A reaction-based fluorescence for monitoring β-Gal in living cells and BALB/c nude mice

Abstract

β-Galactosidase (β-Gal), as a key biomarker closely associated with colorectal cancer, holds an indispensable position in the early diagnosis and therapeutic efficacy assessment of cancer. MQ-βGal achieves highly specific targeted binding with β-Gal and demonstrates outstanding comprehensive performance, featuring a broad linear response range, high detection sensitivity and robust interference resistance. Benefiting from MQ-βGal’s favorable biocompatibility and low cytotoxicity, it has been successfully applied to real-time tracking of dynamic β-Gal activity changes within living cell. Building upon this, MQ-βGal’s application has been further extended to the in vivo level, successfully achieving in situ monitoring of endogenous β-Gal activity in BALB/c nude mouse models, thereby validating its suitability for in vivo biological imaging. More significantly, clinical sample validation experiments demonstrate that MQ-βGal effectively distinguishes biological samples from healthy individuals and colorectal cancer patients, specifically identifying abnormal β-Gal expression patterns during colorectal cancer development. These findings fully highlight the potential application value and translational prospects of this fluorescent diagnostic system in the early clinical diagnosis and disease assessment of colorectal cancer.

Supplementary Information

The online version contains supplementary material available at https://doi.org/10.1038/s41598-026-58121-w.

Introduction

In the field of life sciences, the precise detection of biomarkers remains a pivotal aspect of research, playing a crucial role in deepening our understanding of biological processes, as well as in the early diagnosis and treatment of diseases^1–3. β-galactosidase (β-Gal), as an important biomarker, has garnered significant attention from researchers in recent years^4,5. It plays an indispensable role in numerous physiological processes, such as participating in intracellular carbohydrate metabolism, catalyzing the hydrolysis of lactose into glucose and galactose, thereby providing the essential energy and material foundation for normal cellular physiological activities^6–10. Moreover, β-Gal also plays a key role in cellular physiological and pathological processes, with changes in its expression levels and activity closely associated with the onset and progression of various diseases^11–14. During cellular senescence, β-Gal activity significantly increases, establishing it as a prominent marker of cellular ageing^15–19. Monitoring its activity provides crucial insights for research into age-related diseases, aiding in early detection and intervention to delay the ageing process and enhance human health^20–22. In malignant tumours such as colorectal cancer, β-Gal expression is markedly elevated compared to normal tissues, making it a potential biomarker for early diagnosis and disease monitoring. Accurate measurement of β-Gal levels and activity holds significant importance for colorectal cancer early screening, precise diagnosis and treatment planning^23,24. It enables clinicians to detect tumours promptly, assess disease severity and deliver more effective therapeutic interventions for patients.

Traditional β-Gal detection methods, such as enzyme-linked immunosorbent assays (ELISA) and high-performance liquid chromatography (HPLC), can detect β-Gal to a certain extent²⁵. However, these methods generally suffer from drawbacks including cumbersome procedures, lengthy detection times as well as the requirement for specialized equipment and technical personnel²⁶. Consequently, there exists an urgent practical need to develop a rapid, sensitive, and straightforward β-Gal detection method²⁷. Fluorescent probe detection technology, as an emerging analytical approach, has demonstrated significant advantages and potential in biomolecular detection in recent years. It relies on specific interactions between fluorescent substances and target molecules, enabling qualitative and quantitative analysis through monitoring changes in fluorescent signals. Fluorescent probe detection technology exhibits high sensitivity, capable of detecting target molecules at extremely low concentrations, thereby enabling trace detection of β-Gal^28,29. Its excellent selectivity allows specific recognition of β-Gal, effectively avoiding interference from other biomolecules and ensuring accurate results³⁰. Furthermore, it enables real-time monitoring, facilitating dynamic tracking of β-Gal activity changes and providing a powerful tool for investigating its mechanisms in physiological and pathological processes²⁵. Based on this, we previously designed the CS-βGal for real-time and efficient monitoring of β-Gal. It possesses excellent water solubility, enabling adaptation to complex and diverse detection environments, thereby significantly enhancing compatibility across application scenarios³¹. Consequently, applying fluorescent probe detection technology to β-Gal assessment holds promise in overcoming limitations of conventional methods, offering a more efficient and precise approach for β-galactosidase research and related disease diagnostics.

This study employed the detection system established under Scheme 1, utilizing the novel fluorescent probe MQ-βGal to achieve specific detection of β-Gal. Mechanistic studies confirmed that co-incubation of MQ-βGal with β-Gal induces a specific enzymatic reaction in the probe molecule, accompanied by a conformational shift. This triggered the specific emission of an orange fluorescent signal, providing a direct signaling basis for the visual detection of β-Gal. MQ-βGal can dynamic visual monitor endogenous and exogenous β-Gal expression levels in living cells and BALB/c nude mouse as vivo model. More importantly, MQ-βGal effectively differentiated bio-samples between healthy subjects and colorectal cancer patients, particularly in accurately identifying abnormal β-Gal expression patterns associated with colorectal cancer development. These results confirmed that the diagnostic system developed based on MQ-βGal holds potential translational application value in the clinical diagnosis of colorectal cancer, providing crucial evidence for subsequent clinical translation studies.

Scheme 1.

The design strategy of MQ-βGal against β-Gal activity.

Experiment section

Materials and instruments

The chromatographic-grade solvents and chemicals used were procured from Energy Chemical Co., Ltd. The fluorescence probe (MQ-βGal) was procured from Beyotime Co., Ltd. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from KeyGEN BioTECH Co., Ltd. The UV-vis absorption spectrum was measured with UNICO UV-4802 spectrophotometer and the fluorescence spectra as well as relative fluorescence intensity were recorded with F97XP (Lengguang tech CO., Ltd.). pH meter (METTLER TOLEDO 98 FE28-Micro) was used in the preparation of PBS buffers of different pH. Fluorescence imaging was performed with an Olympus FV-1000 confocal microscope (Japan). In vivo fluorescent imaging of HepG2-tumor-bearing nude mice model was tested by using the IVIS Lumina Ill In Vivo Imaging System (PerkinElmer).

Fluorescence spectroscopy

MQ-βGal was dissolved in DMSO at a stock concentration of 5 mM. Various disrupters were all prepared in deionized water in advance. In PBS buffer solution (10 mM, pH 7.4), the test solution was incubated at 37 ℃ and then the emission wavelength was recorded in the range from 570 to 700 nm by an excitation wavelength of 560 nm with a slit width at 10 × 10 nm. Each experiment was conducted three times to ensure the credibility of the experimental data.

Measurement of detection limit

The detection limit means that an analytical method can detect the maximum or minimum amount of the substance to be tested from the sample within a given degree of reliability. The detection limit (C_DL) was calculated using the following Eq. (1):

1

where, S_b is standard deviation according to 10 blank measurements, m is the slope of the analytical standard curve over a low concentration range.

Cell cultures and staining

Cell culture: HeLa and LoVo cells were cultured in low glucose DMEM medium containing 10% FBS and antibiotics (100 units/mL penicillin and 100 µg/mL streptomycin) in a 5% CO₂ humidity incubator at 37 ℃. Imaging experiments were performed when cells reached 80% confluence (approximately 5 × 10⁴ cells on 12-well plates).

Cell staining experiments: MQ-βGal was dissolved in DMSO at a stock concentration of 5 mM. HeLa and LoVo cells were placed on glass coverslips and allowed to adhere for 12 h. For imaging endogenous β-Gal, HeLa and LoVo cells were incubated with MQ-βGal (10 µM) in DMEM at 37 ℃ for 30 min, then washed and taken photos. The exogenous β-gal group of LoVo cells was pre-incubated 10 U/mL β-Gal for 30 min, and then 10 µM MQ-βGal was added and cultured for another 30 min. The suppression group of LoVo cells was pre-incubated with 50 µM D-galactose for 30 min, and then 10 µM MQ-βGal was added and cultured for another 30 min. Fluorescence imaging was taken by Olympus FV-1000 confocal microscope. The MQ-βGal’s red channel (580–660 nm) was collected with excitation at 560 nm. The above experiments can qualitatively detect endogenous and exogenous enzymes in cells. HeLa and LoVo cells were purchased directly from KeyGEN BioTECH Co., Ltd. and adhere to American Type Culture Collection. The research program was approved by Nanjing Jiangbei Hospital affiliated to Nantong University Biosafety and Ethics Committee (Approval number: AF/SC-08/02.0).

Cytotoxicity assay

The cytotoxicity of MQ-βGal in LoVo and HeLa cells was measured by MTT assay. First, LoVo and HeLa cells were seeded at a density of 1 × 10⁵ cells/mL in 96-well plates with 5% CO₂ at 37 °C. After cultivating for 24 h, cells were incubated with various concentrations (from 1 to 50 µM) of MQ-βGal (dissolved in DMSO) and cultured for another 24 h, respectively. Then 10 µL MTT was added to each well and adequate reaction with cells. The control experiments were conducted under the same conditions but without adding MQ-βGal. Measure the absorbance of the sample and control wells at 570 nm through the microplate reader and then calculate the cell viability by the absorbance ratio of the sample hole to the control cell. The cell viability was expressed by the mean values ± standard deviation (SD). Each experiment was conducted five times to ensure the credibility of the experimental data.

Detail of PBMCs

Peripheral blood mononuclear cells were isolated with histopaque-1077 gradient [SIGMA] through density gradient centrifugation. PBMCs were washed in ice cold PBS for two times and resuspended in the same with a cell density of 3 × 10⁶ cells/mL. PBMCs were treated with MQ-βGal (10 µM) and incubated for 30 min at 37℃ in dark. The endogenous fluorescence intensity (570–620 nm) was measured by Olympus FV-1000 confocal microscope under 40X magnification with excitation at 560 nm. The relative fluorescence intensities were quantitated using ImageJ software. The research program was approved by Nanjing Jiangbei Hospital affiliated to Nantong University Biosafety and Ethics Committee (Approval number: AF/SC-08/02.0). All methods were performed in accordance with the relevant guidelines and the Declaration of Helsinki.

Living mice imaging

BALB/c mice (6 − 8 weeks old, average body weight 16–18 g) were purchased from KeyGEN BioTECH Co., Ltd. All animals were acclimatized to the animal facility for one week prior to experimentation and housed under pathogen-free conditions, fed under conditions of 25 °C and 55% of humidity and allowed free access to standard laboratory water and chow. Before imaging, MQ-βGal (100 µM) was injected intraperitoneal into the BALB/c nude mice and fluorescent images were captured by the Lumina III at 0, 10, 20, 30, 40 50 and 60 min after injections. The imaging experiment were carried out under the 560 nm excitation filter and 580–660 nm emission filter. The experiments were approved by Animal Ethical Experimentation Committee of Nantong University in accordance with the requirements of the China National Act on the use of experimental animals (Approval number: SCXK2024-0011). All experiments were performed in accordance with relevant guidelines and regulations, as well as the IUCN Policy Statement on Research Involving Species at Risk of Extinction and the Convention on the Trade in Endangered Species of Wild Fauna and Flora.

Result and discussion

Spectral properties of MQ-βGal

First, to validate the detection sensitivity of MQ-βGal, we measured its photoluminescence (PL) emission spectra under the action of β-Gal at different activity, with the results shown in Fig. 1. As previously described, when 10 µM MQ-βGal was co-incubated with β-Gal at an excitation wavelength of 560 nm, virtually no fluorescence signal was detected in the system. However, at a β-Gal activity of 12 U/mL, the fluorescence signal at 589 nm exhibited a significant enhancement, increasing by a factor of 17.2. Further experiments demonstrated a good linear relationship between fluorescence signal intensity and β-Gal activity within the range of 0–12 U/mL (R² = 0.99272). According to the IUPAC (CDL= 3Sb/m), the detection limit of MQ-βGal for β-Gal was calculated to be 0.021 U/mL. The aforementioned excellent linear correlation and extremely low detection limit, serving as key indicators of sensitivity and confirming MQ-βGal’s outstanding detection performance. To validate the mechanism of action of this sensing system, this study conducted MS spectral analysis of the reaction products between MQ-βGal and β-Gal. As expected, Fig. 2 presented the mass spectrum signal for [MQ-βGal + H]⁺ with a mass-to-charge ratio (m/z) of 591.4322. Following the reaction between MQ-βGal and β-Gal, a novel characteristic peak [MQ + H]⁺ appeared in the spectrum, corresponding to a mass-to-charge ratio (m/z) of 429.2871. This result is fully consistent with data reported in the literature³². In summary, the proposed sensing reaction mechanism in this study is corroborated by both experimental data and relevant literature.

Fig. 1.

(A) Fluorescence intensity of MQ-βGal cultured with various concentrations of β-Gal (0–15 U/mL) for 20 min. (B) The fluorescence spectra (I₅₈₉) as a function of the concentrations of β-Gal within the scope of 1–12 U/mL. Concentration: 10 µM MQ-βGal, E_ex = 560 nm.

Fig. 2.

Mechanism of validation in MS.

Subsequently, we optimized the response time of the MQ-βGal assay for detecting β-Gal. In the experiment, after incubating 12 U/mL β-Gal with the MQ-βGal system, the fluorescence signal intensity at 589 nm was recorded every 2.5 min, with results shown in Fig. 3. The data indicate that the fluorescence signal gradually intensified with prolonged incubation time, with a marked recovery in fluorescence observed only upon the addition of β-Gal. Further observation revealed that the fluorescence signal reached a stable peak after 20 min of incubation. Based on this, 20 min was determined to be the optimal incubation time for this detection system.

Fig. 3.

(A) Fluorescence responses of MQ-βGal against β-Gal (12 U/mL) at various time points. (B) The fluorescence spectra (I₅₈₉) of MQ-βGal cultured with β-Gal (12 U/mL) as a function of time. Concentration: 10 µM MQ-βGal, E_ex = 560 nm.

Selectivity was one of the core metrics for evaluating the performance of fluorescent probes. Therefore, we further investigated the recognition specificity of MQ-βGal towards β-Gal and common biological interferents. Experiments employed common physiological environment substances such as cations, silver ions, amino acids, and various enzymes as interfering agents. Changes in fluorescence signals following co-incubation of MQ-βGal with these substances and β-Gal were detected, with results presented in Fig. 4. The data indicated that the fluorescent signal of the system exhibited no significant fluctuation after interaction with various interfering substances. However, a marked enhancement in the fluorescent signal was observed only when co-incubated with β-Gal. These results confirm that MQ-βGal possesses excellent recognition selectivity towards β-Gal, effectively mitigating the influence of common interfering substances found in biological systems.

Fig. 4.

The fluorescence spectra (I₅₈₉) of MQ-βGal to assorted frequently-used compounds existing in our body. (1: MQ-βGal alone; 2, AlCI₃; 3, CaCl₂; 4, NO³⁻; 5, ONOO⁻; 6, ClO⁻; 7, NaHS; 8, L-cysteine; 9, glutathione; 10, β-glucuronidase; 11, lysozyme; 12, esterase; 13 H₂O₂; 14, H₂S; 15, β-Gal. Concentration: 10 µM MQ-βGal, E_ex = 560 nm.

Given that MQ-βGal has demonstrated rapid responsiveness, excellent quantitative detection capabilities as well as high selectivity, this study systematically investigated its photostability and pH tolerance to further validate its suitability for detection in complex physiological environments. The influence of pH values ranging from 4.0 to 10.0 on the detection system was first investigated. As depicted in Fig. 5A, the fluorescence intensity of MQ-βGal remained stable throughout this pH range, regardless of whether β-Gal treatment had been applied. Photostability was assessed through comparative stability experiments: under identical experimental conditions, continuous fluorescence monitoring was conducted on both MQ-βGal with or without β-Gal and the commonly used organic dye fluorescein, with fluorescence intensity recorded every 25 min, as shown in Fig. 5B. The data indicate that regardless of β-Gal presence, the fluorescence intensity of MQ-βGal exhibited virtually no significant fluctuation throughout the monitoring period, whereas that of fluorescein showed marked attenuation, demonstrating the excellent photostability of MQ-βGal.

Fig. 5.

(A) The fluorescence spectra (I₅₈₉) of MQ-βGal in the absence (black square) and presence (red cycle) of β-Gal versus different pH from 4.0 to 10.0 in PBE buffer solution. (B) Time-based fluorescence spectra of MQ-βGal and fluorescein over a course of 6 h. (C) The fluorescence spectrum of MQ-βGal in water−glycerol mixtures with glycerol from 0 to 100%. (D) The fluorescence spectrum of MQ-βGal in different organic solvents and buffer solutions. Concentration: 10 µM MQ-βGal, E_ex = 560 nm.

Subsequently, to further investigate response of MQ-βGal for viscosity, we measured the fluorescence spectra in different ratios of glycerol-water systems. As illustrated in Fig. 5C, the fluorescence intensity of the MQ-βGal was gradually enhanced with the increase of glycerol ratio, exhibiting merely a 1.1-fold enhancement in pure glycerol compared with pure water. Finally, the fluorescence spectrum of MQ-βGal (10 µM) was tested in different organic solvents and buffer solutions, as we can see from Fig. 5D, the fluorescence spectrum of MQ-βGal was relatively stable with the changes of polarity in organic solvent. These results confirmed that MQ-βGal possesses remarkable photostability and pH adaptability as well as outstanding resistance to interference from polarity and viscosity, providing crucial support for its practical application in bioimaging and complex physiological sample detection.

Fluorescence imaging studies of MQ-βGal

The unique imaging properties of MQ-βGal, coupled with its high selectivity and specificity in vitro, established a foundation for its application in bioimaging. Consequently, we further explored its feasibility for application at the cellular level. First, MTT assays were employed to evaluate MQ-βGal’s cytotoxicity, verifying its suitability for cellular analysis: LoVo and HeLa cells were co-incubated with varying MQ-βGal activity (0, 1, 5, 10, 25, 50 µM) for 24 h, followed by cell viability assessment. Results are presented in Fig. 6. Experimental data indicate a slight increase in cytotoxicity with rising MQ-βGal activity. However, even at the highest concentration (50 µM), cell viability remained above 86.21%. Based on these low-cytotoxicity characteristics, MQ-βGal is confirmed as suitable for detecting β-Gal activity in LoVo and HeLa cells.

Fig. 6.

Cell viability (% of control) of LoVo and HeLa cells incubated in the presence of MQ-βGal for 24 h.

To evaluate the specificity of MQ-βGal in intracellular β-Gal detection, this study employed HeLa cells (human cervical carcinoma cells) with low β-Gal expression and LoVo cells (human colon carcinoma cells) with high β-Gal expression as model cells. Results demonstrated that a pronounced fluorescent signal was observable in LoVo cells (Fig. 7(d)-(f)), whereas HeLa cells exhibited virtually no fluorescence signal (Figs. 7(a)-(c)). This discrepancy stems from the distinct β-Gal expression characteristics of the two cell lines: LoVo cells, derived from human colon cancer tissue, exhibit β-Gal overexpression; conversely, HeLa cells, originating from human cervical cancer tissue, display extremely low β-Gal expression levels. This confirmed that MQ-βGal can precisely distinguish cells with differing expression levels by specifically recognizing β-Gal and generating red fluorescence. To further validate that the fluorescence signal specifically originates from the interaction between β-Gal and MQ-βGal, an exogenous intervention experiment was conducted: LoVo cells were pre-incubated with the addition of either 20 U/mL exogenous β-Gal or 50 µM D-galactose (β-Gal inhibitor) for 0.5 h, followed by the addition of 10 µM MQ-βGal for 30 min before fluorescence imaging. Results demonstrated (Fig. 7(g)-(i)) that exogenous β-Gal significantly enhanced intracellular fluorescence intensity, whereas D-galactose addition caused a marked reduction in cellular fluorescence signals (Fig. 7(j)-(l)). Furthermore, by performing a quantitative analysis of the red fluorescence signal as shown in Fig. 7B, it was possible to reveal the differences in fluorescence intensity and the trends between the groups in a more intuitive and quantitative manner. These findings confirmed that MQ-βGal not only specifically detected endogenous β-Gal activity in living cells but also responds to changes in exogenous β-Gal expression, demonstrating exceptionally high specificity for intracellular β-Gal recognition.

Fig. 7.

(A) Fluorescence images of HeLa and LoVo cells incubated with MQ-βGal (10 µM) for 30 min: (a)–(c) HeLa cells, and (d)–(f) LoVo cells, (g)-(i) LoVo cells pretreated with an exogenous β-Gal for 0.5 h, (j)–(l) LoVo cells pre-treated with an inhibitor (50 µM) for 0.5 h. (B) Quantitative fluorescence intensities in red channel for the detection experiment. Scale bar = 50 μm. (E_ex= 560 nm).

β-Gal was frequently overexpressed in colorectal cancer cells, whereas MQ-βGal specifically responded to LoVo colorectal cancer cells exhibiting high β-Gal expression, generating strong fluorescent signals. Based on this characteristic, this study hypothesized that the MQ-βGal held potential for distinguishing between healthy individuals and colorectal cancer patients. To validate this hypothesis, a fluorescence detection system based on MQ-βGal was established to quantitatively analyze endogenous β-Gal expression levels in human peripheral blood mononuclear cells (PBMCs). The specific experimental procedure was as follows: purified PBMCs were seeded into centrifuge tubes, supplemented with MQ-βGal at a final concentration of 10 µM and incubated at 37 °C for 30 min. Subsequently, ImageJ software was employed to quantify the average fluorescence intensity of the cells. As illustrated in Fig. 8, results demonstrated significantly higher fluorescence signals in PBMCs from early-stage colorectal cancer patients compared to both normal controls and polyp groups (P < 0.05). This indicated that MQ-βGal can serve as a specific fluorescent probe responsive to endogenous β-Gal, suitable for early colorectal cancer screening and identification. Fluorescence signals in patients with mid-to-late colorectal cancer were further elevated compared to early-stage patients, with an increase of approximately one-fold. This result confirmed MQ-βGal’s excellent discriminatory efficacy across different disease progression stages. In summary, MQ-βGal, as a novel fluorescent detection tool, achieved highly efficient early detection of colorectal cancer through its specific targeting of endogenous β-Gal. This provided new technical support and research approaches for the early clinical diagnosis of colorectal cancer.

Fig. 8.

The mean fluorescence intensity of human PBMCs treated with Normal control (N), Polyps group (P), Early-stage colorectal cancer group (EM), Mid-stage colorectal cancer group (MM) and Late-stage colorectal cancer group (LM) with 10 µM MQ-βGal. Values represent means ± SD (n = 5), bars with different characters are statistically different at *p < 0.05, **p < 0.01, ***p < 0.001.

Bio-imaging of β-Gal in BALB/c nude mice

Leveraging the high-fidelity imaging performance of MQ-βGal at the cellular level, we further explored its potential for real-time in vivo monitoring of β-Gal activity. Benefiting from the MQ-βGal’s highly efficient enzymatic conversion properties, rapid fluorescence signals were generated in mice following intraperitoneal injection of MQ-βGal. As depicted in Fig. 9, a distinct fluorescent signal emerged throughout the entire peritoneal cavity 10 min post-injection, with signal intensity progressively increasing over time. Thirty minutes after injection, the fluorescent signal continued to rise without exhibiting rapid decay. These results confirm that MQ-βGal possessed core advantages of rapid in vivo response (effective within 10 min) and high sensitivity, enabling efficient detection of β-Gal activity in vivo. Furthermore, its time-dependent enhancement of fluorescence signals and excellent signal stability indicate significant application potential for real-time dynamic monitoring and long-term bioimaging. In summary, MQ-βGal serves as an effective tool for time-dependent and long-term visualization of β-Gal enzyme activity in vivo. This provides crucial technical support for investigating the physiological and pathological functions of β-Gal and for the clinical diagnosis of related diseases.

Fig. 9.

In vivo fluorescence imaging of MQ-βGal (100 µM) in BALB/c nude mice after abdominal cavity injection. The images were taken at different times with the excitation of 560 nm and emission collected of 580–660 nm.

Conclusion

An enzyme-activated fluorescent probe, MQ-βGal, had been innovatively developed, providing a new tool for detecting β-Gal activity. Its outstanding performance (detection limit as low as 0.021 U/mL, rapid enzymatic response, excellent photostability and pH tolerance) ensured precise detection of β-Gal activity under physiological conditions. At the application level, MQ-βGal has successfully undergone multi-dimensional scenario validation: at the cellular level, it can specifically distinguish between β-Gal high/low expression cells (LoVo/HeLa), clearly revealing differences in endogenous enzyme activity; At the in vivo level, following intraperitoneal injection in mice, it generates stable fluorescent signals within 10 min and exhibits long-term dynamic monitoring capability without significant signal decay; At the clinical translation level, this probe effectively distinguishes biological samples from healthy individuals and colorectal cancer patients, precisely identifying tumour-associated β-Gal abnormal expression patterns, providing direct evidence for disease-specific biomarker detection. In summary, MQ-βGal developed in this study not only provided an excellent tool for investigating the physiological and pathological functions of β-Gal but also established a potential diagnostic system spanning basic research to clinical translation. In future studies, our team will further investigate the tissue distribution patterns and in vivo pharmacokinetic behaviors of MQ-βGal, and systematically evaluated its in vivo metabolism, circulation retention and targeted accumulation properties, thereby providing a more comprehensive theoretical basis and experimental support for the subsequent biomedical applications and clinical translation research of this fluorescent probe.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

#The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Xiaoli Huang and Shuyu Liu contributed equally.

Funding

This research work was supported by Nanjing City Special Fund Project for Health Technology Development (YKK23262) and Medical Research Project Fund of Jiangsu Provincial Health Commission (M2022031).

Data availability

The datasets used and/or analyzed during the current study available from the corresponding author on reasonable request.

Declarations

Competing interests

The authors declare no competing interests.

Ethical approval

The experiments were approved by Animal Ethical Experimentation Committee of Nantong University in accordance with the requirements of the China National Act on the use of experimental animals (Approval number: SCXK2024-0011). All experiments were performed in accordance with relevant guidelines and regulations, as well as the IUCN Policy Statement on Research Involving Species at Risk of Extinction and the Convention on the Trade in Endangered Species of Wild Fauna and Flora.Venous blood (3mL) was obtained by venipuncture from volunteer donors with informed consent. The research program was approved by Nanjing Jiangbei Hospital affiliated to Nantong University Biosafety and Ethics Committee (Approval number: AF/SC-08/02.0). All methods were performed in accordance with the relevant guidelines and the Declaration of Helsinki.