Precise AIE-Based Ternary Co-Assembly for Saccharide Recognition and Classification

Abstract

Saccharides are involved in nearly all life processes. However, due to the complexity and diversity of saccharide structures, their selective recognition is one of the most challenging tasks. Distinct from conventional receptor designs that rely on delicate and complicated molecular structures, here a novel and precise ternary co-assembled strategy is reported for achieving saccharide recognition, which originates from a halogen ions-driven aggregation-induced emission module called p-Toluidine, N, N'-1-propen-1-yl-3-ylidene hydrochloride (PN-Tol). It exhibits ultra-strong self-assembly capability and specifically binds to 4-mercaptophenylboronic acid (MPBA), forming highly ordered co-assemblies. Subsequent binding of various saccharides results in heterogeneous ternary assembly behaviors, generating cluster-like, spherical, and rod-like microstructures with well-defined crystalline patterns, accompanied by significant enhancement of fluorescence. Owing to the excellent expandability of the PN module, an array sensor is constructed that enables easy classification of diverse saccharides, including epimer and optical isomers. This strategy demonstrates wide applicability and paves a new avenue for saccharide recognition, analysis, and sequencing.

Keywords: aggregation‐induced emission; array sensor; co‐assembly; saccharide recognition.

Figure 1

Significance of saccharide recognition and our design idea. a) Graphic illustration of glycan compositions and structures and their significance in biological systems; b) Representative synthetic ligands used for saccharide recognition; c) Illustration of sequential co‐assembly among PN‐Tol, MPBA, and saccharides; d) Construction of assay fluorescent sensors for saccharide differentiation and classification.

Figure 2

AIE properties of PN‐Tol. a) Synthetic route of PN‐Tol, inset displays fluorescence image of PN‐Tol crystal and crystal morphology; b) Fluorescence emission spectra of PN‐Tol (33 µm) in different solvents at 25 °C, excitation wavelength (Ex): 293 nm; c,d) Fluorescence spectra (c) and changes in fluorescent intensity at 565 nm (d) of PN‐Tol (33 µm) in THF/n‐hexane mixed solution with different volume fraction of n‐hexane, inset shows the Tyndall effect; e) Fluorescence decay curves and corresponding lifetime values of PN‐Tol (150 µm) in n‐hexane and THF at 25 °C, Ex: 293 nm; f) Photograph of PN‐Tol (0.1 mM) in different ratios of THF/n‐hexane mixed solutions under 365 nm ultraviolet (UV) light irradiation; g,h) Crystal packing models of PN‐Tol viewed from different angles, where gray, white, and blue colors represent C, H, and N atoms, respectively, and the blue‐green spheres represent Cl ions. The d1, d2, and d3 represent the bond lengths.

Figure 3

Selective binding of PN‐Tol to MPBA. a) Relative fluorescent intensity (I/I₀) of PN‐Tol (33 µm) with addition of different amino acids or MPBA analogs (0.3 mm) at 25 °C, Ex: 293 nm; b) Fluorescence spectra after addition of different concentrations of MPBA (0–39 µm); c) Kinetic curve of fluorescence intensity changes (at 565 nm) of PN‐Tol (33 µm) after addition of MPBA (66 µm). To slow down the complexation process, the slowest stirring rate of 30 rounds per minute was set in this test; d) Fluorescence decay curves and corresponding lifetime values of PN‐Tol (0.2 mm) and its mixture with MPBA, detected by time‐resolved spectroscopy. For these fluorescence tests, Tris‐HCl buffers (10 mm, pH 6.8) are used as the solutions and the test temperature is 25 °C. e) Hydrogen nuclear magnetic resonance (¹H NMR) spectra of PN‐Tol (40 mg·mL⁻¹), MPBA (40 mg mL⁻¹), and PN‐Tol after adding 0.5 equiv. and 1 equiv. MPBA in d₆ ‐DSMO at 25 °C, respectively; f) 2D COSY ¹H–¹H spectrum of PN‐Tol mixed with equimolar MPBA (0.4 M) in d₆ ‐DSMO at 25 °C; g)¹¹B NMR spectra of PN‐Tol and its mixture with equimolar MPBA (0.6 m) in d₆ ‐DSMO; h) The crystal structure and binding mode of the complex formed by PN‐Tol and thiophenol, where green represents chlorine atoms and yellow represents sulfur atoms.

Figure 4

Self‐assembly morphology study. a) Schematic diagram of the self‐assembly process of PN‐Tol and its complex with MPBA; b,d,e) SEM images of the assemblies formed by PN‐Tol (b) or its complex with equimolar MPBA (d, e) on the silicon wafer surface, c) Atomic force microscopy imaging of the assemblies formed by PN‐Tol on mica sheet surface; the concentrations of the PN‐Tol solutions are 2.5 mM in CH₃OH contain 10% DMSO, test temperature: 25 °C; f) Powder X‐ray diffraction pattern of PN‐Tol (black) and its complex with MPBA (red), scan speed: 5^omin⁻¹; scan range: 2.5–50^o; g) Structure factors of PN‐Tol (black) and it's complex with MPBA (red) calculated from experimental SAXS data, inset shows the corresponding SAXS scattering curves; h) Particle size distribution of PN‐Tol (upper panel) and it's complex with MPBA (lower panel) in water (1 mm), measured by dynamic light scattering at 25 °C.

Figure 5

Different fluorescent responses to saccharides. a) The ratio of change in fluorescent intensity (at 565 nm) of PN‐Tol@MPBA (33 µM) after adding various saccharides (66 µm); All values are shown as mean ± SE, and significance analysis was calculated by the origin 2021 software. Lowercase letters indicate the significant statistical differences by one‐way ANOVA with the Tukey test (P < 0.05). There is no significant difference when the mark contains the same letter [a, b, c, d, e, f, g, and h (P < 0.05)]. b) Fluorescent intensity changes (at 565 nm) of PN‐Tol@MPBA (33 µM) upon addition of different concentrations of Mal (blue) and Tre (purple), (n.s. not significant, ^* P < 0.05, ^** P < 0.01, ^*** P < 0.001, versus Mal/Tre group); c,f) Fluorescence emission spectra of PN‐Tol@MPBA (33 µm) after adding different concentrations of Neu5Ac (c) or Fru (f), respectively; d, e) SEM image, schematic diagram (inset), and LCSM image (e) of the co‐assemblies of PN‐Tol@MPBA (5.0 mm in CH₃OH solution containing 10% DMSO) with equimolar Neu5Ac on silicon wafer surface; For these fluorescent tests, Tris‐HCl buffers (10 mm, pH 6.8) are used as the solutions and the test temperature is 25 °C.

Figure 6

Self‐assembly morphology of PN‐Tol@MPBA with various saccharides. a) Schematic diagram of the ternary co‐assembly process and the typical characteristic of each assembly. b–j) SEM images of ordered assemblies on silicon surface formed by PN‐Tol@MPBA with D‐Fru (b), Ara (c), Lyx (d), Man (e), Glc (f), Gal (g), Suc (h), Tre (i) and Mal (i), respectively. The inset of b represents the twisting direction of the spiral. The concentrations of these saccharides are 2.5 mm, HPLC grade CH₃OH was used as the solvent contains 10% DMSO, and the temperature is 25 °C. Here Tris‐HCl buffer was not used because salts could crystallize on the surface, which strongly impacts on observation of the co‐assemblies. The inset of (c–f,h–j) represents the LCSM image of the corresponding assembly in quartz plate surface (2.5 mm in CH₃OH contains 10% DMSO), Ex: 405 nm.

Figure 7

Fluorescent array sensors and data analysis. a) Schematic diagram of the array sensor; b) Fluorescence responses (I) upon addition of Ala, Lyx, Fru, Gal, and Man to the array sensor, I₀ represents the initial fluorescent intensity. Each component in the array displays different fluorescence responses, enabling multivariate analysis for distinguishing the saccharide categories. c) PCA scores plot generated from the data in (b). d) PCA scores plot of Gen, Mal, Tre, using PN‐Tol, PN‐TPE, PN‐Boctyl, PN‐BP, and PN‐BIP as the optimal array combination. The inset shows the confusion matrix of the PCA analysis. Correct predictions are represented by on‐diagonal blue squares, while incorrect predictions are represented by off‐diagonal white squares. e) PCA scores plot of mono‐, di‐, and tri‐saccharide, using PN‐Tol, PN‐TPE, PN‐Boctyl, PN‐BP, and PN‐BA as the optimal array combination. Error bars in (b) indicate the standard deviation of six repeated measurements. f) Confusion matrix of the PCA analysis of mono‐, di‐, and tri‐saccharides. g) Canonical scores plot resulting from CDA of the individual responses from the five dyes to the 36 samples grouped by category. The test solution was Tris‐HCl buffer (10 mm, pH 6.8), with a volume of 200 µL containing PN‐Tol (2.5 µm), MPBA, and saccharides (5.0 µm). The testing temperature was 27 °C. Ex: 385 nm, Em: 565 nm. h) Schematic diagram of the array sensor, executed in a mixed sample; i) PCA scores plot of Ala, Lyx, Fru, Gal, and Man (5.0 µm) in BSA mixture (0.31 mg mL⁻¹ in Tris‐HCl buffer (10 mm, pH 6.8)); j) PCA scores plot of Fru, Gen, Maltotriose, Isomaltotriose, Man, Suc (5.0 µm) in serum mixture with a total protein content of 1.53 mg mL⁻¹.