An AIE-Active NIR Fluorescent Probe with Good Water Solubility for the Detection of Aβ1–42 Aggregates in Alzheimer’s Disease

Alzheimer’s disease (AD), an amyloid-related disease, seriously endangers the health of elderly individuals. According to current research, its main pathogenic factor is the amyloid protein, which is a kind of fibrillar aggregate formed by noncovalent self-assembly of proteins. Based on the characteristics of aggregation-induced emission (AIE), a bislactosyl-decorated tetraphenylethylene (TPE) molecule TMNL (TPE + malononitrile + lactose), bearing two malononitrile substituents, was designed and synthesized in this work. The amphiphilic TMNL could self-assemble into fluorescent organic nanoparticles (FONs) with near-infrared (NIR) fluorescence emission in physiological PBS (phosphate buffered saline), achieving excellent fluorescent enhancement (47-fold) upon its combination with Aβ1–42 fibrils. TMNL was successfully applied to image Aβ1–42 plaques in the brain tissue of AD transgenic mice, and due to the AIE properties of TMNL, no additional rinsing process was necessary. It is believed that the probe reported in this work should be useful for the sensitive detection and accurate localization mapping of Aβ1–42 aggregates related to Alzheimer’s disease.


Introduction
Alzheimer's disease, an incurable neurodegenerative disease, seriously endangers the physical and psychological health of elderly individuals [1][2][3]. One of the pathological features of Alzheimer's disease is the abnormal deposition and accumulation of β-amyloid outside neurons in the cerebral cortex [4][5][6]. When the environment of the protein is changed, such as the temperature, pH, etc., or the protein is misfolded, its biological activity will decrease or even disappear, and the inactive protein aggregates, forming amyloids [7,8]. The most common types of β-amyloids in human bodies are Aβ 1-40 and Aβ  , and Aβ  polypeptides are more prone to aggregate and deposit into fibrillar aggregates [9][10][11][12].
Developing sensitive and efficient tools for the accurate sensing of Aβ polypeptides is of great importance to the diagnosis and intervention of AD in its early stage. At present, most reported fluorescent probes used for Aβ imaging, such as Thioflavin T (ThT), Thioflavin S (ThS), BODIPYs, and oxazines [13][14][15], lead to serious self-fluorescence quenching due to aggregation at the Aβ binding site, and the rinse process also needs to be repeatedly performed during real-time imaging to overcome the disadvantages of the aggregation-caused quenching (ACQ) effect. In addition, ThT and Congo red (CR) fluorophores with short emission wavelengths and small Stokes shifts are not suitable for imaging in vivo [16,17]. Several fluorescent probes with D-π-A structures and the

Results and Discussion
According to the reported literature [20], the malononitrile substituent could be used as an acceptor in D-π-A type NIR fluorophores for the detection of Aβ fibrils, and the π bridge could enhance the fluorescence emission of the probe and increase its redshift. In addition, lactose is a highly biocompatible and water-soluble substance, and it is a good choice for use as the hydrophilic unit.
Herein, we designed and synthesized a bislactosyl-decorated tetraphenylethylene (TPE) molecule, TMNL (Scheme 1), with typical AIE fluorescence characteristics, which contained two malononitrile substituents in the molecule. The amphiphilic TMNL could self-assemble into fluorescent organic nanoparticles (FONs) with NIR fluorescence emission in PBS buffer solution (pH 7.4) and could achieve excellent no-rinsing fluorescence imaging of Aβ 1-42 fibrils through the combination of malononitrile groups with Aβ 1-42 fibrils. TMNL was bestowed on the following extraordinary features. (i) The water-soluble lactose units would increase the water solubility and biocompatibility of TPE. (ii) The AIE-active TPE unit would overcome the ACQ effect of traditional fluorophores. (iii) The malononitrile substituent with an electron-withdrawing effect could extend the conjugated system of TPE, which redshifted the emission wavelength to the NIR region. Compared to the reported AIE-type NIR fluorescence probes for the detection of the Aβ amyloid (Tables S1 and S3), TMNL was the first amphiphilic and water-soluble AIE-active NIR fluorescent probe with a large Stokes shift for the detection of Aβ 1-42 and high-fidelity in situ mapping of Aβ 1-42 plaques.
Molecules 2023, 28, x FOR PEER REVIEW 3 of 13 Scheme 1. The synthesis route of TMNL.

Synthesis
The details of the synthetic routes of TMNL are shown in Scheme 1. Briefly, the tetraphenylethene derivative, Compound 9, was synthesized with 5 and 6 as starting materials by McMurry coupling reaction, radical substitution reaction, and Kornblum oxidation reaction. The double bond was introduced to Compound 9 by the Witting reaction and then coupled to malononitrile by the Knoevenagel reaction to afford 13. Finally, TMNL was obtained by the click reaction of lactose derivative 4 and TPE derivative 13. The chemical structures of TMNL and the synthetic intermediates were characterized by 1 H NMR, 13 C NMR, and HRMS ( Figures S1-S5).

Synthesis
The details of the synthetic routes of TMNL are shown in Scheme 1. Briefly, the tetraphenylethene derivative, Compound 9, was synthesized with 5 and 6 as starting materials by McMurry coupling reaction, radical substitution reaction, and Kornblum oxidation reaction. The double bond was introduced to Compound 9 by the Witting Molecules 2023, 28, 5110 4 of 13 reaction and then coupled to malononitrile by the Knoevenagel reaction to afford 13. Finally, TMNL was obtained by the click reaction of lactose derivative 4 and TPE derivative 13. The chemical structures of TMNL and the synthetic intermediates were characterized by 1 H NMR, 13 C NMR, and HRMS (Figures S1-S5).

Photophysical Characterization of TMNL
We first investigated the photophysical properties of TMNL under physiological conditions (PBS buffer, pH 7.4). TMNL showed weak absorption at approximately 320-380 nm, and the maximum emission wavelength was 645 nm (Figure 1). The large Stokes shift of approximately 285 nm could reduce the self-absorption effect and make TMNL have a strong ability to resist background interference [41,42].
Molecules 2023, 28, x FOR PEER REVIEW

Photophysical Characterization of TMNL
We first investigated the photophysical properties of TMNL under physiologi ditions (PBS buffer, pH 7.4). TMNL showed weak absorption at approximately 3 nm, and the maximum emission wavelength was 645 nm (Figure 1). The large Stok of approximately 285 nm could reduce the self-absorption effect and make TMNL strong ability to resist background interference [41,42].  Initial background minimization and fidelity signal amplification of TMNL w sential for ultrasensitive and accurate detection of Aβ1-42 fibrils; therefore, it was ne to choose a suitable detection concentration to avoid a "false-positive" AIE sign fluorescence spectra of TMNL itself as a function of concentration were first me As shown in Figure 2, when the concentration of TMNL was less than 0.752 µM, orescence intensity in PBS buffer solution (pH 7.4, 10 mM) did not change obvious increasing concentration. Therefore, the critical micelle concentration (CMC) was ured at 0.752 µM, and TMNL could self-assemble to form FONs when the concen exceeded 0.752 µM. To make the fluorescence intensity of TMNL as low as possible binding to Aβ1-42 fibrils, 1 µM was selected as the test concentration.   Initial background minimization and fidelity signal amplification of TMNL were essential for ultrasensitive and accurate detection of Aβ 1-42 fibrils; therefore, it was necessary to choose a suitable detection concentration to avoid a "false-positive" AIE signal. The fluorescence spectra of TMNL itself as a function of concentration were first measured. As shown in Figure 2, when the concentration of TMNL was less than 0.752 µM, the fluorescence intensity in PBS buffer solution (pH 7.4, 10 mM) did not change obviously with increasing concentration. Therefore, the critical micelle concentration (CMC) was measured at 0.752 µM, and TMNL could self-assemble to form FONs when the concentration exceeded 0.752 µM. To make the fluorescence intensity of TMNL as low as possible before binding to Aβ 1-42 fibrils, 1 µM was selected as the test concentration.

Photophysical Characterization of TMNL
We first investigated the photophysical properties of TMNL under physiological conditions (PBS buffer, pH 7.4). TMNL showed weak absorption at approximately 320-380 nm, and the maximum emission wavelength was 645 nm (Figure 1). The large Stokes shift of approximately 285 nm could reduce the self-absorption effect and make TMNL have a strong ability to resist background interference [41,42].  Initial background minimization and fidelity signal amplification of TMNL were essential for ultrasensitive and accurate detection of Aβ1-42 fibrils; therefore, it was necessary to choose a suitable detection concentration to avoid a "false-positive" AIE signal. The fluorescence spectra of TMNL itself as a function of concentration were first measured. As shown in Figure 2, when the concentration of TMNL was less than 0.752 µM, the fluorescence intensity in PBS buffer solution (pH 7.4, 10 mM) did not change obviously with increasing concentration. Therefore, the critical micelle concentration (CMC) was measured at 0.752 µM, and TMNL could self-assemble to form FONs when the concentration exceeded 0.752 µM. To make the fluorescence intensity of TMNL as low as possible before binding to Aβ1-42 fibrils, 1 µM was selected as the test concentration.

The Performance of Aβ1-42 Fibrils Detection
The binding properties of TMNL to Aβ1-42 fibrils were mainly characterized by fluorescence spectroscopy in PBS buffer solution (pH 7.4, 10 mM). The Aβ1-42 species included monomers, oligomers, and aggregates, and different aggregation degrees could have a critical influence on the change in fluorescence intensity [39]. The Aβ1-42 fibrils were prepared from the Aβ1-42 peptide by coincubation in PBS buffer solution (pH 7.4, 10 mM) at

The Performance of Aβ 1-42 Fibrils Detection
The binding properties of TMNL to Aβ 1-42 fibrils were mainly characterized by fluorescence spectroscopy in PBS buffer solution (pH 7.4, 10 mM). The Aβ 1-42 species included monomers, oligomers, and aggregates, and different aggregation degrees could have a critical influence on the change in fluorescence intensity [39]. The Aβ 1-42 fibrils were prepared from the Aβ 1-42 peptide by coincubation in PBS buffer solution (pH 7.4, 10 mM) at 37 • C for seven days (please refer to Section 3.3 for the detailed steps). ThT is generally used as an authoritative standard probe for the detection of the aggregation state of amyloid [37,[43][44][45][46]. As shown in Figure 3, upon coincubation with treated Aβ 1-42 fibrils, the fluorescence intensity of the excitation spectra and the emission spectra sharply enhanced, accompanied by a redshift, which indicated that the Aβ 1-42 protein was in a good aggregation state and could be used for the following detection.
Molecules 2023, 28, x FOR PEER REVIEW 5 of fluorescence intensity of the excitation spectra and the emission spectra sharply enhance accompanied by a redshift, which indicated that the Aβ1-42 protein was in a good aggreg tion state and could be used for the following detection. To clarify the saturation time of the interaction between TMNL and Aβ1-42 fibrils, th "Time-Fluorescence Intensity" experiment was performed in PBS buffer solution (pH 7. 10 mM). After coincubation with Aβ1-42 fibrils, the fluorescence emission intensity at 64 nm was slightly enhanced, but a very obvious emission peak appeared at 496 nm an increased rapidly with longer coincubation time, which can be attributed to the RIM effe by the binding of TMNL with the Aβ1-42 fibrils. The fluorescence intensity at 496 nm b came constant within 60 min, and the color change in the solution before and after th addition of Aβ1-42 fibrils could be clearly distinguished with the naked eye (from red yellow) under UV illumination at 365 nm ( Figure 4, insert). When the coincubation tim was fixed at 60 min, the fluorescence intensity of TMNL at approximately 496 nm in creased gradually and finally tended to flatten with increasing Aβ1-42 fibril concentratio The increased fluorescence intensity was up to 47-fold ( Figure 5). Moreover, the line relationship between the Aβ1-42 fibril concentration and the fluorescence intensity TMNL at 496 nm was obtained in the concentration range of 0-45 µg·mL −1 (R 2 = 0.995 Figure 5), indicating that the concentration of Aβ1-42 fibrils can be quantitatively estimate by TMNL. Then, the UV absorption spectra of TMNL in the presence and absence of Aβ 42 fibrils were also determined. As shown in Figure 6a, the absorption band at 265-360 nm gradually increased with increasing concentrations of Aβ1-42 fibrils, which also indicate that the binding of TMNL and Aβ1-42 fibrils changed the conjugation system of the TMN molecule. These results suggested that TMNL could act as an efficient and sensitive to for the detection of Aβ1-42 fibrils. To clarify the saturation time of the interaction between TMNL and Aβ 1-42 fibrils, the "Time-Fluorescence Intensity" experiment was performed in PBS buffer solution (pH 7.4, 10 mM). After coincubation with Aβ 1-42 fibrils, the fluorescence emission intensity at 645 nm was slightly enhanced, but a very obvious emission peak appeared at 496 nm and increased rapidly with longer coincubation time, which can be attributed to the RIM effect by the binding of TMNL with the Aβ 1-42 fibrils. The fluorescence intensity at 496 nm became constant within 60 min, and the color change in the solution before and after the addition of Aβ 1-42 fibrils could be clearly distinguished with the naked eye (from red to yellow) under UV illumination at 365 nm ( Figure 4, insert). When the coincubation time was fixed at 60 min, the fluorescence intensity of TMNL at approximately 496 nm increased gradually and finally tended to flatten with increasing Aβ 1-42 fibril concentration. The increased fluorescence intensity was up to 47-fold ( Figure 5). Moreover, the linear relationship between the Aβ 1-42 fibril concentration and the fluorescence intensity of TMNL at 496 nm was obtained in the concentration range of 0-45 µg·mL −1 (R 2 = 0.9955, Figure 5), indicating that the concentration of Aβ 1-42 fibrils can be quantitatively estimated by TMNL. Then, the UV absorption spectra of TMNL in the presence and absence of Aβ 1-42 fibrils were also determined. As shown in Figure 6a, the absorption band at 265-360 nm gradually increased with increasing concentrations of Aβ 1-42 fibrils, which also indicated that the binding of TMNL and Aβ 1-42 fibrils changed the conjugation system of the TMNL molecule. These results suggested that TMNL could act as an efficient and sensitive tool for the detection of Aβ 1-42 fibrils. Figure 5), indicating that the concentration of Aβ1-42 fibrils can be quantitatively estimated by TMNL. Then, the UV absorption spectra of TMNL in the presence and absence of Aβ1-42 fibrils were also determined. As shown in Figure 6a, the absorption band at 265-360 nm gradually increased with increasing concentrations of Aβ1-42 fibrils, which also indicated that the binding of TMNL and Aβ1-42 fibrils changed the conjugation system of the TMNL molecule. These results suggested that TMNL could act as an efficient and sensitive tool for the detection of Aβ1-42 fibrils.

Selectivity Study
We also screened a series of sulfur-containing substances to verify the selectivity of TMNL for Aβ1-42 fibrils, including L(+)-cysteine (Cys), glutathione (GSH), HSO3 − , SO3 2− , and BSA. TMNL was incubated with various high-concentration interfering substances. As shown in Figure 7, the fluorescence intensity of TMNL was nearly unchanged in the presence of other sulfur-containing compounds. Although BSA could produce a weak flu-

Selectivity Study
We also screened a series of sulfur-containing substances to verify the selectivity of TMNL for Aβ1-42 fibrils, including L(+)-cysteine (Cys), glutathione (GSH), HSO3 − , SO3 2− , and BSA. TMNL was incubated with various high-concentration interfering substances. As shown in Figure 7, the fluorescence intensity of TMNL was nearly unchanged in the presence of other sulfur-containing compounds. Although BSA could produce a weak flu-

Selectivity Study
We also screened a series of sulfur-containing substances to verify the selectivity of TMNL for Aβ 1-42 fibrils, including L(+)-cysteine (Cys), glutathione (GSH), HSO 3 − , SO 3 2− , and BSA. TMNL was incubated with various high-concentration interfering substances. As shown in Figure 7, the fluorescence intensity of TMNL was nearly unchanged in the presence of other sulfur-containing compounds. Although BSA could produce a weak fluorescence response, it was far lower than the 47-fold increase produced by Aβ 1-42 fibrils, which suggested that TMNL could be used for highly selective detection of Aβ 1-42 fibrils under complex physiological conditions. Compared to the reported probes (Table S2), TMNL had no inferior selectivity for Aβ 1-42 fibrils.

The Dissociation Constant Study
Binding affinity was a crucial factor for TMNL to efficiently trace the Aβ1-42 fibrils, and a saturation binding experiment was performed to quantitatively evaluate the binding ability of TMNL to Aβ1-42 fibrils. The fluorescence intensity was measured by incubating Aβ1-42 fibrils with different concentrations of TMNL, as shown in Figure 8. The dissociation constant Kd in this process was calculated to be 410.4 nM, which indicated that TMNL was a good substrate for Aβ1-42 fibrils and could be well applied to Aβ1-42 fibril detection. Compared to the reported AIE-type NIR fluorescence probes for the detection of Aβ amyloid (Tables S1 and S3), TMNL had a moderate binding affinity.

Appearance Observations
To intuitively clarify the binding appearance of TMNL with Aβ1-42 fibrils, transmission electron microscopy (TEM) images of TMNL binding to Aβ1-42 fibrils were obtained. As shown in Figure 9a

The Dissociation Constant Study
Binding affinity was a crucial factor for TMNL to efficiently trace the Aβ 1-42 fibrils, and a saturation binding experiment was performed to quantitatively evaluate the binding ability of TMNL to Aβ 1-42 fibrils. The fluorescence intensity was measured by incubating Aβ 1-42 fibrils with different concentrations of TMNL, as shown in Figure 8. The dissociation constant K d in this process was calculated to be 410.4 nM, which indicated that TMNL was a good substrate for Aβ 1-42 fibrils and could be well applied to Aβ 1-42 fibril detection. Compared to the reported AIE-type NIR fluorescence probes for the detection of Aβ amyloid (Tables S1 and S3), TMNL had a moderate binding affinity.

The Dissociation Constant Study
Binding affinity was a crucial factor for TMNL to efficiently trace the Aβ1-42 fibrils, and a saturation binding experiment was performed to quantitatively evaluate the binding ability of TMNL to Aβ1-42 fibrils. The fluorescence intensity was measured by incubating Aβ1-42 fibrils with different concentrations of TMNL, as shown in Figure 8. The dissociation constant Kd in this process was calculated to be 410.4 nM, which indicated that TMNL was a good substrate for Aβ1-42 fibrils and could be well applied to Aβ1-42 fibril detection. Compared to the reported AIE-type NIR fluorescence probes for the detection of Aβ amyloid (Tables S1 and S3), TMNL had a moderate binding affinity.

Appearance Observations
To intuitively clarify the binding appearance of TMNL with Aβ1-42 fibrils, transmission electron microscopy (TEM) images of TMNL binding to Aβ1-42 fibrils were obtained.

Appearance Observations
To intuitively clarify the binding appearance of TMNL with Aβ 1-42 fibrils, transmission electron microscopy (TEM) images of TMNL binding to Aβ 1-42 fibrils were obtained. As shown in Figure 9a,b, TMNL could self-assemble into small spherical nanostructures 30-100 nm in diameter. After coincubation with Aβ 1-42 fibrils in PBS buffer solution (pH 7.4, 10 mM) for 60 min, as shown in Figure 9c,d, TMNL aggregates were attached to the intertwined Aβ 1-42 fibrils, which indicated that TMNL and Aβ 1-42 fibrils could combine to make TPE aggregate and to emit strong fluorescence.

In Vitro Mapping with High-Fidelity Aβ1-42 Plaque Information
To evaluate the performance of TMNL in bioimaging-related fields, TMNL was ap plied to stain Aβ1-42 plaques in the brain tissue of AD transgenic mice (App, 13 month old). As shown in Figure 10, TMNL bound to and stained Aβ1-42 plaques in the brain tissue of AD transgenic mice, as observed by an Axio Observer Z1 microscope. Due to the AIE properties of TMNL, no additional rinsing or processing was necessary. These results pre liminarily indicated that TMNL had a good affinity for Aβ1-42 plaques in biological tissues which was suitable and convenient for further clinical application.

In Vitro Mapping with High-Fidelity Aβ 1-42 Plaque Information
To evaluate the performance of TMNL in bioimaging-related fields, TMNL was applied to stain Aβ 1-42 plaques in the brain tissue of AD transgenic mice (App, 13 months old). As shown in Figure 10, TMNL bound to and stained Aβ 1-42 plaques in the brain tissue of AD transgenic mice, as observed by an Axio Observer Z1 microscope. Due to the AIE properties of TMNL, no additional rinsing or processing was necessary. These results preliminarily indicated that TMNL had a good affinity for Aβ 1-42 plaques in biological tissues, which was suitable and convenient for further clinical application.
To evaluate the performance of TMNL in bioimaging-related fields, TMNL was applied to stain Aβ1-42 plaques in the brain tissue of AD transgenic mice (App, 13 months old). As shown in Figure 10, TMNL bound to and stained Aβ1-42 plaques in the brain tissue of AD transgenic mice, as observed by an Axio Observer Z1 microscope. Due to the AIE properties of TMNL, no additional rinsing or processing was necessary. These results preliminarily indicated that TMNL had a good affinity for Aβ1-42 plaques in biological tissues, which was suitable and convenient for further clinical application. Figure 10. (a,b) Fluorescence staining images of TMNL in brain tissue sections of AD transgenic mice. Figure 10. (a,b) Fluorescence staining images of TMNL in brain tissue sections of AD transgenic mice.

Preparation of AD Transgenic Mouse Brain Tissue Paraffin Sections
Brain tissue paraffin sections of AD transgenic mice (App, 13 months old) were obtained from the Institute of Laboratory Animals Science, CAMS & PUMC (Beijing, China). Pretreatment of the brain tissue paraffin sections of AD transgenic mice (App, 13 months old) included soaking the sections in dimethylbenzene solution for 5 min to dewax, rinsing with ethanol and secondary water, and finally air-drying for further use. The brain tissue paraffin section images were observed on an Axio Observer Z1 microscope (Carl Zeiss AG, Oberkochen, Germany).

Coincubation Preparation Process of TMNL with Aβ 1-42 Fibrils
The TMNL was mixed with Aβ 1-42 aggregate solution and then coincubated at 37 • C for 1 h at 120 r·min −1 in a constant temperature oscillator for further testing.

Synthesis of Compound 4
Compounds 1 to 3 were synthesized according to the methods reported in a previous article [47].
Compound 4: Compound 3 (0.1 g, 0.054 mmol) was dissolved in dry methanol (5.42 mL), and a solution of MeONa/MeOH (5.4 M, 19 µL) was added with a microsyringe, and then the mixture was stirred for 4 h at room temperature. The reaction was neutralized with an IR-120 hydrogen ion resin to adjust the pH of the mixture to approximately 7. Then, the mixture was filtered, and the solvent was evaporated. The crude compound was purified by Bio-Gel ® P-2 gel (pure H 2 O) to obtain Compound 4 (41 mg, 40% yield). 1

Synthesis of Compound 13
Compounds 5 to 12 were synthesized according to the methods reported in a previous article [47].
Compound 13: Compound 12 (50 mg, 0.096 mmol), malononitrile (51 mg, 0.77 mmol), and ethanol (4 mL) were added in a round bottom flask in that order. The mixture was then stirred at 80 • C until the reactant disappeared. After the reaction was accomplished, the reaction was cooled to room temperature, and the solvent was evaporated. The residue was purified by column chromatography (Hexane:EtOAc = 3:1, v/v) to obtain Compound 13 (18 mg, 31% yield). 1

Synthesis of Compound TMNL
TMNL: Compound 13 (0.42 g, 0.11 mmol) and Compound 4 (30 mg, 0.049 mmol) were added to a double neck bottle and dissolved in THF (3 mL) under N 2 . Then, sodium ascorbate (0.019 M, 1 mL, aq.) and CuSO 4 (0.096 M, 1 mL, aq.) were added successively and stirred at 60 • C for 4 h. Then, the reaction was cooled to room temperature, extracted with DCM, washed with saturated aqueous sodium chloride, and dried over Na 2 SO 4 . After filtration, the solvent of the mixture was evaporated. The crude product was purified by Bio-Gel P-2 gel (pure H 2 O) to obtain TMNL (0.14 g, 45% yield). 1

Conclusions
In this work, an AIE-active water-soluble and near-infrared fluorescent luminogen, TMNL, was successfully designed, synthesized, and well characterized; it contained hydrophilic lactose units, hydrophobic malononitrile, and a TPE derivative moiety. TMNL could self-assemble into fluorescent organic nanoparticles in aqueous solution and was used for the detection of Aβ 1-42 fibrils and the NIR imaging of Aβ 1-42 plaques sensitively and selectively. After coincubation with Aβ 1-42 fibrils, the fluorescence intensity of TMNL at 496 nm increased up to 47-fold, and it also had excellent selectivity for Aβ 1-42 fibrils.
The K d value (410.4 nm) indicated the good affinity between TMNL and Aβ 1-42 fibrils, which could also be observed by TEM. TMNL could be utilized for the imaging of Aβ 1-42 plaques in brain tissue accurately and conveniently, which could be an alternative to commercial probes. Although TMNL still had some characteristics to be further improved, such as complex preparation, this work was expected to facilitate relevant studies on Alzheimer's disease.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/molecules28135110/s1, Table S1: AIE-type fluorescent probes for the detection of Aβ amyloid; Table S2: The selectivity comparison of AIE-type fluorescent probes for the detection of Aβ amyloid; Table S3: The molecular structures of the corresponding probes in Tables S1 and S2; Figure S1: 1 H NMR spectra of compound 4; Figure S2: 1 H NMR spectra of compound 13; Figure S3: 13 C NMR spectra of compound 13; Figure S4

Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.

Data Availability Statement:
The data that support the findings of this study are available from the corresponding author upon reasonable request.