and hence reduces the free energy barrier [22]. A comparison of the catalytic activity of the synthesized PBAs with the reported PBAs is presented in Table S2. The study of the reaction kinetics is crucial to understand the OER activity, which is investigated by the electrochemical impedance spectroscopy (EIS). The EIS analyses for SF[Fe-Tol-M] modified electrodes have been performed in phosphate buffer at pH 7 to obtain the Nyquist plot at in the frequency range from 100 mHz to 100 kHz providing a small AC signal of 5 mV (Figure 3e) [43]. Finally, to evaluate the long-term stability, chronopotentiometric measurement was carried out for SF[Fe-Tol-Co] under the steady generation of oxygen, as given in Figure 3f. The stability test was performed by applying constant current corresponding to j value of 5 mA·cm −2 and the corresponding potential was measured. The electrocatalyst showed good stability in the galvanostatic measurements for 24 h during continuous OER.

Conclusions
Many non-oxo bridged transition metal coordination networks of hexa-and pentacyanometalates are well known for their electrocatalytic OER performances. In this work, the surfactant-assisted PBAs of earth-abundant transition metals as heterogeneous electrocatalysts have been synthesized by exploiting a binder-free approach. This strategy employed cationic surfactant to facilitate the modification of GCE by PBAs film that subsequently boosts their electrochemical properties towards OER electrocatalysis, as compared to the conventional PBAs. The catalytic current density of 10 mA·cm −2 was achieved at much lower overpotential i.e., 610 mV for SF[Fe-Tol-Co], while 1 mA cm −2 was achieved at 480 mV, 570 mV for SF[Fe-Tol-Ni] and SF[Fe-Tol-Mn] modified electrodes, respectively. The high current density, significantly lower over-potential value, and high ECSA of SF[Fe-Tol-Co] compared to the recently reported PBAs, can be attributed to the binder-free fabrication approach. The fabrication procedure reported herein unveils a facile route for designing advanced binder-free electrocatalysts with easily available transition metals with tuned performance. Further, the strategy will be used to introduce a series of robust and efficient catalysts to the field of water oxidation and can be extended to other materials and substrates as well.


s2fieldsofstudy: Chemistry Biology

text:
Folding and Duplex Formation in Sequence-Defined Aniline Benzaldehyde Oligoarylacetylenes

In all known genetic polymers, molecular recognition via hydrogen bonding between complementary subunits underpins their ability to encode and transmit information, to form sequence-defined duplexes, and to fold into catalytically active forms. Reversible covalent interactions between complementary subunits provide a different way to encode information, and potentially function, in sequence-defined oligomers. Here, we examine six oligoarylacetylene trimers composed of aniline and benzaldehyde subunits. Four of these trimers self-pair to form two-rung duplex structures, and two form macrocyclic 1,3-folded structures. The equilibrium proportions of these structures can be driven to favor each of the observed structures almost entirely depending upon the concentration of trimers and an acid catalyst. Quenching the acidic trimer solutions with an organic base kinetically traps all species such that they can be isolated and characterized. Mixtures of complementary trimers form exclusively sequence-specific 3-rung duplexes. Our results suggest that reversible covalent bonds could in principle guide the formation of more complex folded conformations of longer oligomers.

. Relevant section of the 1 H NMR spectra showing the conversion of ss-BAB to ds-BAB at different [BAB] after treatment with TFA (2 mM) and quenching with TEA (6 mM). The CHN imine peaks for ds-BAB, the CHO aldehyde peaks for ds-BAB and ss-BAB, and the aromatic peaks from the A subunit of ss-BAB are labeled as such.  Figure S2. Relevant section of the 1 H NMR spectra showing the conversion of ss-BAB to ds-BAB after treatment with different amounts of TFA. The CHN imine peaks for ds-BAB, the CHO aldehyde peaks for ds-BAB and ss-BAB, and the aromatic peaks from the A subunit of ss-BAB are labeled as such.  Figure S4. Relevant section of the 1 H NMR spectra showing the conversion of ss-ABA to ds-ABA after treatment with different amounts of TFA and after quenching the reaction with a 3-fold excess of TEA relative to the [TFA]. The CHN imine peaks for ds-ABA and the CHO aldehyde peaks for ss-ABA are labeled as such.  2M+Na/+1 S19 Figure S18. ESI-TOF Mass spectra for ds-, and fold-ABOs. a These peaks are distinguishable from the corresponding fold-ABOs by their isotopic ratios indicating a +2 charge state. b These peaks are not distinguishable from the M+Na/+1 ions of the corresponding ds-ABOs, however NMR data confirms these solutions do not contain significant amounts of ds-ABO and so it can be concluded that these peaks arise from 2M+Na/+1 ions of the fold-ABO.  . Concentration dependence of the 1 H NMR chemical of shift of 3-rung duplexes in CDCl3 at rt and 50 °C for three resonances: blue, the internal imine (NHC), red the terminal imines (NHC), and yellow the aromatic protons meta to the ester functional groups. Curves represent the line of best fit to the equal K model of indefinite association. The error is the standard error estimated from the variance-covariance matrix of the least-squares fitted parameters.

EXPERIMENTAL SECTION
General. All solvents and catalysts, as well as starting materials 3-ethnylbenzlaldhye 1 and 3-ethnylaniline 4 were purchased from commercial sources and used without further treatment. Diiodide 2, [1] 3,5-diethnylbenzalehyde, [2] and 3,5-diethnylaniline [3] were prepared via previously reported methods. "Dry" CDCl3 was prepared by treating and storing commercially available CDCl3 over 4 Å molecular sieves. THF was purchased as a solution stabilized with 250 ppm BHT and fractionally distilled before use. Removal of peroxides from the THF was crucial to the solution stability of the ABOs. MM2 energy minimum calculations for 1,3-folded AAB and 1,2-folded AAB were accomplished using the Perkin Elmer Chem3D software package. 1 H NMR and 13 C NMR spectra were recorded at 93.94 kG ( 1 H 400 MHz, 13 C 100 MHz) at 25 °C. Hydrogen chemical shifts are expressed in parts per million (ppm) relative to the residual protio-solvent resonance: CDCl3 δ 7.26. For 13 C spectra, the centerline of the solvent signal was used as internal reference: CDCl3 δ 77.16. Unless otherwise noted, each carbon resonance represents a single carbon (relative intensity).
ESI-TOF high resolution mass spectrometric data were obtained on a ToF (time-of-flight) Agilent Technologies system. Samples were injected as 10 uM solutions in 8:2 THF:H2O with 20 uM sodium acetate. The MS settings were: capillary voltage 4500 V, desolvation temperature 300 °C, fragmentor voltage 450 V. Matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry experiments were carried out as follows: 0.5 uL of a 100 uM solution of 2,5-dihydroxybenzoic acid matrix (Sigma) in methanol was deposited on a MTP 384 polished steel BC target plate (Bruker), and the solvent was allowed to evaporate. A single 0.5 μL aliquot of analyte in CDCl3 was then added on top of the 2,5-dihydroxybenzoic acid film and the sample analyzed with an autoflextreme MALDI-TOF mass spectrometer (Bruker). For Ke determination, a global fit of three protons to the equal K model of indefinite association was preformed using the MATLAB software package the multiple curve fitting with common parameters tool. [4]

ss-AAA.
To a 20 mL scintillation vial AsPh3 (0.026 mmol, 7.9 mg) and Pd2(dba)3 (0.0033 mmol, 3.0 mg, 10 mol%) were added and dissolved in anhydrous toluene (0.5 mL). The head space of the vial was purged with argon then the vial sealed with a screw on cap. The purple solution was gently swirled until the Pd2(dba)3 dissolved and the color changed to bright yellow (approx. 10 min). In a separate vial equipped with a stir bar, 2,6-diethynylaniline (9.2 mg, 0.065 mmol, 1 equiv.) and aryl-iodide 5 (2 equiv., 60 mg, 0.13 mmol) were dissolved in anhydrous toluene (1.3 mL) and DIPEA (12 equiv., 1.6 mmol, 271 uL). While stirring under a stream of argon the catalyst solution was added dropwise to the solution containing 2,6-diethynylaniline and 5. The vessel was sealed with a screw on cap and heated to 40 °C. After stirring overnight, the reaction was cooled to rt, transferred to a round bottom flask, and diluted with DCM (approx. 50 mL) and TEA (approx. 1 mL). Silica gel (approx. 2 g) was added to the vessel and the crude reaction mixture was concentrated onto the silica gel. Silica gel flash chromatography (hexane with a 70-100% gradient of EtOAC over 5 minutes, then held at 100% EtOAc for 25 min, both organic solvents were basified with 0.1 % TEA) gave ss-AAA (34 mg, 0.042 mmol, 65%) as a white solid after concentration in vacuo. 1

ss-BBB.
To a 20 mL scintillation vial AsPh3 (0.19 mmol, 19 mg) and Pd2(dba)3 (0.0075 mmol, 6.8 mg, 5 mol%) were added and dissolved in anhydrous toluene (1.0 mL). The head space of the vial was purged with argon then the vial sealed with a screw on cap. The purple solution was gently swirled until the Pd2(dba)3 dissolved and the color changed to bright yellow (approx. 10 min). In a separate vial equipped with a stir bar, 2,6-diethynylbenzaldehyde (19 mg, 0.12 mmol, 1.0 equiv.) and aryl-iodide 3 (2.5 equiv., 143 mg, 0.3 mmol) were dissolved in anhydrous toluene (3.0 mL) and DIPEA (12 equiv., 1.5 mmol, 261 uL). While stirring under a stream of argon the catalyst solution was added dropwise to the solution containing 2,6-diethynylbenzaldehyde and 3. The vessel was sealed with a screw on cap and stirred at rt. After stirring overnight, the crude reaction mixture was transferred to a round bottom flask and diluted with DCM (approx. 50 mL) and TEA (approx. 1 mL). Silica gel (approx. 5 g) was added to the vessel and the crude reaction mixture was concentrated onto the silica gel. Silica gel flash chromatography (hexane with a 20-90% gradient of EtOAC) gave pure ss-BBB (98 mg, 0.11 mmol, 94%) as a white solid after concentration in vacuo. 1

ss-ABA.
To a 20 mL scintillation vial AsPh3 (0.02 mmol, 6.2 mg) and Pd2(dba)3 (0.0025 mmol, 2.2 mg, 10 mol%) were added and dissolved in anhydrous toluene (0.5 mL). The head space of the vial was purged with argon then the vial sealed with a screw on cap. The purple solution was gently swirled until the Pd2(dba)3 dissolved and the color changed to bright yellow (approx. 10 min). In a separate vial equipped with a stir bar, 2,6-diethynylbenzaldehyde (7.7 mg, 0.05 mmol, 1 equiv.) and aryl-iodide 5 (2 equiv., 47 mg, 0.1 mmol) were dissolved in anhydrous toluene (1 mL) and DIPEA (12 equiv., 0.6 mmol, 104 uL). While stirring under a stream of argon the catalyst solution was added dropwise to the solution containing 2,6-diethynylbenzaldehyde and 5. The vessel was S32 sealed with a screw on cap and heated to 40 °C. After stirring at overnight, the reaction was cooled to rt, transferred to a round bottom flask, and diluted with DCM (approx. 50 mL) and TEA (approx. 1 mL). Silica gel (approx. 2 g) was added to the vessel and the crude reaction mixture was concentrated onto the silica gel. Silica gel flash chromatography (hexane with a 70-100% gradient of EtOAC over 5 minutes, then held at 100% EtOAc for 10 min, both organic solvents were basified with 0.1 % TEA) gave ss-ABA (31 mg, 0.037 mmol, 74%) as a white solid after concentration in vacuo. 1

ds-ABA.
A 7.5 mM solution of ss-ABA (3.7 mg, 4.5 µmol) in dry CDCl3 (600 uL) was prepared in an NMR tube. An initial 1 H NMR spectrum of ss-ABA was acquired and then 10 uL of a solution of 1% TFA in CDCl3 (v/v) was added. The capped NMR tube was immediately inverted several times to mix, and then the solvent evaporated under a stream of Ar. Dry CDCl3 (600 uL) was added to the NMR tube and the yellow residue allowed to dissolve (approx. 5 min). Another 1 H NMR spectrum was acquired, and then the acidic solution was quenched by adding TEA (0.5 uL) directly to the NMR tube. Another 1 H NMR spectrum was acquired showing complete conversion to ds-ABA. Using DCM (~4 mL) the solution was transferred to a test tube and the organic layer washed with water (4 mL) 3 times. The cloudy organic layer was then concentrated in vacuo to give ds-ABA (3.7  A 7.5 mM solution of ss-BAB (7.6 mg, 9 µmol) in dry CDCl3 (1.2 mL) was prepared in an NMR tube. An initial 1 H NMR spectrum of ss-BAB was acquired and then 10 uL of a solution of 1% TFA in CDCl3 (v/v) was added. The capped NMR tube was immediately inverted several times to mix, and then the solvent evaporated under a stream of Ar. Dry CDCl3 (1.2mL) was added to the NMR tube and the yellow residue allowed to dissolve (approx. 5 min). Another 1 H NMR spectrum was acquired, and then the acidic solution was quenched by adding TEA (1 uL) directly to the NMR tube. Another 1 H NMR spectrum was acquired showing nearly complete conversion to ds-BAB. Using DCM (~4 mL) the solution was transferred to a test tube and the organic layer washed with water (4 mL) 3 times. The cloudy organic layer was then concentrated in vacuo to give ds-BAB (7.

ds-AAB.
A 7.5 mM solution of ss-AAB (3.7 mg, 4.5 umol) in dry CDCl3 (600 uL) was prepared in an NMR tube. An initial 1 H NMR spectrum of ss-AAB was acquired and then TFA was added (2 uL). The capped NMR tube was immediately inverted several times to mix.
Another 1 H NMR spectrum was acquired, and then the acidic solution was quenched by adding TEA (6 uL) directly to the NMR tube. Another 1 H NMR spectrum was acquired showing nearly complete conversion to ds-AAB. Using DCM (~4 mL) the solution was transferred to a test tube and the organic layer washed with water (4 mL) 3 times. The cloudy organic layer was then concentrated in vacuo to give ds-AAB (3.7 mg, 2.3 µmol, 100%) as a white solid. 1

ds-ABB.
A 7.5 mM solution of ss-ABB (7.6 mg, 9 umol) in dry CDCl3 (1.2 mL) was prepared in an NMR tube. An initial 1 H NMR spectrum of ss-ABA was acquired and then 10 uL of a solution of 1% TFA in CDCl3 (v/v) was added. The capped NMR tube was immediately inverted several times to mix, and then the solvent evaporated under a stream of Ar. Dry CDCl3 (600 uL) was added to the NMR tube, 5 uL of a solution of 1% TFA in CDCl3 (v/v) was added, and the yellow residue allowed to dissolve (approx. 5 min). Another 1 H NMR spectrum was acquired, and then the acidic solution was quenched by adding TEA (1 uL) directly to the NMR tube.

fold-AAB.
A 3.9 mM solution of ss-AAB (6.4 mg, 7.7 umol) in dry CDCl3 (2 mL) was prepared in a 100 mL flask, and then a 5 uL of a solution of 1% TFA in CDCl3 (v/v) was added. The solvent was evaporated under a stream of Ar, then the residue was dissolved in 80 mL dry CDCl3 (100 uM AAB), and then 5 uL of a solution of 1% TFA in CDCl3 (v/v) was added. The solution was allowed to stand for 5 min and then the acid was quenched with 6 uL TEA. An 1 H NMR spectrum of an aliquot of the solution showed complete


Aptamer-Adjusted Carbon Dot Catalysis-Silver Nanosol SERS Spectrometry for Bisphenol A Detection

Carbon dots (CDs) can be prepared from various organic (abundant) compounds that are rich in surfaces with –OH, –COOH, and –NH2 groups. Therefore, CDs exhibit good biocompatibility and electron transfer ability, allowing flexible surface modification and accelerated electron transfer during catalysis. Herein, CDs were prepared using a hydrothermal method with fructose, saccharose, and citric acid as C sources and urea as an N dopant. The as-prepared CDs were used to catalyze AgNO3–trisodium citrate (TSC) to produce Ag nanoparticles (AgNPs). The surface-enhanced Raman scattering (SERS) intensity increased with the increasing CDs concentration with Victoria blue B (VBB) as a signal molecule. The CDs exhibited a strong catalytic activity, with the highest activity shown by fructose-based CDs. After N doping, catalytic performance improved; with the passivation of a wrapped aptamer, the electron transfer was effectively disrupted (retarded). This resulted in the inhibition of the reaction and a decrease in the SERS intensity. When bisphenol A (BPA) was added, it specifically bound to the aptamer and CDs were released, recovering catalytical activity. The SERS intensity increased with BPA over the concentration range of 0.33–66.67 nmol/L. Thus, the aptamer-adjusted nanocatalytic SERS method can be applied for BPA detection.

Introduction
Because of their high selectivity, high affinity, and low concentration dissociation, aptamer (Apt) reactions have been widely used in biomedicine, analytical chemistry, and clinical examination [1][2][3][4][5][6][7][8]. Through surface-enhanced Raman scattering (SERS), an enhanced Raman signal is obtained for the molecules adsorbed on or close to the metal surface and activated by its local surface plasma resonance (LSPR) [9][10][11][12][13]. With the development of nanoparticle preparation technology, SERS substrates have become more flexible and inexpensive, allowing them to be modified and fixed on slides or optical fibers [14,15] or used directly in colloids [16]. These advantages have resulted in the widespread use of SERS nanosubstrates with local surface plasmon effects [17][18][19][20]. In addition, nanocatalysis has been conducted to generate noble metal nanoparticles. The generated nanoparticles have been subsequently used as a direct SERS substrate based on the LSPR effect and subjected to the Apt reaction to establish an analysis platform [21][22][23].
Carbon dots (CDs) typically exhibit good biocompatibility and have been widely used as bioimaging probes and biosensors [24,25]. CDs have different preparation methods and numerous sources (including carbohydrates, amino acids, and organic acids) that promote flexible structure modification [26][27][28][29]. In addition, CDs exhibit good electronic transfer ability and can be used to catalyze redox reactions [30][31][32][33] and to establish analysis methods. Long groups [31] prepared CDs with lampblack followed by reduction with NaBH 4 to form r-CDs, which was then used to catalyze the reaction of hydrogen peroxide with 3,3 ,5,5 -tetramethylbenzidine for hydrogen peroxide detection (detection range: 0.01-0.1 mM). Wang and coworkers [32] synthesized CDs containing O, N, Fe, S, and C by the hydrothermal treatment of animal blood. The as-prepared CDs exhibited excellent peroxidase-like catalytic activity and could be mixed with glucolase to determine glucose by colorimetry with a detection range of 0.2-2.5 mM. However, the combination of Aptadjusted CD catalysis and Ag nanosol SERS for the detection of bisphenol A (BPA), which is an endocrine disruptor, has not been extensively investigated.
Endocrine-disrupting chemicals (EDCs) can damage human and ecosystem health by inhibiting reproduction as well as causing birth defects, dysplasia, and metabolic disorders. Prolonged exposure to EDCs may cause obesity, diabetes, cardiovascular disease, carcinogenesis, and neurotoxicity [34]. BPA is a common industrial material that has been detected as a potential risk for human diseases, as evaluated by supervision organizations and health agencies [35][36][37]. BPA threatens health through food, when it is present in a container during food heating [38]. The main methods of detecting BPA include chromatography [39,40], absorption spectroscopy [41,42], fluorimetry [43,44], electrochemistry methods [45], resonance Rayleigh scattering (RRS) [46,47], and surface-enhance Raman spectroscopy [48,49]. However, these methods exhibit low selectivity and low sensitivity or require precious and expensive instrumentation. Thus, real-time detection of BPA is difficult. Herein, an SERS method for BPA detection was developed using as-prepared Ag nanoparticles (AgNPs) with a strong surface plasmon resonance effect as an SERS substrate.

Principle
The carbon dots (CDs) surface contains abundant electrons, which can accelerate the electron transfer between the oxidant and the reductant, allowing the redox reaction to proceed more easily. At a certain concentration of silver nitrate (AgNO 3 )-trisodium citrate (TSC), effective collisions occur infrequently between citrate and silver ions. When CDs are added, they adsorb silver ions and citrate molecules on the surface and rapidly transfer electrons from citrate to silver ions, resulting in the generation of elemental silver, 1,3acetonedicarboxylic acid, and CO 2 . The as-prepared Ag nanoparticles (AgNPs) increased with increasing CDs loading ( Figure 1). The SERS signal of the system was strong with AgNPs as the SERS substrate and Victoria blue B (VBB) as the molecular probe. When an aptamer (Apt) enwrapped the surface of the CDs, the absorption of citrate and silver ions on the CDs was blocked, inhibiting the catalytic activity. This resulted in a decrease in the SERS intensity. In the presence of BPA, a specific bonding with the Apt was achieved, resulting in CDs exposed to the reaction system and recovering the catalytic activity. The generated AgNPs increased with BPA loading, and the SERS signals were linearly increased, allowing for the development of an SERS method for BPA detection.

Article
and silver ions on the CDs was blocked, inhibiting the catalytic activity. This resulted in a decrease in the SERS intensity. In the presence of BPA, a specific bonding with the Apt was achieved, resulting in CDs exposed to the reaction system and recovering the catalytic activity. The generated AgNPs increased with BPA loading, and the SERS signals were linearly increased, allowing for the development of an SERS method for BPA detection.

SERS Spectra
At 85 °C, the reaction of silver nitrate-TSC was blocked, but in the presence of CDs, the redox reaction proceeded to produce yellow AgNPs. When VBB was used as a signal molecule, four enhanced SERS peaks were observed at 1614, 1394, 1200, and 795 cm −1 . The

SERS Spectra
At 85 • C, the reaction of silver nitrate-TSC was blocked, but in the presence of CDs, the redox reaction proceeded to produce yellow AgNPs. When VBB was used as a signal molecule, four enhanced SERS peaks were observed at 1614, 1394, 1200, and 795 cm −1 . The 795 cm −1 peak can be attributed to the inner surface deformation of the ring. The 1200 cm −1 peak was attributed to the outside surface deformation of NH 2 , while the 1394 cm −1 peak was assigned to C-H of C=C and C-H bending vibration. The 1614 cm −1 peak was assigned to the C=C and C=N stretching vibration of the benzene ring [50]. In addition, the 1614 cm −1 peak was the most intense and linearly increased with the increasing CD concentration. Various C sources were selected to prepare CDs (glucose, fructose, sucrose, and citric acid), and urea was used as an N source to prepare N-CDs for catalysis investigation (Figures 2 and S1-S3). In the presence of the Apt, the CD surfaces were enwrapped and isolated from the catalytic system, suppressing the CD catalytic activity and the SERS intensity (Figures 3 and S4). When BPA was added, it specifically conjugated with the Apt, releasing CDs and recovering catalytic activity. With the increasing BPA loading, the amount of released CDs increased, and the generated AgNPs

The Catalytic Effect of CDs and the Inhibition of the Apt
Under the optimal conditions, AgNO3 slowly reacted with TSC. When the nanocatalyst was added, the small particle size, high surface energy, and high surface electron density allowed silver ions and citrate to absorb on its surface, facilitating electron

The Catalytic Effect of CDs and the Inhibition of the Apt
Under the optimal conditions, AgNO 3 slowly reacted with TSC. When the nanocatalyst was added, the small particle size, high surface energy, and high surface electron density allowed silver ions and citrate to absorb on its surface, facilitating electron transfer. The reaction generated yellow AgNPs, and the system SERS intensity increased rapidly due to the accelerated electron transfer. The catalytic activities of various catalysts, i.e., the as-prepared CDs using glucose, fructose, sucrose, citric acid, and the corresponding N dopants, as well as AgNPs, were investigated. The CDs produced with pure citric acid as a C source showed no catalysis, while the others had strong catalysis with N doping further increasing the catalytic activity (Table 1). This indicated that N atoms in the CD crystal lattice facilitated the incorporation between CDs and -COOH or -NH 2 by non-covalent hydrogen bonds and Van der Waals forces. The as-prepared AgNPs caused the SERS value to increased (Table S1). In addition, AgNPs could catalyze this reaction even at the concentration of 13.33 nmol/L, indicating that the as-prepared AgNPs were autocatalytic ( Figure S8).

Scanning Electron Microscopy (SEM)
The reaction solution was diluted up to 10 times for final BPA concentrations of 0, 3.33, and 13.33 nmol/L. Subsequently, a 10 µL sample solution was dropped onto a silicon wafer and dried naturally before conducting SEM. As can be seen in Figure 5a, in the absence of BPA, few AgNPs with a mean grain size of 100 nm were present in the reaction solution. Upon BPA addition, the catalytic activity was recovered, and AgNPs were formed by aggregation, with a mean grain size of 70 nm (Figure 5b,c), as corroborated by the laser scattering image ( Figure S9).
The reaction solution was diluted up to 10 times for final BPA concentrations of 0, 3.33, and 13.33 nmol/L. Subsequently, a 10 μL sample solution was dropped onto a silicon wafer and dried naturally before conducting SEM. As can be seen in Figure 5a, in the absence of BPA, few AgNPs with a mean grain size of 100 nm were present in the reaction solution. Upon BPA addition, the catalytic activity was recovered, and AgNPs were formed by aggregation, with a mean grain size of 70 nm (Figure 5b,c), as corroborated by the laser scattering image ( Figure S9).

Optimization of Catalysis Conditions
The effect of the reagent concentration on the determination was studied. With the increasing AgNO 3 concentration, the amount of generated AgNPs increased with the SERS value, while the I value was largest at the AgNO 3 concentration of 1.33 mmol/L ( Figure  S10). When the AgNO 3 concentration increased continuously, the SERS value decreased conversely, because the AgNPs aggregated excessively and the control test also reacted. Therefore, 1.33 mmol/L AgNO 3 was chosen for subsequent use. With the increasing TSC concentration, the amount of generated AgNPs increased, the SERS value increased, and the I value was maximized at the TSC concentration of 4.67 mmol/L ( Figure S11). Thus, 4.67 mmol/L TSC was selected as optimal. The reaction temperature significantly influenced the generated AgNPs, and at 85 • C for 21 min, the I value was maximized. Thus, 85 • C and 21 min were chosen as the optimal conditions for the reaction (Figures S12 and S13). The effect of Apts was also studied, and the I value reached the maximum at the Apt concentration of 13.33 nmol/L ( Figure S14). In this examination, some time was required for the combination of the Apt with fullerol. With the increasing of the binding time, the combination strengthened within 8 min ( Figure S15). With the increasing time, the I value was maintained; thus, to ensure sufficient stability, a binding time of 10 min was selected as optimal.

Working Curve
Under the optimal conditions, the working curves were prepared according to the relationship between C (BPA) and the corresponding ∆I 1614 cm −1 values (Figures 6 and S16-S19), and the analytical characteristics are listed in Table 1. The SERS method showed the maximum slope of 54.50 with a limit of detection of 0.1 nmol/L. These methods were compared with previously reported methods for BPA determination. The newly developed method was simple and showed high sensitivity and good selectivity. Therefore, it can be used to detect residues BPA in plastic products.
The effect of the reagent concentration on the determination was studied. With the increasing AgNO3 concentration, the amount of generated AgNPs increased with the SERS value, while the △of 13.33 nmol/L ( Figure S14). In this examination, some time was required for the combination of the Apt with fullerol. With the increasing of the binding time, the combination strengthened within 8 min ( Figure S15). With the increasing time, the △I value was maintained; thus, to ensure sufficient stability, a binding time of 10 min was limit of detection of 0.1 nmol/L. These methods were compared with previously reported methods for BPA determination. The newly developed method was simple and showed high sensitivity and good selectivity. Therefore, it can be used to detect residues BPA in plastic products.

Influence of Substances
According to the procedure, CD-FN was used as a catalyst, and the influence of the coexisting interfering substances on the determination of 3.33 nmol/L BPA was tested. The common substances tested did not interfere with the determination with a relative error of ±10% (Table S2).

Sample Analysis
Different brands of plastic films and polythene bags, unbranded grocery bag, and 2 disposable plastic drinking cup brands were purchased from the market and snipped. Then, 0.4 g of the samples was soaked for 48 h in ethyl alcohol. The extracts were then airdried in a well-ventilated area, subsequently dissolved in 100 mL of double-distilled water and stored at 4 • C. According to the procedure, 50 µL of the samples were used to detect BPA. A known amount of BPA was added to the sample, and recoveries of 98.5-105.4% were obtained (Table S3).

Apparatus
A model DXR smart Raman spectrometer (Thermo Company, Waltham, MA, USA) with a laser wavelength of 633 nm and a laser power of 3 mW, a Cary Eclipse fluorescence spectrophotometer (Varian Company, Palo Alto, CA, USA), a TU-1901 double-beam UVvisible spectrophotometer (Beijing General Instrument Co., LTD, Beijing, China), and an FEI Quanta 200 FEG field-emission scanning electron microscope (FEI Company, Hillsboro, OR, USA) were used.

Reagents
Apt with a sequence of 5 -3 GGG CCG TTC GAA CAC GAG CAT G N 60 GG ACA GTA CTC AGG TCA TCC TAG G (Sangon Biotech (Shanghai) Co., Ltd., China). 1.0 × 10 −3 mol/L BPA: 22.8 mg BPA were dissolved with 2.0 mL ethanol and then diluted to 100 mL with water (0.1 mol/L, measured by the HPLC method [51]). The solution was diluted and used step by step. 0.01 mol/L silver nitrate (Sinopharm chemical reagent Co. Ltd., China); 0.1 mol/L TSC (Xilong Scientific Co., Ltd., Shantou, China); 10 −3 mol/L VBB solution: 25 mg VBB were dissolved with 5.0 mL ethanol and then diluted to 50 mL with water. The solution was diluted and used step by step. glucose; fructose; sucrose; citric acid; urea; and Ca(OH) 2 (Sinopharm chemical reagent Co. Ltd., Shanghai, China). All reagents were analytically pure, and water was double-distilled.
Preparation of N-CDs (CD-GN): 1 g glucose and 1 g urea ultrasonic dissolved in 30 mL water (N: 11.6%) to form a yellow solution. The mixture was transferred into a highpressure reaction kettle heated with polytetrafluoroethylene lining for 180 • C for 5 h and then air-cooled to room temperature. The reaction mixture was a brown yellow solution. Then, it was dialyzed for 12 h with an MWCO 3500Da dialysis bag, and the water was changed at every 2 h until the dialysate was colorless. The CDs were adjusted to neutral with 50 mmol/L NaOH and then diluted to 30 mL with water. The CD GN concentration was 0.025 g/mL.
Preparation of N-CDs (CD-FN): 1 g fructose and urea (0, 0.2, 0.5, 1.0, 1.5, and 2.0 g) ultrasonically dissolved in 30 mL water to form a yellow solution, marked as CD-FN0, CD-FN1, CD-FN2, CD-FN3, CD-FN4, and CD-FN5, respectively. The mixture was transferred into a high-pressure reaction kettle heated with polytetrafluoroethylene lining at 180 • C for 5 h and then air-cooled to room temperature. The reaction mixture was a brown yellow solution. Then, it was dialyzed for 12 h with an MWCO 3500Da dialysis bag, and the water was changed at every 2 h until the dialysate was colorless. The CDs were adjusted to neutral with 50 mmol/L NaOH and then diluted to 30 mL with water. The CD-FN concentration was 0.025 g/mL.
Preparation of N-CDs (CD-SN): 1 g sucrose and urea (0, 0.2, 0.5, 1.0, 1.5, 2.0 g) ultrasonically dissolved in 30 mL water to form a yellow solution, marked as CD-SN0, CD-SN1, CD-SN2, CD-SN3, CD-SN4, and CD-SN5, respectively. The mixture was transferred into a high-pressure reaction kettle heated with polytetrafluoroethylene lining at 180 • C for 5 h and then air-cooled to room temperature. The reaction mixture was a brown yellow solution. Then, it was dialyzed for 12 h with an MWCO 3500Da dialysis bag, and the water was changed at every 2 h until the dialysate was colorless. The CDs were adjusted to neutral with 50 mmol/L NaOH and then diluted to 30 mL with water. The CD-SN concentration was 0.025 g/mL.
Preparation of Ca-CDs (CD Ca ): 1 g citric acid and 0.4 g Ca(OH) 2 were dissolved in a reaction kettle with 10 mL water, and then, 500 µL ethidene diamine were added slowly and mixed well. The mixture was heated at 200 • C for 4 h in a muffle furnace. Then, the reaction mixture was centrifuged at 10,000 rad/s for 10 min. The precipitate dissolved in water and adjusted to pH 7.5 with 50 mmol/L NaOH and then diluted to 10 mL with water. The CD Ca concentration was 0.1 g/mL.
Preparation of N-CDs (CD-CN): 1 g citric acid and urea (0, 0.2, 0.5, 1.0, 1.5, 2.0 g) ultrasonically dissolved in 30 mL water to form a yellow solution, marked as CD-CN0, CD-CN1, CD-CN2, CD-CN3, CD-CN4, and CD-CN5, respectively. The mixture was transferred into a high-pressure reaction kettle heated with polytetrafluoroethylene lining at 180 • C for 5 h and then air-cooled to room temperature. The reaction mixture was a brown yellow solution. Then, it was dialyzed for 12 h with an MWCO 3500Da dialysis bag, and the water was changed at every 2 h until the dialysate was colorless. The CDs solution was adjusted to neutral with 50 mmol/L NaOH and then diluted to 30 mL with water. The CD-CN was 0.025 g/mL.

Procedure
First, 20 µL of 1.5 µmol/L Apt, a certain amount of BPA, and 15 µL of a 0.02 g/L CD solution were added to a 5 mL graduated tube, mixed well and reacted for 20 min. Then, 200 µL of 0.01 mol/L AgNO 3 and 70 µL of 0.1 mol/L TSC were added, and the mixture was diluted to 1.5 mL with water. The mixture was subsequently heated for 21 min in an 85 • C water bath and cooled with ice water. Next, 50 µL of 1.0 × 10 −5 mol/L VBB and 40 µL of 1 mol/L NaCl were added and mixed well. The SERS spectra were recorded using a Raman spectrometer. The reaction solution SERS intensity at 1614 cm −1 (I 1614 cm −1 ) and that of a blank solution without BPA (I 0 ) were recorded, allowing the value of I = I 1614 cm −1 −I 0 to be calculated.

Conclusions
The prepared CDs had a high surface effect and effectively catalyzed the reaction of TSC and silver nitrate to produce yellow AgNPs. The generated AgNPs showed strong SERS effects, with the SERS intensity linearly increased with the CD loading. When CDs were enwrapped using an Apt, the CD-silver ion binding was blocked, suppressing the catalytic activity. BPA specifically conjugated with the Apt, releasing the CDs and recovering the catalytic activity. The system SERS intensity linearly increased with the increasing BPA content. Therefore, an Apt-adjusted nanocatalysis and an SPR effect spectral analysis for BPA detection were established with excellent sensitivity, selectivity, simplicity, and rapidness.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/10 .3390/nano12081374/s1, Figure S1: SERS spectra of CD GN -AgNO 3 -TSC, Figure S2: SERS spectra of CD Ca -AgNO 3 -TSC, Figure S3: SERS spectra of CD-CN-AgNO 3 -TSC, Figure S4 SERS spectra of Apt-CD-CN-AgNO 3 -TSC, Figure S5: SERS spectra of BPA-Apt-CD-GN-AgNO 3 -TSC, Figure S6: SERS spectra of BPA-Apt-CD Ca -AgNO 3 -TSC, Figure S7: SERS spectra of BPA-Apt-CD-CN-AgNO 3 -TSC, Figure S8: The effect of AgNPs on the SERS intensity, Figure S9: Laser scattering image of Apt-CD-FN-AgNO 3 -TSC-BPA system, Figure S10: Effect of AgNO 3 concentration on the I value, Figure S11: Effect of TSC concentration on the I value, Figure S12: Effect of temperature on the I value, Figure S13: Effect of time on the I value, Figure S14: Effect of Apt on the I value, Figure S15: Effect of binding time on the I value, Figure S16: Working curve for the SERS determination of Apt-CD-GN-AgNO 3 -TSC-BPA, Figure S17: Working curve for the SERS determination of Apt-CD Ca -AgNO 3 -TSC-BPA, Figure S18: Working curve for the SERS determination of Apt-CD CN -AgNO 3 -TSC-BPA, Figure S19: Working curve for the SERS determination of Apt-AgNP -AgNO 3 -TSC-BPA ,  Table S1: The catalytic effects of various catalyst and the inhibiting effect of Apt, Table S2: Selectivity of the analysis of BPA by the SERS method, Table S3: Sample analysis results (n = 5).
Author Contributions: A.L. and Z.J. conceived and designed the experiments; H.O. performed the experiments, analyzed the data and wrote the paper; Y.X., L.M. and S.L. collected data and revised the paper. All authors have read and agreed to the published version of the manuscript.