A NIR ratiometric fluorescent biosensor for sensitive detection and imaging of α‐L‐fucosidase in living cells and HCC tumor‐bearing mice

Detection and imaging of α‐L‐fucosidase (AFU) is of great value to understand its roles in hepatocellular carcinoma (HCC) and tumor early diagnosis, but ideal assays are still lacking. Herein, a near‐infrared (NIR) fluorescent biosensor (α‐Fuc‐DCM) was elaborately designed and synthesized for rapid and ratiometric detection of AFU activity in cells and HCC tumor mouse models. In the presence of AFU, this biosensor shows an enhancement in NIR emission in a ratiometric manner, which significantly improves the detection accuracy with the limit of detection as low as 4.8 mU/mL. Taking advantage of these merits, the activity of AFU in lysosomes could be visualized using ratiometric and NIR dual modality in living cells. Furthermore, its remarkable application for monitoring of endogenous AFU activity in HCC tumor‐bearing mouse model is also demonstrated with bright fluorescence signal, which indicated that the biosensor could clearly monitor the liver tumor in the early stage. Importantly, the α‐Fuc‐DCM probe can be utilized to detect the AFU in serum from HCC patients. This strategy offers a promising biosensor system for early diagnosis of HCC and studying the roles of AFU in cancers.


S C H E M E 1
Schematic illustration of the proposed detection mechanism for AFU activation of α-Fuc-DCM, and its specific detection and bio-imaging of AFU activities in living cells and hepatocellular carcinoma (HCC) tumor-bearing nude mice detection methods to screen the HCC in the early stage, which could lead to early therapy and thus improve the chances of survival. [4] Among the variously developed methodologies, the detection of HCC-specific serum biomarkers has been regarded as the most promising method for earlier diagnosis of HCC. In clinic, the primary tumor marker for HCC is α-fetoprotein (AFP), which is a single polypeptide chain of glycoprotein. [5] Through monitoring the level of AFP in HCC screening program, a number of HCC patients have been screened out. However, the sensitivity and the specificity of AFP in the diagnosis of HCC are still insufficient, [6,7] because AFP is not secreted in all cases of HCC, and the level of AFP might be normal or acceptable in 40% patients with early HCC. [7,8] As a member of the glycosidase family, AFU, which is widely presented in various tissues, cells and fluids at low concentrations, could hydrolyze the terminal α-L-fucose monosaccharide residue on glycolipids or glycoproteins in lysosome, and it is highly active within a pH range from 4 to 6.5. [9] Previous study showed that the detection of the overexpression of AFU in serum is able to diagnose 85% of HCC patients up to 6 months before it is detectable by ultrasonography. [4] So, real-time detection of AFU would be a promising strategy for early diagnosis of HCC.
Numerous strategies have been developed to detect AFU, which include colorimetry, [10,11] fluorescent method, [4,[12][13][14][15][16][17][18][19][20][21][22] immunoassay, [23] and chromatography techniques. [24,25] Among them, colorimetric and fluorescence assays were based on the hydrolysis property of AFU to detect the activity of AFU. Colorimetric assay, as the standard method, is commonly used in clinical setting. [26] However, due to the innate nature, the disadvantages of this method is low sensitivity, heavy interference by circumstances, and limitation of usage in animals. [27] To improve the detection accuracy, sensitivity, and scope of application, the fluorescence detection method, as a convenient and noninvasive way, has been employed to detect or monitor the onset and progression of HCC in real-time in living organisms. Up to now, several light-up fluorescent biosensors have been reported to detect AFU. [15,[18][19][20]22] However, most of them cannot be used for in vivo imaging, because of the short emission wavelengths (<600 nm), which cause strong background interference arising from inferior tissue penetration and high tissue autofluorescence.
Herein, we reported an AFU responsive, turn-on, ratiometric and near-infrared (NIR) fluorescent biosensor (α-Fuc-DCM) with large Stokes shift, which was applied to detect the AFU activity in living cells and HCC tumor-bearing mice (Scheme 1). The near-infrared (NIR) fluorescence imaging can effectively reduce autofluorescence and optical absorption in tissue, which results in high penetration depth and sensitivity. The ratiometric property of the biosensor enables reducing the impact of environmental fluctuation and increasing the detection accuracy. α-Fuc-DCM was assembled of α-L-fucose monosaccharide, as the AFU-selective recognition unit, with a dicyanomethylene-4H-pyran (DCM) chromophore, as the fluorogenic reporter owing to its excellent sensing properties. [28,29] When exposure to AFU, the biosensor exhibited a gradual increase of fluorescence intensity in the NIR region. By using this biosensor, the intracellular distribution of endogenous AFU was successfully detected. In addition, the α-Fuc-DCM probe allows for the in situ visualization of AFU activity in a murine tumor model and serum directly and accurately. . MeONa (7 mg, 0.13 mmol, 0.5 equiv) was added to a solution of Ac 3 α-Fuc-DCM (150 mg, 0.26 mmol, 1 equiv) in DCM and MeOH (12 mL, v/v = 1:2). The mixture was stirred for 30 min, at room temperature and monitored by TLC (DCM:MeOH = 10:1). Upon completion, the reaction mixture was evaporated under reduced pressure. The orange residue was purified by column chromatography on silica gel (DCM:MeOH = 10:1) affording the target product α-Fuc-DCM as an orange solid (105.8 mg, 90% yield). 1 3.59 (s, 1H), 1.08 (d, J = 6.5 Hz, 3H). 13

Cellular fluorescence imaging
HepG2 cells and LO2 cells were seeded and cultured in confocal imaging chambers. Subsequently, the cells were washed three times with 1× phosphate buffered saline (PBS) and incubated with or without 50 μM deoxynojirimycin hydrochloride (prepared in serum-free medium) for 60 min at 37 • C. Next, the cells were treated with α-Fuc-DCM nanoparticle solution (10 μM) in serum-free cell culture medium at 37 • C for 120 min. After washing three times with PBS, the cells were imaged using a laser scanning confocal microscope (Olympus, Tokyo, Japan).

Fluorescence imaging of AFU in HCC tumor-bearing mice
All animal studies were performed in compliance with the guidelines set by Tianjin Committee of Use and Care of Laboratory Animals and the overall project protocols were approved by the Animal Ethics Committee of Nankai University. Eight-week-old BALB/c mice were obtained from Beijing Vital River Laboratory Animal Technology Co. Ltd. (Beijing, China). To establish the xenograft HepG2 tumor-bearing mouse model, HepG2 cancer cells (3 × 10 6 ) suspended in 50 μL of DMEM were injected subcutaneously into the right axillae space of the mouse. When the tumors diameter reached approximately 1 mm, the mice were used for in vivo imaging study. α-Fuc-DCM nanoparticle solution (10 μL, 1 mg/mL) was in situ injected into the HepG2 tumor in tumor-bearing nude mice. Fluorescence imaging of cancer tissues in tumor-bearing mice was performed using an in vivo imaging system (IVIS Spectrum, PerkinElmer). For the in vivo imaging, λ ex = 530 ± 10 nm and λ em = 700 ± 15 nm.

Data and statistical analysis
All data were collected in triplicates or more and are presented as the mean ± standard deviation.

RESULTS AND DISCUSSION
The synthetic route of α-Fuc-DCM is shown in Scheme 2. Firstly, the starting substrate of Ac 3 α-Fuc-Ph-CHO was synthesized based on the reported literature, [30] which could efficiently offer the product with α glycosidic bond. Then, it was coupled to DCMC, as a superior electron acceptor, under the catalysis of piperidine to afford the intermediate of Ac  Figure S9, which indicated that this biosensor has good photostability.
Upon incubation with AFU (0.2 U) in PBS buffer (pH 6.0) at 37 • C, the absorption spectra of the biosensor were periodically recorded. The absorption peak of α-Fuc-DCM at 440 nm decreased, simultaneously, a new absorption peak at 535 nm appeared obviously, as shown in Figure 1A. Because of the large red-shift (approximate 100 nm) in the absorption spectra, the obvious color change from faint yellow to rose red could be employed to detect the AFU with colorimetric method ( Figure 1A, inset). Followed by the measurement of the fluorescence spectra of the biosensor, when excited at 440 nm, the emission band ranging from 550 to 750 nm increased sharply. Meanwhile, the emission band at 500 nm decreased gradually with linear relationship, as shown in Figure S10A, which indicated a distinct ratiometric fluorescent property (I 680 nm /I 500 nm ) for this α-Fuc-DCM biosensor ( Figure 1B). Upon excitation at 535 nm, a remarkable enhancement of the NIR fluorescence intensity at 680 nm was observed ( Figure 1C), which would facilitate the biosensor to image in vivo. The results of the time dependent experiments indicated that the enzymatic reaction was almost completed in 150 min; however, in the absence of AFU, there was no obvious change in the ratio of fluorescence intensity at 680 and 500 nm ( Figure 1D).
The fluorescence spectra of the α-Fuc-DCM biosensor solution, incubated with various AFU concentrations for 150 min, were also recorded. When excited at 440 nm, the distinct ratiometric fluorescent property was also presented in the fluorescence spectrum (Figure 2A and Figure S10B). And based on the FL intensity ratio of I 680 nm /I 500 nm and AFU concentrations, the standard curve was established, as shown in Figure 2B. Moreover, there is a good linear relationship between the ratio value of I 680 nm /I 500 nm and the AFU level. With the AFU concentration ranging from 0 to 160 mU/mL, the limit of detection for detecting AFU was calculated based on the 3σ/k method to be 4.8 mU/mL, as shown in Table S1, which indicated that the α-Fuc-DCM biosensor was sensitive enough to be used for AFU detection in biological samples. When excited at 535 nm, the FL intensities enhanced gradually with the increasing of AFU concentration ( Figure 2C). The fluorescence intensity at 680 nm was linearly associated with the AFU concentrations ranging from 0 to 120 mU ( Figure 2D). The absorption and emission spectra of DCM-O¯were measured, as shown in Figure S11, which are same as the spectra profiles of the enzyme hydrolysis product in Figures 1A-C and 2A,C. The spectra similarity indicated the production of DCM-O¯from α-Fuc-DCM.
The α-Fuc-DCM probe was docked with the fucosidase crystal structures, which includes the a humam lysosomal fucosidase (7PM4) [31] and a bacterial fucosidase (4WSK) [32] . The results of molecular docking (Schrodinger software) are shown in Figure 3 and Figure S12. The absolute scores of α-Fuc-DCM docked with 7PM4 and 4WSK are 5.527 and 3.993, respectively, which indicates that α-Fuc-DCM probe can be recognized and hydrolyzed efficiently by fucosidases from different sources.
The high-performance liquid chromatography (HPLC) analysis was performed to investigate the mechanism of activation of α-Fuc-DCM with AFU. HPLC profiles validated that α-Fuc-DCM could be cleaved by AFU and generate DCM-O¯, which gradually increased with prolonged incubation time ( Figure 4A). The new product chromatographic peak appeared in the incubation solution of α-Fuc-DCM biosensor and AFU was collected for mass spectrometric measurement, which was found at m/z 311.0814 ( Figure S13), consistent with the calculated molecular mass of DCM-O¯(311.0821).
To investigate whether the α-Fuc-DCM biosensor could be used in complex biological systems, the selectivity of the biosensor was measured by the fluorescence changes in the presence of different biological substances. As shown in Figure 4B, only AFU induces a prominent enhancement of the I 680 nm /I 500 nm value, and there was no obvious influence by the other species, which include ions (such as Ca 2+ , Zn 2+ , Mg 2+ , Mn 2+ , Fe 2+ , and Fe 3+ ), small biological molecules (such as α-L-fucose, glucose, and cysteine), protein enzymes (such as alkaline phosphatase (ALP), carboxylesterase, deacetylase, γ-glutamyltranspeptidase), and others (bovine serum albumin, glutathione, and H 2 O 2 ).
The results of the selectivity indicated that the biosensor could specifically respond to AFU with good selectivity and anti-interference.
In order to increase the solubility in aqueous and reduce the latent toxicity of organic solvent, a nanoprecipitation method with DSPE-PEG 2000 as matrix was applied to fabricate the nanoparticle to investigate the performance of the α-Fuc-DCM biosensor for ratiometric tracking and imaging of endogenous AFU activity in living cells. Dynamic light scattering measurement indicated an average hydrodynamic diameter of ∼150 nm ( Figure 5A). Their sizes were very close to the results revealed by transmission electron microscopy image ( Figure 5B), which also indicate successful nanoparticle formation with uniform spherical morphologies.
The cytotoxicity of the α-Fuc-DCM nanoparticle was tested with HepG2 and LO2 cell lines using a 3-(4,5-dimethylthiazol-2-yl)-3,5-di-phenytetrazolium bromide (MTT) assay, and the results indicated that the α-Fuc-DCM nanoparticle has negligible toxicity and possesses superior cytocompatibility toward both of the cell lines ( Figure 6A). And then, we employed the biosensor for AFU imaging in HepG2 cell line with overexpressed AFU under the ratiometric modality. After incubation with α-Fuc-DCM nanoparticle (10 μM) at 37 • C for 2 h, the HepG2 cells showed a reduced fluorescence in the green channel and an accompanied rising in the red channel ( Figure 6D, a-c), which is corresponding to the formation of DCM-O¯. Meanwhile, the HepG2 cells, which were pretreated with the AFU inhibitor deoxynojirimycin hydrochloride (50 μM) for 1 h, exhibited both bright fluorescence in the green channel and little fluorescence in the red channel ( Figure 6D, d-f). For comparison, LO2 cell line, which does not over-express AFU, was selected as another control. Similar to the HepG2 with inhibitor, the LO2 cells also showed the brighter fluorescence in the green channel than that in the red channel ( Figure 6D, g-i). The ratiometric imaging of endogenous AFU activity was performed based on the FL intensities in both red and green channels. The average ratios of the FL intensities from both channels of the HepG2, HepG2 with inhibitor, and LO2 cells were calculated to be 2.5, 1.0, and 0.86, respectively ( Figure 6B). Therefore, the quantitative ratiometric imaging of endogenous AFU activity has been successfully demonstrated.
Moreover, because of the much more release of DCM-Ob y intracellular AFU in hepG2 cells, when excited at 560 nm, a much brighter NIR fluorescence image was captured in HepG2 cells than that in HepG2 with inhibitor and LO2 cells ( Figure 6D, j-l). And the average FL intensities of the HepG2, HepG2 with inhibitor, and LO2 cells were calculated to be 122.6, 58.2, and 60.1, respectively ( Figure 6C). These results aforementioned suggested that α-Fuc-DCM nanoparticle can be activated specifically in living cells, which could be used as both ratiometric and light-up NIR detection tools for quantitative tracking and visualization of the endogenous AFU.
After we verified that the α-Fuc-DCM nanoparticle had negligible in vivo toxicity against healthy mice and good bio-compatibility ( Figure S14), the animal studies were started to perform. In order to investigate the ability of α-Fuc-DCM nanoparticle for real-time imaging of AFU activity in vivo, the AFU-overexpressed tumor-bearing mice models were established through subcutaneous injection of HepG2 cells ( Figure 7A). After 5 days, the α-Fuc-DCM biosensor solution was injected intratumorally and subcutaneously. As shown in Figure 7B, we can see an obvious NIR fluorescence signal at the tiny tumor (∼1 mm) site following in situ injection of α-Fuc-DCM nanoparticle (10 μL, 1 mg/mL). The H&E histological staining of the light-up region indicated that the excised tissue was tumor ( Figure 7C).
When α-Fuc-DCM was injected into tumor, after merely 5 min, the tumor has already been lit up by the NIR fluorescence signal at 680 nm, which was emitted from the enzymolysis product (DCM-O¯) of α-Fuc-DCM biosensor. After gradually increasing for 180 min, the fluorescence intensity reached maximum, then slowly decayed with time. In comparison, there was minimal light-up fluorescence following subcutaneous injection of α-Fuc-DCM nanoparticle ( Figure 7D,E). Moreover, the ex vivo fluorescence images of tumor and other organs, which were collected immediately after sacrificing the mice, were recorded ( Figure 7F). It confirmed that the NIR fluorescent signal was indeed emitted from the product of DCM-O¯. We successfully completed the in situ fluorescence imaging to monitor AFU activity in HCC tumor, which might offer an efficient method for HCC early diagnosis.
In order to investigate the AFU detection capacity of α-Fuc-DCM in serum, 24 serum samples from healthy volunteers and 24 serum samples from HCC patients were collected and measured, and the detection is based on the MG-CNPF-α-Fuc probe used in clinic and α-Fuc-DCM designed by ourselves. The samples were measured based on α-Fuc-DCM probe, and the results was shown in Figure 8A,B. The median of the FL intensity from HCC patients was 1.89 × 10 7 , which was statistically higher than that (1.44 × 10 7 ) of healthy volunteers (p < 0.0001). The same samples were measured again based on 2-chloro-4nitrophenyl-α-L-fucopyranoside (MG-CNPF-α-Fuc) probe, and the detection result is presented as the concentration of AFU. As shown in Figure 8C, the median of the AFU concentration from HCC patients was 34.7 U/L, which was statistically higher than that (28.2 U/L) of healthy volunteers

CONCLUSIONS
In summary, we have successfully developed a NIR fluorescent ratiometric biosensor (α-Fuc-DCM) based on DCM-Of uorophore with large Stokes shift for AFU activity detection, which was assembled by DCM-O¯and α-L-fucose residues as the AFU-specific trigger. This biosensor could detect AFU in aqueous media with high sensitivity and selectivity owing to the ratiometric property. It was confirmed that the AFU could efficiently catalyze the cleavage of the α-L-fucose residue to light up the biosensor. In addition, the results in living cells demonstrated that α-Fuc-DCM nanoparticle could enable visualization of AFU activity under both ratiometric and NIR fluorescence modalities. Furthermore, α-Fuc-DCM nanoparticle could be used as a robust biosensor for detection of endogenous AFU activity in HCC tiny tumor-bearing mouse models at the early stage. Importantly, the α-Fuc-DCM probe can be utilized to detect the AFU in serum from HCC patients. All in all, this strategy provides an effective approach to track AFU activity in vitro and in vivo, which may be beneficial to early diagnosis of HCC.

C O N F L I C T O F I N T E R E S T
The authors declare no competing financial interest.