Time-encoded bio-fluorochromic supramolecular co-assembly for rewritable security printing

Innovative fluorescence security technologies for paper-based information are still highly pursued nowadays because data leakage and indelibility have become serious economic and social problems. Herein, we report a novel transient bio-fluorochromic supramolecular co-assembly mediated by a hydrolytic enzyme (ALP: alkaline phosphatase) towards rewritable security printing. A co-assembly based on the designed tetrabranched cationic diethynylanthracene monomer tends to be formed by adding adenosine triphosphate (ATP) as the biofuel. The resulting co-assembly possesses a time-encoded bio-fluorochromic feature, upon successively hydrolyzing ATP with ALP and re-adding new batches of ATP. On this basis, the dynamic fluorescent properties of this time-encoded co-assembly system have been successfully enabled in rewritable security patterns via an inkjet printing technique, providing fascinating potential for fluorescence security materials with a biomimetic mode.


Introduction
Paper document security is of paramount importance even in the electronic information world, because the widespread medium for documentation is still paper. [1][2][3][4][5][6][7] Developing innovative condentiality methods for genuine documents is in great demand. [8][9][10] In this respect, researchers are interested in utilizing uorescent security inks to achieve this goal, because of their visibility only under UV light. [11][12][13][14][15] Nevertheless, most of the reported uorescent inks are poor in complexity and tunability, which are relatively easy to replicate. Additionally, the printed information is usually not erasable, and the paper can only be used once. Hence, it is highly desirable to develop advanced uorescent materials with security printing and rewritable features.
To attain these objectives, a feasible strategy is to exploit various uorophores with stimuli-responsiveness by chemical synthesis. [16][17][18][19] However, the disadvantage of this strategy is that rare responsive units are available. An ingenious approach is to dynamically regulate the packing mode of p-conjugated molecules in a noncovalent self-assembly way, resulting in a reversible change of uorescence intensity and wavelength. [20][21][22][23] In this respect, uorochromic supramolecular assemblies triggered by heat, light, solvent, vapor and force have drawn great enthusiasm. [24][25][26][27][28] Among them, bio-uorochromic assembly is of particular interest, because of its possible transient characteristics in response to biological sources, which can be regarded as a superior candidate for smart uorescent materials. Nevertheless, reports highlighting bio-uorochromic assembly and biofueled transient assembly [29][30][31][32][33][34] in one system have been rarely exploited so far, but need to be addressed because they are closer to the naturally occurring system and more efficient in some specic scenarios. Here, we propose a simple co-assembly strategy to build a novel transient bio-uorochromic system, where the emission signals of p-conjugated units can be reversibly modulated on a time scale, nicely meeting the color variability and repeatability requirements for exploiting in rewritable security printing.
As a proof of concept, we present a transient bio-uorochromic supramolecular co-assembly that can be mediated by an enzyme with a tunable emission signal. Specically, a tetrabranched cationic diethynylanthracene monomer 1 has been rstly designed and synthesized (Fig. 1). It can selfassemble into nano-micelles in aqueous medium, accompanied by dramatically red-shied uorescence. Upon adding a biofuel of adenosine triphosphate (ATP) into 1, supramolecular co-assembly 1/ATP tends to form, accompanied by the quenching of the emission signal. Gratifyingly, the co-assembly 1/ATP can be transiently destroyed by temporally regulating the consumption of ATP using a hydrolytic enzyme (ALP: alkaline phosphatase), which is similar to the naturally occurring selfassembled cytoskeleton proteins in living cells. 35,36 Thus, timeencoded uorescence is generated in the synthetic assembled system, via repeatedly adding new batches of ATP (Fig. 1). On this basis, the resulting transient bio-uorochromic supramolecular assembly can be used as a security ink to develop rewritable security printing materials with a unique timeencoded feature, which highly improves the security and repeatability of paper-based condential information.

Monomer design
The structure of desired monomer 1 is shown in Fig. 1, which contains two sets of symmetrical propyl ammonium salt groups that are linked to the periphery of the 9,10-bis(phenylethynyl) anthracene unit via amide bonds. The branched ammonium cation is in charge of solubility in aqueous solution. More importantly, it favors the formation of electrostatic interaction and hydrogen bonding between the R-NH 3 + of positively charged 1 and R-PO 3 2À of negatively charged ATP, resulting in the formation of supramolecular co-assembly with chirality transfer from ATP to 1. 9,10-Bis(phenylethynyl)anthracene, the core of 1, possesses red-shied uorescence in the aggregated states, which is suitable for being employed to construct uorochromic materials. 37,38 Detailed synthetic procedures and characterization results are shown in the ESI. †

Self-assembly behaviors of 1
Initially, the supramolecular self-assembly behavior of 1 is carefully investigated. In dilute aqueous solution, the UV-Vis spectra of 1 show two major absorption bands in the regions of 300-325 nm and 350-500 nm with vibronic ne structures, indicative of molecularly dissolved states (ESI Fig. S1A, † black line). 39,40 Upon gradually increasing the concentration of 1, the molar extinction coefficient (3) at around 460 nm gradually decreases, while a red-shied shoulder band located between 470 and 530 nm appears (ESI Fig. S1A †). Two distinctive isosbestic points at 471 and 375 nm suggest the transition from a molecularly dissolved state to a self-assembled state. The concentration-dependent uorescence spectra of 1 are then studied. An emission band centered at 484 nm, along with a vibronic peak at 513 nm, is observed at a diluted concentration (green emission color, Fig. 2A), which is a typical emission attributed to the monomeric 9,10-bis(phenylethynyl)anthracene units. Upon increasing the concentration of 1, the emission band at 484 nm increases rstly, and then decreases to disappear, and a structureless emission band located at 517 nm gradually turns into the main band ( Fig The self-assembly mechanism of 1 is further elucidated via temperature-dependent UV-Vis spectral measurements. In detail, the absorption spectra of 1 (1.2 Â 10 À4 M) display two isosbestic points (452 and 469 nm) upon heating (Fig. 2B, inset), suggesting the transition between self-assembled and monomeric states. When monitoring the fraction of aggregated species (a agg , l ¼ 490 nm) versus temperature, a sigmoidal melting curve is obtained (Fig. 2B, and ESI Fig. S3 †), which demonstrates the involvement of an isodesmic mechanism in the self-assembly process of 1. [41][42][43] Non-linear tting of the melting curve affords a DH (enthalpy release upon aggregation) value of À87.5 kJ mol À1 , together with a T m (melting temperature at which a agg is 0.5) value of 306.2 K (Table S1 †). According to the modied van't Hoff plot, the DG (Gibbs free energy) value of the self-assembly process of 1 is calculated to be À24.9 kJ mol À1 at 298 K (ESI Fig. S4 †). Thus, combining with density functional theory (DFT) calculations at the level of B3LYP/6-31G(d) (Fig. 2B, inset), we rationalized that, driven by the intermolecular p-p stacking and hydrophobic interactions, 44-46 1 self-assembles into small nano-aggregates in aqueous solution via an isodesmic mechanism.

ATP-driven supramolecular co-assembly
Aer elucidating the self-assembly behaviors of 1 (1.2 Â 10 À4 M), we then sought to investigate the co-assembly of 1 with ATP. Intriguingly, as ATP is progressively introduced into the solution of 1 (2.0 Â 10 À5 M), 47 the original absorption bands located between 350 and 550 nm gradually decrease, while a new set of bands centered at 457 nm emerges with an isosbestic point at 479 nm (ESI Fig. S5 †). Simultaneously, the addition of ATP to 1 reduces the uorescence intensity until complete quenching (Fig. 3A), which is a result of large aggregate formation of 1/ATP (vide infra). 48 Detailed titration experiments reveal that around one equivalent of ATP is required to bind with 1 (Fig. 3A, inset, and ESI Fig. S5 †). These phenomena evidence that ATP is able to induce the formation of co-assembly of 1/ATP in aqueous media.
To get further insights into the co-assembly process of 1/ATP, various experimental techniques are then employed. Upon mixing 1 and ATP equivalently in D 2 O (2.00 mM for each component), the resonances on 1 and ATP shi up-eld (Dd ¼ 0.57, 0.52, 0.25, and 0.26 for H a-d on 1, and 0.91 ppm for H 3 on ATP, respectively), while the well-dened signals become broad (ESI Fig. S6A †). 1 H NMR titration measurements show that the aromatic protons on 1 gradually up-eld shi aer doping with ATP (Fig. 3B, and ESI Fig. S6B and C †). Only one set of aromatic signals for 1 is present, revealing the involvement of fastexchanging noncovalent interactions between 1 and ATP on the NMR time scale. 49 Circular dichroism (CD) is then considered to reveal the gradual evolution of Cotton effects of 1/ATP. The CD signals progressively reach a plateau with an isodichroic point at 383 nm (for 457 nm, D3 ¼ À59.6 L mol À1 cm À1 and anisotropy factor g ¼ À0.0062; for 489 nm, D3 ¼ À37.6 L mol À1 cm À1 and g ¼ À0.0042; for 521 nm, D3 ¼ 43.0 L mol À1 cm À1 and g ¼ 0.0122, Fig. 3C and ESI Fig. S7A †), which suggests that ATP induces a preferred supramolecular chirality to the resulting co-assembly of 1/ATP. A negligible linear dichroism (LD) signal can be detected for 1/ATP (ESI Fig. S7B †), indicating that the measured CD signals are real to reect the supramolecular chirality, without the interference of LD. 50 Additionally, DLS spectra titration reveals that the size gradually increases when continually adding ATP into 1, accompanied by the emergence of the Tyndall effect (Fig. 3D, and inset). TEM and scanning electron microscopy (SEM) images show that the morphologies of 1/ATP are nanostructures with a width of 40 nm and mutually entangled to form large-sized aggregates (Fig. 3D, inset, and ESI Fig. S8 †), which is in stark contrast to the morphological size of the self-assembly of 1.
For the purpose of obtaining deeper mechanistic insight into the ATP-driven co-assembly with 1, molecular dynamics (MD) simulations are performed. The simulation box of the coassembly 1/ATP containing 14 monomers 1 and 14 ATP molecules in explicit water solvent is calculated based on a CHARMM36 force eld at 298 K for 20 ns to equilibrate the initial conguration (ESI Fig. S9 †). As shown in the side and top views of 1/ATP in the nal snapshots ( Fig. 3E and F), the supramolecular M-helical structure is composed of the 9,10bis(phenylethynyl)anthracene planar cores and the chiral adenosine lying at the periphery of the aggregates, through the intermolecular electrostatic and hydrogen-bonding interactions between the R-NH 3 + units in 1 and phosphates in ATP. All three of the phosphates in ATP participate in the binding with 1 (the zoomed image in Fig. 3E, shown as red dashed lines). We thus rationalized that the binding of one equivalent of ATP causes the core of 1 to stack with each other by p-p interactions (average value of p-p distances is found to be 3.7Å, Fig. 3G), which synergistically stabilizes the co-assemblies 1/ATP and endows them with well-dened structures. Compared to the co-assemblies of 1/ATP induced by ATP, the addition of similar diphosphate (ADP: adenosine diphosphate) or monophosphate (AMP: adenosine monophosphate) leads to a different co-assembly behavior from that of 1/ATP. Specically, around two equivalents of ADP are required to totally change the emission and absorbance signals (Fig. 3A, inset, and ESI Fig. S10A and B †). Although ADP can also induce CD signals with an isodichroic point at 397 nm, they are less intense than that of ATP (Fig. 3C, inset, and ESI Fig. S10C †). As both of the negative and positive maxima of CD signals are located in the same region for 1/ADP and 1/ATP, respectively, 1/ADP should possess the same helical handedness with 1/ATP. Only small aggregations are formed for the resulting co-assembly 1/ADP, as evidenced by the TEM and DLS measurements (ESI Fig. S10D †). Unfortunately, even though an excess of AMP is added into 1, insignicant CD and emission spectral changes are observed (ESI Fig. S11 †). Considering that the monophosphate AMP cannot induce ordered supramolecular structures, 33,51 it is thus envisioned that the multivalent and chelation effect of noncovalent interactions between the positively charged ammonium groups of 1 and the negatively charged phosphates of ATP play a crucial role in inducing structurally well-dened supramolecular co-assembly 1/ATP.

Transient bio-uorochromic assembly
Before investigating the transient bio-uorochromic coassembly of 1/ATP mediated by an enzyme, we rstly explore the enzyme induced disassembly capacity upon in situ decomposition of ATP. ALP, a kind of enzyme obtained from bovine intestinal mucosa, is chosen as the enzymatic cleavage reagent, because it can hydrolyse ATP to adenosine and three equivalents of monophosphates (Pi) under mild conditions. 52 Inasmuch as the hydrolysates cannot induce 1 to assemble into coassemblies, 1/ATP would disassemble along with the hydrolysis of ATP. When ALP is added to the co-assembly of 1/ATP, the intensity of uorescence spectra gradually increases up to the initial intensity of that of monomer 1, indicative of the ALP induced disassembly and release of free monomers (Fig. 4A). Simultaneously, the emission color of the solution mixture recovers to the original green (Fig. 4B). Further evidence for ALP induced disassembly is observed by DLS measurements, showing a signicant decrease in the aggregate size (Fig. 4C). Time-dependent CD spectra show a weakening trend during the enzymatic process of 1/ATP by ALP (Fig. 4D, ESI Fig. S12A and B †), which clearly indicates the gradual disassembly of the chiral aggregates.
Furthermore, the kinetics of the disassembly process of 1/ ATP with varying enzyme units presents accelerated emission recovery with increasing concentration of ALP. When the amount of ALP increases from 0.32 U mL À1 to 0.63 U mL À1 and 0.94 U mL À1 , the recovery half-life time (dened as the time required to reach 50% of the uorescence intensity) shortens from 14.5 min via 7.3 min to 3.6 min (Fig. 4A, and ESI  Fig. S12C †). The corresponding rate constants of disassembly are calculated to be 0.06 min À1 , 0.12 min À1 , and 0.29 min À1 . The results unambiguously prove that the rate of the enzymatic hydrolysis reaction for ATP directly reects the progress of the disassembly process. Additionally, the rate of the disassembly process of 1/ATP could also be regulated by the temperature of enzymatic hydrolysis, which demonstrates a faster disassembly process at a higher temperature (Fig. 4E).
With the re-addition of ATP into the solution mixture, the green emission color is immediately quenched, accompanied by uorescence spectra disappearance (Fig. 4B). Meanwhile, the DLS hydrodynamic diameter and Cotton effects are totally restored to the initial state (Fig. 4C, and ESI Fig. S12D †). Such phenomena manifest that the co-assembly 1/ATP is re-formed. More interestingly, the uorescent signal reappears spontaneously with time increasing, which is ascribed to the presence of ALP in the solution system. Subsequently, with the addition of ATP again, the same trend as the previous cycle is observed. With continuously adding new batches of ATP, the emission signal can be reversibly switched for multiple cycles (Fig. 4F). Furthermore, the transient bio-uorochromic cycles of coassembly 1/ATP are conrmed at a different order of ALP units. Upon increasing the amount of prestored ALP in 1/ATP, the shortened half-life times of the transient cycles are observed from the time-dependent uorescence spectra (Fig. 4F). The same trend is also obtained from the absorption signals (ESI Fig. S13 †). Hence, a transient bio-uorochromic co-assembly mediated by an enzyme is successfully established, with timeencoded dynamic uorescent properties.

Rewritable security printing
Considering that the emission signals of 1/ATP can be netuned with ALP, it can be employed for developing time-encoded bio-uorochromic materials. To verify this assumption, a 3D model of uorescence changing with time is fabricated rstly. In detail, 2 wt% agarose aqueous solution is heated to clarify, and then transferred into a 3D printed mold (Fig. 5A). A non-uorescent star-shaped model is obtained aer immersing it into the mixed solution of 1/ATP/ALP for several minutes. When the model is standing at room temperature, the green emission color gradually emerges within 20 min (Fig. 5B). This spontaneous emission variation of the hydrogel system on a time scale is highly plausible, since 1/ATP disassembles in the hydrogel along with the enzyme-catalyzed hydrolysis process of ATP. 53 Thus, it is evident that the ALP mediated transient assembly of 1/ATP can be regarded as an effective strategy for the construction of time-encoded emission systems.
On this basis, we then turn to explore its potential application in rewritable security printing. Specically, the aqueous solution of 1 is rstly lled into the customized cartridge of an inkjet printer (Fig. 5C and D). A square yellow emission area is printed on a non-uorescent paper. Then, a QR code pattern can be printed in this yellow emission area, by loading with the mixture of ATP/ALP aqueous solution as an ink. The phenomenon is attributed to the co-assembling of 1 and ATP, resulting in the uorescence quenching of the printed location on the paper. The QR code is invisible under natural light, but can be decoded under UV light by using a smartphone. Interestingly, the temporary QR code is gradually erased with time increasing, which fails to be recognized aer 10 min. Such results are ascribed to the presence of ALP in the printed area, which destroyed the co-assembly 1/ATP by hydrolysing ATP. Thus, the ATP/ALP aqueous solution can be used as a transient ink to transfer messages with time-encoded features in security papers. Furthermore, upon reprinting with ATP/ALP inks, the writing/erasing process can be performed multiple times (Fig. 5D). Moreover, the erasing time of the rewritable information can be regulated according to the concentration of ALP inks, endowing with anti-counterfeiting ability in the time dimension (ESI Fig. S14 and S15 †). The rewritable security paper possesses enough stability under ambient conditions, as evidenced by the unchanged emission color for at least half a year (ESI Fig. S16 †). Overall, the time-encoded bio-uorochromic co-assembly 1/ATP possesses temporal emission signals mediated by ALP on a time scale, which is suitable for multiple-time uses in security printing.

Conclusions
In summary, we have successfully established a transient bio-uorochromic supramolecular system through the electrostatic and hydrogen bonding interactions between the tetrabranched cationic diethynylanthracene monomer 1 and anionic biofuel ATP. The nonequilibrium state of co-assembly 1/ATP can be nely controlled in aqueous medium by temporally regulating the consumption of ATP using its hydrolytic enzyme ALP, accompanied by uorescence signal variations. Furthermore, the resulting bio-uorochromic system is exploited to achieve rewritable security printing, by taking advantage of its time-encoded multiple-cycle emission switching features. Hence, the present work not only opens up a new avenue towards articial functional materials with a biomimetic mode, but also greatly improves the reusability and security level of paper-based condential information.

Data availability
Detailed synthetic procedures, analytical data, and computational methods are provided in the ESI.

Author contributions
Z. G. and W. T. conceived the idea for this project. S. Q., F. Y. and S. Z. performed the experiments, analyzed the data, and produced the artwork under the direction of Z. G. and W. T. All authors contributed to the manuscript preparation.

Conflicts of interest
There are no conicts to declare.