Next generation single-molecule techniques: Imaging, labeling, and manipulation in vitro and in cellulo.

Owing to their unique abilities to manipulate, label, and image individual molecules in vitro and in cellulo, single-molecule techniques provide previously unattainable access to elementary biological processes. In imaging, single-molecule fluorescence resonance energy transfer (smFRET) and protein-induced fluorescence enhancement in vitro can report on conformational changes and molecular interactions, single-molecule pull-down (SiMPull) can capture and analyze the composition and function of native protein complexes, and single-molecule tracking (SMT) in live cells reveals cellular structures and dynamics. In labeling, the abilities to specifically label genomic loci, mRNA, and nascent polypeptides in cells have uncovered chromosome organization and dynamics, transcription and translation dynamics, and gene expression regulation. In manipulation, optical tweezers, integration of single-molecule fluorescence with force measurements, and single-molecule force probes in live cells have transformed our mechanistic understanding of diverse biological processes, ranging from protein folding, nucleic acids-protein interactions to cell surface receptor function.

Generation of site-specifically labelled fluorescent human XPA to investigate DNA binding dynamics during nucleotide excision repair.

Nucleotide excision repair (NER) promotes genomic integrity by removing bulky DNA adducts introduced by external factors such as ultraviolet light. Defects in NER enzymes are associated with pathological conditions such as Xeroderma Pigmentosum, trichothiodystrophy, and Cockayne syndrome. A critical step in NER is the binding of the Xeroderma Pigmentosum group A protein (XPA) to the ss/ds DNA junction. To better capture the dynamics of XPA interactions with DNA during NER we have utilized the fluorescence enhancement through non-canonical amino acids (FEncAA) approach. 4-azido-L-phenylalanine (4AZP or pAzF) was incorporated at Arg-158 in human XPA and conjugated to Cy3 using strain-promoted azide-alkyne cycloaddition. The resulting fluorescent XPA protein (XPACy3) shows no loss in DNA binding activity and generates a robust change in fluorescence upon binding to DNA. Here we describe methods to generate XPACy3 and detail in vitro experimental conditions required to stably maintain the protein during biochemical and biophysical studies.

Polystyrene-Based Matrix to Enhance the Fluorescence of Aggregation-Induced Emission Luminogen for Fluorescence-Guided Surgery.

Achieving ultrabright fluorogens is a key issue for fluorescence-guided surgery (FGS). Fluorogens with aggregation-induced emission (AIEgens) are potential agents for FGS on the benefit of the bright fluorescence in physiological conditions. Herein, the fluorescence brightness of AIEgen is further improved by preparing the nanoparticle using a polystyrene-based matrix and utilizing it for tumor FGS with a high signal-to-background ratio. After encapsulating AIEgen into polystyrene-poly (ethylene glycol) (PS-PEG), the fluorescence intensity of the prepared AIE@PS-PEG nanoparticles is multiple times that of nanoparticles in 1, 2-distearoyl-sn-glycero-3-phosphoethanolamine-poly (ethylene glycol) (DSPE-PEG), a commonly used polymer matrix for nanoparticle preparation. Molecular dynamics simulations suggest that higher free energy is required for the outer rings of AIEgen to rotate in polystyrene than in the DSPE, indicating that the benzene rings in polystyrene can restrict the intramolecular motions of AIEgen better than the alkyl chain in DSPE-PEG. Fluorescence correlation microscopy detections suggest that the triplet excited state of AIEgens is less in PS-PEG than in DSPE-PEG. The restricted intramolecular motions and suppressed triplet excited state result in ultrabright AIE@PS-PEG nanoparticles, which are more conducive to illuminating tumor tissues in the intestine for FGS. The illumination of metastatic tumors in lungs by AIE@PS-PEG nanoparticles is also tried.

Enhancing Fluorescence in Both Solution and Solid States Induced by Imine Cage Formation.

Developing luminescent materials that exhibit strong emissions in both solution and solid phases is highly desirable and challenging. Herein, we report imine-bond directed formation of a rigid organic cage (TPE-cage) that was synthesized by [2+4] imine condensation of a TPE-cored tetra-aldehyde (TPE-TA) with a clip-like diamine (XA) to illustrate confinement-induced fluorescence enhancement. Compared to the non-emissive TPE-TA (ϕF =0.26 %) in the dichloromethane (DCM) solution, the TPE-cage achieved a remarkable (~520-fold) emission enhancement (ϕF =70.38 %). In contrast, a monomeric tetra-imine model compound (TPE-model) showed only a minor enhancement (ϕF =0.56 %) in emission compared to the parent tetra-aldehyde TPE-TA. The emission of TPE-cage was further enhanced by ~1.5-fold (ϕF =80.96 %) in the aggregated state owing to aggregation-induced emission enhancement (AIEE). This approach establishes the potential for synthesizing luminescent materials with high emission in both solution and solid-state by employing a single-step imine condensation reaction.

Density Functional Theory Calculations on Fluorescence-Enhanced Mechanisms of the Optical Sensor for Zinc Ions, ADPA.

N-(9-anthracenylmethyl)-N-(2-pyridinylmethyl)-2-pyridinemethanamine (ADPA) as a specific ion sensor for Zn2+ has been widely applied. Although the photo-induced electron transfer (PET) mechanism was proposed previously, its fluorescence-enhanced effect still remains somewhat ambiguous, according to unknown influences of non-radiative energy decay pathways, such as intersystem crossing and internal conversion. Herein, a thorough study using density functional theory has been performed for low-lying electronic states of the ADPA monomer and hydrated ADPA-Zn2+ complex. Based on interfragment charge transfer analyses, we quantitatively calculated the amount of transferred electrons in the monomer and complex, providing solid evidences for the PET mechanism and in line with the conclusion of frontier molecular orbital analyses. Moreover, the ISC process of S1→T2 was confirmed to play a considerable role in the excitation energy relaxation process of the ADPA monomer, but this influence was significantly suppressed in the hydrated ADPA-Zn2+ complex. These results provide additional clues for the design of new metal ion-specific fluorescence probes.

Giant enhancement of fluorescence resonance energy transfer based on nanoporous gold with small amount of residual silver.

Fluorescence resonance energy transfer (FRET) was found strongly enhanced by plasmon resonance. In this work, Nanoporous Gold with small amount of residual silver was used to form nanoporous gold/organic molecular layer compound with PSS and PAH. The ratio of its specific gold and silver content is achieved by controlling the time of its dealloying. Layered films of polyelectrolyte multilayers were assembled between the donor-acceptor pairs and NPG films to control distance. The maximum of FRET enhancement of 80-fold on the fluorescence intensity between the donor-acceptor pairs (CFP-YFP) is observed at a distance of ∼10.5 nm from the NPG film. This Nanoporous Gold with small amount of residual silver not only enhanced FRET 4-fold more than nanoporous gold of only gold content almost, but also effectively realized the regulation of FRET enhancement. The ability to precisely measure and regulate the enhancement of FRET enables the rational selection of plasmonic nanotransducer dimensions for the particular biosensing application.

A molecularly imprinted ratiometric fluorescent sensor for visual detection of 1-naphthol based on fluorescence-enhanced CdTeS QDs via APTES modification.

CdTeS quantum dots (CdTeS QDs) were synthesized using the hydrothermal method and subsequently modified with (3-aminopropyl)triethoxysilane (APTES). This modification resulted in a significant enhancement of the fluorescence intensity, which was observed to be five times stronger than that of unmodified CdTeS QDs at 597 nm. Only after the fluorescence enhancement by APTES modification, the material showed a response to 1-naphthol (1-NP). Based on this, the molecularly imprinted polymers (MIPs) with ratiometric fluorescence were developed for the detection of 1-NP, that is, the synthetic raw material and the metabolite of the pesticide carbaryl. Under the excitation of 365 nm UV, the bright orange-red fluorescence (597 nm) of CdTeS QDs encapsulated in MIPs was quenched by 1-NP in the suspension, and 1-NP showed a gradually increasing blue emission (460 nm) with the increase of its concentration. This sensor has a good linear relationship between fluorescence intensity ratio (F460/F597) and 1-NP concentration (C1-NP) in a large concentration range (6.0-140.0 µM, LOD=0.45 µM, RSD<4.41%). It exhibits a visible fluorescence change from orange-red to blue-purple. Excellent recoveries in real samples were obtained by simulating carbaryl metabolism and demonstrated its potential in detection of 1-NP and carbaryl.

Plasmonic-Enhanced Bright Single Spin Defects in Silicon Carbide Membranes.

Optically addressable spin defects in silicon carbide (SiC) have emerged as attractable platforms for various quantum technologies. However, the low photon count rate significantly limits their applications. We strongly enhanced the brightness by 7 times and spin-control strength by 14 times of single divacancy defects in 4H-SiC membranes using a surface plasmon generated by gold film coplanar waveguides. The mechanism of the plasmonic-enhanced effect is further studied by tuning the distance between single defects and the surface of the gold film. A three-energy-level model is used to determine the corresponding transition rates consistent with the enhanced brightness of single defects. Lifetime measurements also verified the coupling between defects and surface plasmons. Our scheme is low-cost, without complicated microfabrication and delicate structures, which is applicable for other spin defects in different materials. This work would promote developing spin-defect-based quantum applications in mature SiC materials.

Alkyl-Doping Enables Significant Suppression of Conformational Relaxation and Intermolecular Nonradiative Decay for Improved Near-Infrared Fluorescence Imaging.

Despite various efforts to optimize the near-infrared (NIR) performance of perylene diimide (PDI) derivatives for bio-imaging, convenient and efficient strategies to amplify the fluorescence of PDI derivatives in biological environment and the intrinsic mechanism studies are still lacking. Herein, we propose an alkyl-doping strategy to amplify the fluorescence of PDI derivative-based nanoparticles for improved NIR fluorescence imaging. The developed PDI derivative, OPE-PDI, shows much brighter in n-Hexane (HE) compared with that in other organic media, and the excited state dynamics investigation experimentally elucidates the solvent effect-induced suppression of intermolecular energy transfer and intramolecular nonradiative decay as the underlying mechanism for the fluorescence improvement. Theoretical calculations reveal the lowest reorganization energies of OPE-PDI in HE among various solvents, indicating the effectively suppressed conformational relaxation to support the strongest radiative decay. Inspired by this, an alkyl atmosphere mimicking HE is constructed by incorporating the octadecane into OPE-PDI-based nanoparticles, permitting up to 3-fold fluorescence improvement compared with the counterpart nanoparticles. Owing to the merits of high brightness, anti-photobleaching, and low biotoxicity for the optimal nanoparticles, they have been employed for probing and long-term monitoring of tumor. This work highlights a facile strategy for the fluorescence enhancement of PDI derivative-based nanoparticles.

Fluorescence Enhancement of Adamantane-Modified Dyes in Aqueous Solution via Supramolecular Interaction with Methyl-ß-cyclodextrin and Their Application in Cell Imaging.

The fluorescence of functional dyes was generally quenched in aqueous solution, which hindered their application in water-bearing detections. In this work, a novel strategy based on host-guest interaction was provided for the purpose of fluorescence enhancement in aqueous solution and cell imaging. Three adamantane-modified fluorescent dyes (Coum-Ad, NP-Ad, NR-Ad) with coumarin, 1,8-naphthalimide and Nile Red as fluorophores were initially designed and prepared. The ((adamantan-1-yl)methyl)amino group, as the auxochrome of those dyes, complexed with methylated ß-cyclodextrin (M-ß-CD) via supramolecular interaction, and then fluorescent supramolecular nanoparticles (FSNPs) were formed by self-assembly in water. The inclusion equilibrium constant (K) could be as high as 3.94×104  M-1 . With the addition of M-ß-CD, fluorescence quantum yields of these dyes were separately improved to 69.8 %, 32.9 % and 41.3 %. Inspired by the above satisfactory results, six adamantane-modified probes organelle-NPAds with organelle-targeting capability were further obtained. As the formation of hydrogen bonds between organelle-NPAd2 and M-ß-CD verified by theoretical calculation, K of organelle-NPAd2 (5.13×104  M-1 ~4.53×105  M-1 ) with M-ß-CD was higher than that of organelle-NPAd1 (1.15×104  M-1 ~3.66×104  M-1 ) and their fluorescence quantum yields increased to 32.8 %~83.6 % in aqueous solution. In addition, fluorescence enhancement was realized in cell imaging with the addition of M-ß-CD.

Synthesis, Characterization, Analytical Application, and Theoretical Studies of a Schiff Base, (E)-2-(2-aminophenylthio)-N-(Thiophen-2-yl-methylene) Benzenamine.

In this study, a new Schiff base, (E)-2-(2-aminophenylthio)-N-(thiophen-2-yl-methylene) benzenamine was synthesized for selective detection of Hg2+. This Schiff base was characterized by proton nuclear magnetic resonance (1HNMR), carbon-13 nuclear magnetic resonance (13CNMR), and Fourier-transform infrared (FTIR) spectroscopy. Binding interaction between (E)-2-(2-aminophenylthio)-N-(thiophen-2-yl-methylene)benzenamine and various metal ions has been studied by UV-Vis spectroscopic measurements and shows promising coordination towards Hg2+ and almost no interference from other metal ions (Ag+, Mn2+, Fe3+, Al3+, Co2+, Ni2+, Cu2+, Zn2+, Cd2+, Fe2+ and Cr3+).This Schiff base exhibiting detection limit of 3.8 × 10- 8 M. The Schiff base newly synthesized in this study was successfully applied to the determination of Hg2+ in water samples. In addition to the experimental study, a theoretical study was conducted using Gaussian 09 program to support the experimental findings. FTIR, NMR, bond angle, bond length, torsional angles, and structural approximation were studied using theoretical consideration.

Spectroscopic and Molecular Docking Studies of the Interaction of Non-steroidal Anti-inflammatory Drugs with a Carrier Protein: an Interesting Case of Inner Filter Effect and Intensity Enhancement in Protein Fluorescence.

Interaction of diclofenac and indomethacin with lysozyme was studied using several spectroscopic and molecular docking methods. Difference UV-visible spectra showed that the absorption profile of lysozyme changed when both diclofenac and indomethacin were mixed with the former. The sequential addition of both drugs to the lysozyme solution caused the decrease of the intrinsic fluorescence of the latter, however, when the data were corrected for inner filter effect, an enhancement in the fluorescence of lysozyme was detected. Accordingly, the fluorescence enhancement data were analyzed using Benesi-Hildebrand equation. Both, diclofenac and indomethacin showed good interaction with lysozyme, although, the association constants of indomethacin were nearly two-fold higher as compared to that of diclofenac. The binding was slightly more spontaneous in case of indomethacin and the major forces involved in the binding of both drugs with lysozyme were hydrogen bonding and hydrophobic interactions. Secondary structural analysis revealed that both drugs partially unfolded lysozyme. Results obtained through molecular docking were also in good agreement with the experimental outcomes. Both, diclofenac and indomethacin, are bounded at the same site inside lysozyme which is located in the big hydrophobic cavity of the protein.

Ultra-stable perovskite quantum dot composites encapsulated with mesoporous SiO2 and PbBr(OH) for white light-emitting diodes.

Lead halide perovskite quantum dots (QDs) with high fluorescence efficiency and high color purity have a broad application prospect in the field of backlight display, but poor stability has been a key factor limiting their commercialization. Herein, we successfully synthesized CsPbBr3 QDs-KIT-6 (CsPbBr3 -K6) composite by using KIT-6 molecular sieve as the limited template with a simple high temperature solid-phase method. Further, the semi-protected CsPbBr3 QDs in KIT-6 frame will spontaneously hydrolyze when encountering water, and finally the double-encapsulated CsPbBr3 QDs-KIT-6@PbBr(OH) (CsPbBr3 -K6@PbBr(OH)) composite are obtained. CsPbBr3 -K6@PbBr(OH) composite shows excellent green emission properties, including a photoluminescence quantum yield (PLQY) (~73%) and a narrow emission linewidth of 25 nm. It is interesting that, the composite has excellent stability, including water stability without attenuation of fluorescence intensity after soaking in water for 60 days, thermal stability of 120°C heating-cooling cycle, and excellent optical stability without attenuation under continuous ultraviolet irradiation.

A method for ultrafast tissue clearing that preserves fluorescence for multimodal and longitudinal brain imaging.

BACKGROUND: Tissue-clearing techniques have recently been developed to make tissues transparent for three-dimensional (3D) imaging at different scales, including single-cell resolution. However, current tissue-clearing workflows have several disadvantages, including complex protocols, time-consuming application, and fluorescence quenching. Additionally, they can be used mainly for clearing larger-volume samples, preventing wide and easy applicability in conventional experimental approaches. In this study, we aimed to develop a versatile, fast, and convenient method for clearing thin and semi-thick samples, which can be used for three-dimensional imaging of experimental or even clinical samples. RESULTS: We developed an alkaline solution (AKS) containing a combination of 2,2'-thiodiethanol (TDE), DMSO, D-sorbitol, and Tris for tissue clearing, as the alkaline environment is suitable for maintaining the fluorescence of most commonly used fluorescence protein GFP and its variants, and tested its clearing effect on samples from mice and human brains. We assessed the clearing speed, the preservation of fluorescence protein and dyes, and the imaging depth and quality. The results showed that AKS treatment rapidly cleared 300-µm-thick brain slices and 1-mm-thick slices from different organs within 5 min and 1 h, respectively. Moreover, AKS was compatible with a variety of fluorescence proteins and dyes. Most importantly, AKS enhanced the fluorescence of YFP, in contrast to the majority of existing tissue-clearing methods which reduce the fluorescence intensity of fluorescent proteins. Using AKS, we performed long-time high-resolution imaging of weak fluorescent protein-labelled tissues, long-distance fibre tracking, larger-scale 3D imaging and cell counting of the entire brain area, neural circuit tracing, 3D neuromorphic reconstruction, and 3D histopathology imaging. CONCLUSIONS: AKS can be used for simple and rapid clearing of samples from mice and human brains and is widely compatible with a variety of fluorescent dyes. Therefore, AKS has great potential to be used as a broad tissue-clearing reagent for biological optical imaging, especially for time-sensitive experiments.

ß-Cyclodextrin Polymer-Based Fluorescence Enhancement Strategy via Host-Guest Interaction for Sensitive Assay of SARS-CoV-2.

Nucleocapsid protein (N protein) is an appropriate target for early determination of viral antigen-based severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We have found that ß-cyclodextrin polymer (ß-CDP) has shown a significant fluorescence enhancement effect for fluorophore pyrene via host-guest interaction. Herein, we developed a sensitive and selective N protein-sensing method that combined the host-guest interaction fluorescence enhancement strategy with high recognition of aptamer. The DNA aptamer of N protein modified with pyrene at its 3' terminal was designed as the sensing probe. The added exonuclease I (Exo I) could digest the probe, and the obtained free pyrene as a guest could easily enter into the hydrophobic cavity of host ß-CDP, thus inducing outstanding luminescent enhancement. While in the presence of N protein, the probe could combine with it to form a complex owing to the high affinity between the aptamer and the target, which prevented the digestion of Exo I. The steric hindrance of the complex prevented pyrene from entering the cavity of ß-CDP, resulting in a tiny fluorescence change. N protein has been selectively analyzed with a low detection limit (11.27 nM) through the detection of the fluorescence intensity. Moreover, the sensing of spiked N protein from human serum and throat swabs samples of three volunteers has been achieved. These results indicated that our proposed method has broad application prospects for early diagnosis of coronavirus disease 2019.

Photochemically Enhanced Emission by Introducing Rational Photoactive Subunits into an Aggregation-Induced Emission Luminogen.

Herein, we propose a rational design strategy by introducing photoactive thienyl and pyridyl groups into an AIE-active tetraarylethene skeleton to achieve highly efficient photochemistry-activated fluorescence enhancement from dominantly photo-physical aggregation-induced emission behavior, and prove that such photoactivated fluorescence enhancement is perfectly suited for superstable photocontrollable dual-mode patterning applications in both solution and solid matrix. It is found that the photoactivated fluorescence of designed AIEgen is attributed to the irreversible cyclized-dehydrogenation reaction under UV irradiation, and the oxidation product has a brighter fluorescence in both solution and solid states owning to its rigid and planar structure. The overall transformation rate of the AIEgen from its opened form to dehydrogenated form is up to nearly 100 % in a short period of UV irradiation, and the fast transformation and the stable product of this photochemical reaction guarantees super stability of photocontrolled patterning, which can be applied in photoactivated dual-mode patterning and advanced anti-counterfeiting.

An Efficient Inclusion Complex Based Fluorescent Sensor for Mercury (II) and its Application in Live-Cell Imaging.

The formation of an inclusion complex between hydroxypropyl-ß-cyclodextrin (H-CD) and 4-acetylphenyl-4-(((6-chlorobenzo[d]thiazol-2-yl)-imino)-methyl)-benzoate (L) was investigated by FT-IR, 1H-NMR, X-ray diffraction (XRD), FT-Raman, scanning electron microscope (SEM) techniques in the solid-state, absorption and emission spectroscopy in the liquid state and the virtual state as molecular docking technique. The binding properties of the inclusion complex (H-CD: L) with cations in deionized water was observed via absorbance and photoluminescence (PL) emission spectroscopy. The fluorescence probe (H-CD: L) inclusion complex (IC) was examined for several heavy metal cations, and identified that the PL emission wavelength of the complex displayed a continuous rise in the fluorescence intensity for Hg2+. A linearity range of 1 × 10-8 - 11 × 10-8 M and limit of detection value of 2.71 × 10-10 M was found to be achieved for the detection of Hg2+. This outcome proves that the inclusion complex H-CD: L would be a promising material for the development a solid-state fluorescence probe for detecting Hg2+. It also shows application in real sample analysis and cell imaging.

Fluorescence Enhancement Based Quantification of Human Serum Albumin from Biological Sample Using Indole Based Nanosuspension: Molecular Interactions and Molecular Docking Studies.

Fluorescent 3-[(E)-(2-phenylhydrazinylidene) methyl]-1H-indole (PHI) was synthesized by condensation of indole-3-carboxaldehyde and phenyl hydrazine in presence of acetic acid and ethanol and after spectral characterization used further to prepare its aqueous nano suspension by reprecipitation method using polyvinylpyrrolidone (PVP) as stabilizer. The average particle size of nano suspension measured by Dynamic Light Scattering (DLS) was found 77.5 nm while FESEM microphotograph showed spherical morphology. The blue shift in the absorption spectrum and stokes shifted fluorescence of nanosuspension of PHI compared to its monomer spectrum in dilute solution indicate formation of H-type aggregate by face to face overlapping of the molecules.The aggregation induced enhanced emission (AIEE) of PVP capped nanosuspension of PHI is increased appreciably by presence of aqueous solution of human serum albumin (HSA). A suitable mechanism of molecular binding interactions based on complex formation between PHI nanoaggregate and HSA through PVP is proposed. Fluorescence life time, zeta potential and particle size data of PHI nanoparticles (PHINPs) obtained in presence of different amounts of HSA are in support of molecular interactions leading to complex formation. The molecular docking studies showed that HSA and PVP capped PHINPs exhibit strong hydrogen bonding interaction. The fluorescence enhancement effect induced in PHI nanosuspension is used further to develop analytical method for quantitative estimation of HSA in aqueous biological sample solution.

Fluorescence signal amplification by optical reflection in metal-coated nanowells.

A significant amplification in the fluorescence signal is demonstrated when measured in metal (aluminum)-coated fluidic wells with volumes on the order of a nanoliter or smaller (nanowells). Photolithographic and wet etching procedures were used to fabricate these nanowells on glass substrates followed by vapor deposition of an aluminum layer on them. The fluorescence signal recorded in these structures was enhanced due to the reflection of the incident and emitted radiation by the metal layer as well as focusing of this light by the curvature of the well surface. While the first effect amplified the background signal in the entire assay chamber, the latter one produced signal hotspots around the edges and center of the nanowell. In this work, we were able to realize over a 20-fold enhancement in the fluorescence signal upon quantitating it at the central hotspot of an aluminum-coated circular nanowell with a depth and photo-patterned diameter of 30 µm and 38 µm, respectively. More interestingly, our experiments indicate that this enhancement factor may be further improved by optimizing the curvature of the nanowell surface to merge all the signal hotspots within a smaller detection zone. Finally, quantitative assays using horseradish peroxidase samples were performed on the reported signal enhancement platform to further demonstrate its utility for making sensitive analytical measurements.

Rigid Schiff Base Complex Supermolecular Aggregates as a High-Performance pH Probe: Study on the Enhancement of the Aggregation-Caused Quenching (ACQ) Effect via the Substitution of Halogen Atoms.

Optical signals of pH probes mainly driven from the formation or rupture of covalent bonds, whereas the changes in covalent bonds usually require higher chemical driving forces, resulting in limited sensitivity and reversibility of the probes. The exploration of high-performance pH probes has been a subject of intense investigation. Herein, a new pH probe has been developed, with optical property investigation suggesting the probe has excellent signal-to-noise ratio, and fluorescence intensity shows exponential growth, combined with a visible color change, as pH increased from 5.1 to 6.0; Moreover, the probe has outstanding stability and reversibility, with more than 90% of the initial signal intensity remaining after 30 cycles. In order to better understand the special fluorescence behavior of the reported probe, the non-halogenated isomer is introduced for comparison, combined with the results of structural analysis, quantitative calculation and optical experiments, and the possible mechanism of the special supramolecular aggregation-caused quenching effect induced by the halogen atom is discussed.