Hydrogen Peroxide and Hypochlorite Responsive Fluorescent Nanoprobes for Sensitive Cancer Cell Imaging

Accurate diagnosis of cancer cells directly affects the clinical treatment of cancer and can significantly improve the therapeutic effect of cancer patients. Cancer cells have a unique microenvironment with a large amount of peroxide inside, effectively differentiated from relevant microenvironment normal cells. Therefore, designing the high-sensitive probes to recognize and distinguish the special physiological microenvironment of cancer cells can shed light on the early diagnosis of cancers. In this article, we design and construct a fluorescence (FL) contrast agent for cancer cell recognition and imaging analysis. Firstly, luminol-gold NPs (Lum-AuNPs) have been initially built, and then successfully loaded with the fluorescent receptor Chlorin e6 (Ce6) to prepare the luminescent nanoprobes (Ce6@Lum-AuNPs) with green synthesis, i.e., with biocompatible agents and mild temperature. The as-prepared fluorescent Ce6@Lum-AuNPs can efficiently and sensitively realize FL bioimaging of cancer cells. The relevant bio-sensing mechanism pertains to the presence of hypochlorite (ClO−); hydrogen peroxide (H2O2) in cancer cells could readily interact with luminol to produce chemiluminescence, which can activate the Ce6 component to emit near-infrared (NIR) FL. Therefore, this raises the possibility of utilizing the Ce6@Lum-AuNPs as efficient fluorescent nanoprobes for promising cancer early diagnosis and other relevant disease bioanalysis.


Introduction
In recent years, with the continuous progress of nanotechnology, more and more functional nanomaterials have been prepared, some of which have been widely used in biomedical diagnosis and treatment due to their excellent physical and chemical properties [1][2][3]. Nanomaterials, as new materials, are favored by researchers for their high surface-volume ratio unique mechanical and optical properties and are widely used in drug delivery, early diagnosis, and treatment of diseases [4]. Therefore, biomedical application based on nanomaterials has become a new trend in clinical research.
Bioimaging technology has made breakthrough progress in recent years. It has been widely and deeply applied in biomedical, clinical diagnosis, and other fields, supporting theoretical research and practical application [5]. Fluorescence (FL) bioimaging technology is the most widely used technology in optical bioimaging [6][7][8], which can realize real-time, dynamic, accurate, non-invasive monitoring of cancers and provide information on the dynamic changes of biological samples at the cellular and living level, which is favored by researchers and healthcare workers [9][10][11]. At present, more and more fluorescent probes are used in biological imaging technology, including quantum dots, upconversion materials, carbon-based materials, etc. [12][13][14]. However, many fluorescent probes have

Synthesis of Ce6@Lum-AuNPs
The AuNPs were synthesized by modifying our previously reported methods [30]. Luminol (10 mmol L −1 , 3 mL) and HAuCl 4 (50 mmol L −1 , 600 µL) were mixed with the molar ratio of 1:1 in 5 mL Tris and left to set for at least 30 min at 25 • C. The AuNPs were collected by centrifuging the solution at 9600 g for 5 min and dispersed in deionized water under the ultrasonic wave. After four repetitions, the AuNPs were suspended in 1 mL DMSO by sonication dispersion. Then, the AuNPs were added to a mixture (containing 0.67 mg Ce6, 24 mg EDC and 12 mg NHS in 5 mL DMSO and stirred for 12 h at 37 • C in advance) for another 24 h stirring at 37 • C. Finally, the solution was purified by dialysis against water to remove unreacted reagents and by-products until no absorption was observed in the UV-Vis absorption spectrum. Finally, the drying of the solution by a vacuum oven at RT.

Ce6 Loading Capacity Calculation
The FL emission intensity at 667 nm (λ ex = 405 nm) was taken for the Ce6 loading capacity calculation. Since the strong absorption peak of Lum-AuNPs is in the range of 275-450 nm, it is not suitable to use the absorbance of Ce6 at 405 nm to measure its load in nanoparticles. Therefore, detecting the FL emission spectra of Ce6 at different concentrations were exploited, and the relevant FL emission intensity at 667 nm (λ ex = 405 nm) was determined, respectively. The standard curve for the relationship between the FL emission intensity and corresponding concentration was obtained by linear fitting. The related loading of Ce6 was calculated based on the standard curve.

FL Properties of the Ce6@Lum-AuNPs
An FL spectrometer was used to detect the FL properties of Ce6@Lum-AuNPs and the relevant changes of the FL properties in the presence of H 2 O 2 . In one group, 0.5 µL 100 mmol L −1 NaClO solution was added twice into 1 mL 2 µg mL −1 Ce6@Lum-AuNPs solution, the FL intensity-time curve was recorded. In another group, 1 mL 2 µg mL −1 Ce6@Lum-AuNPs solution was added with 1 µL10 mol L −1 H 2 O 2 ten times, followed by adding 0.5 µL 100 mmol L −1 NaClO solution twice, the FL intensity-time curve was recorded as well. After the latter group of the solution was left at room temperature for a week, horseradish peroxidase (HRP) was added for photographs.
An amount of 0.5 µL 100 mmol L −1 NaClO solution was added twice into 1 mL Ce6@Lum-AuNPs solution with a concentration of 2 µg mL −1 , and the FL emission spec-trum was detected after each addition. Add 10 µL 10 mol L −1 H 2 O 2 to 1 mL Ce6@Lum-AuNPs solution at a concentration of 2 µg mL −1 . Then 0.5 µL 100 mmol L −1 NaClO solution was added twice, and the FL emission spectrum was detected after each addition. Finally, HRP was added to the latter solution to detect the FL emission spectrum.

Specificity of the Ce6@Lum-AuNPs for H 2 O 2 and ClO −
The FL intensity of Ce6@Lum-AuNPs with and without H 2 O 2 and NaClO in the presence or absence of various ROS, such as 50 µmol L −1 CO + , O 2 − , 1 O 2 , OH· and ONOO − was collected by FL emission spectra.

Cell Culture
HepG2 cells (Shanghai Institute of Biological Sciences, Chinese Academy of Science, Shanghai, China). L02 (human embryo liver cell strand) (Third Military Medical University, Chongqing, China). The cell was cultured in a culture medium (DMEM with 10% FBS and 1% streptomycin/penicillin).

In Vitro Cytotoxicity Test
The cytotoxicity test was performed by conventional CCK-8 assays. HepG2 and L02 cells were seeded in 96-well plates at 5 × 10 3 cells for each well. After a 24 culture, the cells were rinsed and added 100 µL DMEM with various concentrations (40, 80, 160, 320, 640 µg mL −1 ) of Ce6@Lum-AuNPs as test groups (five wells for each group). Cell-free wells adding 100 µL DMEM with the same concentrations of Ce6@Lum-AuNPs were used as control groups. After 24 h continuous incubation, 10 µL of CCK-8 solution was added and incubated for another 1 h. The suspensions were measured at 450 nm. According to the improved Karber's method, IC 50 values were calculated.

In Vitro Bioluminescence Imaging of Cancer Cells
HepG2 and L02 cells were added into a confocal petri dish with a concentration of 5 × 10 3 cells for each dish. The cells were placed in an incubator containing 5% CO 2 humidification at 37 • C overnight. After culture, the cells were washed with PBS; then the control group was added 2 mL fresh medium with nothing. One experimental group added 2 mL of new medium containing 1.35 µg mL −1 Ce6. Two other groups were added with fresh medium containing 50 µg mL −1 Ce6@Lum-AuNPs. After culturing for 6 h, 0.5 µL 100 mmol L −1 NaClO was added into one of the 50 µg mL −1 Ce6@Lum-AuNPs groups. After further cultivation for 4-6 h, the FL distribution in cells was observed under a confocal laser FL microscope.

Synthesis and Characterization of Ce6@Lum-AuNPs
According to the synthesis method and optimization procedure reported in the literature [30], luminol was used as a reducing agent to reduce HAuCl 4 to AuNPs through redox reaction and luminol coated gold nanoparticles (Lum-AuNPs) were prepared. TEM images showed that Lum-AuNPs and the core of Ce6@Lum-AuNPs had an average size of approximately 2.3 ± 0.4 nm and 143.8 ± 29.7 nm, respectively ( Figure 1). Dynamic light scattering results showed that Ce6@Lum-AuNPs were well-dispersed in water, with a mean hydrodynamic size of 183.9 ± 6.6 nm and a polydispersity index of 0.136.
UV-Vis absorption spectra ( Figure 2A) were used to monitor the dialyzed-out solution (curves a and b). After dialysis, the UV-Vis absorption spectrum of the prepared Ce6@Lum-AuNPs showed two characteristic peaks at 409 nm and 689 nm, respectively (curve e). Compared with Ce6, the UV-Vis absorption spectrum of Ce6@Lum-AuNPs showed significant redshift (curves c and d). The FT-IR spectra showed that the surface of Lum-AuNPs was functionalized with -NH 2 group by 3-aminophthalic acid ( Figure 2B). Then Ce6 was conjugated on the Lum-AuNPs by an amide condensation reaction to obtain the Ce6@Lum-AuNPs. In addition, the FT-IR spectrum of Ce6@Lum-AuNPs showed that characteristic absorption bands of Ce6 and Luminol were detected. These results indicated that luminol and Ce6 were successfully modified on the AuNPs surface.

Synthesis and Characterization of Ce6@Lum-AuNPs
According to the synthesis method and optimization procedure reported in the literature [30], luminol was used as a reducing agent to reduce HAuCl4 to AuNPs through redox reaction and luminol coated gold nanoparticles (Lum-AuNPs) were prepared. TEM images showed that Lum-AuNPs and the core of Ce6@Lum-AuNPs had an average size of approximately 2.3 ± 0.4 nm and 143.8 ± 29.7 nm, respectively ( Figure 1). Dynamic light scattering results showed that Ce6@Lum-AuNPs were well-dispersed in water, with a mean hydrodynamic size of 183.9 ± 6.6 nm and a polydispersity index of 0.136. UV-Vis absorption spectra ( Figure 2A) were used to monitor the dialyzed-out solution (curves a and b). After dialysis, the UV-Vis absorption spectrum of the prepared Ce6@Lum-AuNPs showed two characteristic peaks at 409 nm and 689 nm, respectively (curve e). Compared with Ce6, the UV-Vis absorption spectrum of Ce6@Lum-AuNPs showed significant redshift (curves c and d). The FT-IR spectra showed that the surface of Lum-AuNPs was functionalized with -NH2 group by 3-aminophthalic acid ( Figure 2B). Then Ce6 was conjugated on the Lum-AuNPs by an amide condensation reaction to obtain the Ce6@Lum-AuNPs. In addition, the FT-IR spectrum of Ce6@Lum-AuNPs showed that characteristic absorption bands of Ce6 and Luminol were detected. These results indicated that luminol and Ce6 were successfully modified on the AuNPs surface.
The FL intensity of Ce6 with different concentrations at 667 nm was measured, and the standard curve between FL intensity and concentration was drawn in Figure 2C. Then, based on the emission intensity of Ce6@Lum-AuNPs at 667 nm (λex = 405 nm), the bearing capacity of Ce6 was calculated to be 2.7% (mass ratio) according to the standard curve.

FL Spectroscopy Characterization of Ce6@Lum-AuNPs
The emission spectra of Ce6 and Ce6@Lum-AuNPs were recorded at different excitation wavelengths, and three-dimensional (3D) FL spectra ( Figure 3A,B) were also illustrated. It could be shown that the emission spectra of Ce6@Lum-AuNPs and Ce6 have relevant similar spectroscopic characteristics. FL excitation spectra of Ce6 and Ce6@Lum-  The FL intensity of Ce6 with different concentrations at 667 nm was measured, and the standard curve between FL intensity and concentration was drawn in Figure 2C. Then, based on the emission intensity of Ce6@Lum-AuNPs at 667 nm (λ ex = 405 nm), the bearing capacity of Ce6 was calculated to be 2.7% (mass ratio) according to the standard curve.

FL Spectroscopy Characterization of Ce6@Lum-AuNPs
The emission spectra of Ce6 and Ce6@Lum-AuNPs were recorded at different excitation wavelengths, and three-dimensional (3D) FL spectra ( Figure 3A,B) were also illustrated. It could be shown that the emission spectra of Ce6@Lum-AuNPs and Ce6 have relevant similar spectroscopic characteristics. FL excitation spectra of Ce6 and Ce6@Lum-AuNPs in DMSO were shown in Figure 3C, and the maximum excitation wavelength for both was around 400 nm. Figure 3D showed the FL emission spectra of Ce6 and Ce6@Lum-AuNPs, indicating that the maximum emission wavelength of Ce6@Lum-AuNPs was 670 nm, showing an apparent red shift compared with that of Ce6 at 660 nm. Figure 3E showed that the UV-Vis absorption spectrum of Ce6@Lum-AuNPs was similar to that of Ce6, which again illustrates that Ce6 was successfully loaded on gold nanoparticles. Based on the above observations, Ce6@Lum-AuNPs with NIR FL emissions were successfully obtained and had similar spectroscopic properties to Ce6.  Considering the relatively high concentration of H2O2 in the intracellular microenvironment of cancer cells, the effect of H2O2 on the FL properties of Ce6@Lum-AuNPs was investigated for the possibility to apply it for cell recognition and bio-imaging further. Relevant FL emission spectra under different treatment conditions were detected, as shown in Figure 4A. It was evident that when NaClO was added to Ce6@Lum-AuNPs aqueous solution, the fluorescence intensity was the same for the first time and the second time, but both were stronger than that without NaClO. Figure 4B showed that after Ce6@Lum-AuNPs aqueous solution was pre-mixed with H2O2, the FL intensity of the solution with two NaClO additions was significantly enhanced compared to that with only one addition. As shown in Figure 4C, after adding HRP, the FL emission intensity of the solution decreased significantly. Hypochlorous acid with high reactivity is generated by the neutrophil enzyme myeloperoxidase and is particularly relevant in inflammatory conditions. Meanwhile, hydrogen peroxide is a strong two-electron oxidant, but most of its electron oxidation reactions are too slow to be biologically relevant because of high activation energy. NaClO is applied to simulate the tumor tissue microenvironment and oxidate luminol in collaboration with H2O2 [31]. Considering the relatively high concentration of H 2 O 2 in the intracellular microenvironment of cancer cells, the effect of H 2 O 2 on the FL properties of Ce6@Lum-AuNPs was investigated for the possibility to apply it for cell recognition and bio-imaging further. Relevant FL emission spectra under different treatment conditions were detected, as shown in Figure 4A. It was evident that when NaClO was added to Ce6@Lum-AuNPs aqueous solution, the fluorescence intensity was the same for the first time and the second time, but both were stronger than that without NaClO. Figure 4B showed that after Ce6@Lum-AuNPs aqueous solution was pre-mixed with H 2 O 2 , the FL intensity of the solution with two NaClO additions was significantly enhanced compared to that with only one addition. As shown in Figure 4C, after adding HRP, the FL emission intensity of the solution decreased significantly. Hypochlorous acid with high reactivity is generated by the neutrophil enzyme myeloperoxidase and is particularly relevant in inflammatory conditions. Meanwhile, hydrogen peroxide is a strong two-electron oxidant, but most of its electron oxidation reactions are too slow to be biologically relevant because of high activation energy. NaClO is applied to simulate the tumor tissue microenvironment and oxidate luminol in collaboration with H 2 O 2 [31]. Darkfield microscopy (DFM) was used to further observe the change of Ce6@Lum-AuNPs before and after H2O2 and NaClO treatment. Figure 5A-D were DFM images, and Figure 5D was the DFM scattering spectrum of a single nanoparticle marked with circles in the figures. As shown in the figures, Ce6@Lum-AuNPs without treatment were yellow in the DFM image, and their scattering spectrum had a broad peak. The particles treated with NaClO appear red in the DFM image, and their spectra peaks were narrow at about 625 nm. Finally, three kinds of particles with different scattering spectra appeared in the solution treated with H2O2 and NaClO, and the peaks of scattering spectra were 478 nm, 587 nm and 645 nm, respectively. The results showed that H2O2 and NaClO changed the surface structure of Ce6@Lum-AuNPs, contributing to the effects observed in relevant FL spectra. The change of FL intensity was collected by FL intensity-time curves with an excitation at 405 nm. After adding NaClO to Ce6@Lum-AuNPs aqueous solution, the FL intensity at 670 nm was significantly enhanced, then quickly decreased to the original level ( Figure 4D). When NaClO was added again, the FL intensity did not change ( Figure 4D). When the same aqueous solution was mixed with a certain amount of H 2 O 2 solution beforehand, the FL intensity increased rapidly after adding NaClO ( Figure 4E). Unlike the solution without H 2 O 2 , the FL intensity increased with added NaClO and was maintained for a week without a significant decrease ( Figure 4E inset). It was considered that the addition of H 2 O 2 changes the FL enhancement process. To verify the conjecture, horseradish peroxidase (HRP), which could hydrolyze hydrogen peroxide, was added into the latter solution. As could be seen under the UV lamp, the FL of the solution was significantly reduced, shown in the inset of Figure 4E. These observations were consistent with the results of FL emission spectra, indicating that the addition of H 2 O 2 could readily facilitate the FL enhancement effect of NaClO on Ce6@Lum-AuNPs.
Darkfield microscopy (DFM) was used to further observe the change of Ce6@Lum-AuNPs before and after H 2 O 2 and NaClO treatment. Figure 5A-D were DFM images, and Figure 5D was the DFM scattering spectrum of a single nanoparticle marked with circles in the figures. As shown in the figures, Ce6@Lum-AuNPs without treatment were yellow in the DFM image, and their scattering spectrum had a broad peak. The particles treated with NaClO appear red in the DFM image, and their spectra peaks were narrow at about 625 nm. Finally, three kinds of particles with different scattering spectra appeared in the solution treated with H 2 O 2 and NaClO, and the peaks of scattering spectra were 478 nm, 587 nm and 645 nm, respectively. The results showed that H 2 O 2 and NaClO changed the surface structure of Ce6@Lum-AuNPs, contributing to the effects observed in relevant FL spectra.

Specificity of the Ce6@Lum-AuNPs for H2O2 and ClO −
Considering that there may exist other kinds of ROS in cells, we had investigated the specificity of the Ce6@Lum-AuNPs for H2O2 and NaClO. As shown in Figure 6, comparing the FL intensity of Ce6@Lum-AuNPs with and without H2O2 and NaClO in the presence or absence of CO + , O2 − , 1 O2, OH· and ONOO − , the results illustrated that the presence of these ROS had little or almost no influence on the relevant FL intensity. Meanwhile, our experimental results also indicated that there were few or no fluorescent changes observed upon addition of different concentrations of H2O2 alone to the solution of Ce6@Lum-AuNPs. Thus, it is hard to obtain the corresponding concentration for 50% of maximal effect (EC50) of Ce6@Lum-AuNPs for H2O2. Nevertheless, in the presence of hypochlorite (ClO -), hydrogen peroxide in cancer cells could readily interact with luminol to produce remarkable chemiluminescence (as described later). This raises the possibility of utilizing the relevant hydrogen peroxide and hypochlorite responsive fluorescent nanoprobes for sensitive cancer cell imaging.

Specificity of the Ce6@Lum-AuNPs for H 2 O 2 and ClO −
Considering that there may exist other kinds of ROS in cells, we had investigated the specificity of the Ce6@Lum-AuNPs for H 2 O 2 and NaClO. As shown in Figure 6, comparing the FL intensity of Ce6@Lum-AuNPs with and without H 2 O 2 and NaClO in the presence or absence of CO + , O 2 − , 1 O 2 , OH· and ONOO − , the results illustrated that the presence of these ROS had little or almost no influence on the relevant FL intensity. Meanwhile, our experimental results also indicated that there were few or no fluorescent changes observed upon addition of different concentrations of H 2 O 2 alone to the solution of Ce6@Lum-AuNPs. Thus, it is hard to obtain the corresponding concentration for 50% of maximal effect (EC50) of Ce6@Lum-AuNPs for H 2 O 2 . Nevertheless, in the presence of hypochlorite (ClO -), hydrogen peroxide in cancer cells could readily interact with luminol to produce remarkable chemiluminescence (as described later). This raises the possibility of utilizing the relevant hydrogen peroxide and hypochlorite responsive fluorescent nanoprobes for sensitive cancer cell imaging.

Cytocompatibility Evaluation In Vitro
The CCK-8 assays were used to evaluate the cytotoxicity in vitro. Both HepG2 and L02 cells were co-incubated with Ce6@Lum-AuNPs at different concentrations for 24 h The cell inhibition rates of both cell lines increased with Lum-AuNCs concentration. Fo each concentration of Ce6@Lum-AuNPs, the inhibition rate of HepG2 cells was highe than that of L02 cells. The half-maximal inhibitory concentration (IC50) of Ce6@Lum AuNPs for HepG2 and L02 cells were 73.76 μg mL −1 and 203.64 μg mL −1 , respectively determined by improved Karber's method. The results showed that compared with L0 cells, Ce6@Lum-AuNPs had a relatively more substantial inhibitory effect on HepG2 cell (Figure 7).

High-Resolution FL Imaging of Cells In Vitro
For better application in biomedical imaging, we further examined the FL character istics of Ce6@Lum-AuNPs in cells. Both Ce6@Lum-AuNPs and Ce6 were incubated with HepG2 and L02 cells for 12 h, respectively, and their FL properties and distribution in cell were observed under a confocal FL microscope. As shown in Figure 8A, no obvious FL signal was observed in cancer cells and normal cells in the control group without the ad dition of Ce6@Lum-AuNPs and Ce6. Both cancer cells and normal cells incubated with Ce6 showed an obvious FL signal due to the good FL performance of Ce6, but it could no distinguish cancer cells from normal cells. The Ce6@Lum-AuNPs group had a red FL sig nal in normal cells, while the FL signal in cancer cells was slightly stronger than norma

Cytocompatibility Evaluation In Vitro
The CCK-8 assays were used to evaluate the cytotoxicity in vitro. Both HepG2 and L02 cells were co-incubated with Ce6@Lum-AuNPs at different concentrations for 24 h. The cell inhibition rates of both cell lines increased with Lum-AuNCs concentration. For each concentration of Ce6@Lum-AuNPs, the inhibition rate of HepG2 cells was higher than that of L02 cells. The half-maximal inhibitory concentration (IC 50 ) of Ce6@Lum-AuNPs for HepG2 and L02 cells were 73.76 µg mL −1 and 203.64 µg mL −1 , respectively, determined by improved Karber's method. The results showed that compared with L02 cells, Ce6@Lum-AuNPs had a relatively more substantial inhibitory effect on HepG2 cells (Figure 7).

Cytocompatibility Evaluation In Vitro
The CCK-8 assays were used to evaluate the cytotoxicity in vitro. Both HepG2 and L02 cells were co-incubated with Ce6@Lum-AuNPs at different concentrations for 24 h. The cell inhibition rates of both cell lines increased with Lum-AuNCs concentration. For each concentration of Ce6@Lum-AuNPs, the inhibition rate of HepG2 cells was higher than that of L02 cells. The half-maximal inhibitory concentration (IC50) of Ce6@Lum-AuNPs for HepG2 and L02 cells were 73.76 μg mL −1 and 203.64 μg mL −1 , respectively, determined by improved Karber's method. The results showed that compared with L02 cells, Ce6@Lum-AuNPs had a relatively more substantial inhibitory effect on HepG2 cells (Figure 7).

High-Resolution FL Imaging of Cells In Vitro
For better application in biomedical imaging, we further examined the FL characteristics of Ce6@Lum-AuNPs in cells. Both Ce6@Lum-AuNPs and Ce6 were incubated with HepG2 and L02 cells for 12 h, respectively, and their FL properties and distribution in cells were observed under a confocal FL microscope. As shown in Figure 8A, no obvious FL signal was observed in cancer cells and normal cells in the control group without the addition of Ce6@Lum-AuNPs and Ce6. Both cancer cells and normal cells incubated with Ce6 showed an obvious FL signal due to the good FL performance of Ce6, but it could not distinguish cancer cells from normal cells. The Ce6@Lum-AuNPs group had a red FL signal in normal cells, while the FL signal in cancer cells was slightly stronger than normal

High-Resolution FL Imaging of Cells In Vitro
For better application in biomedical imaging, we further examined the FL characteristics of Ce6@Lum-AuNPs in cells. Both Ce6@Lum-AuNPs and Ce6 were incubated with HepG2 and L02 cells for 12 h, respectively, and their FL properties and distribution in cells were observed under a confocal FL microscope. As shown in Figure 8A, no obvious FL signal was observed in cancer cells and normal cells in the control group without the addition of Ce6@Lum-AuNPs and Ce6. Both cancer cells and normal cells incubated with Ce6 showed an obvious FL signal due to the good FL performance of Ce6, but it could not distinguish cancer cells from normal cells. The Ce6@Lum-AuNPs group had a red FL signal in normal cells, while the FL signal in cancer cells was slightly stronger than normal cells.
After the addition of NaClO, the FL signal in the Ce6@Lum-AuNPs group was significantly higher than that in the normal cells, which may be due to the special microenvironment in the cancer cells, such as a certain amount of H 2 O 2 , enhancing the FL. The relative FL intensities of L02 and HepG2 cell cross-sections were shown in Figure 8B-D. Therefore, we believe that Ce6@Lum-AuNPs can be used as a potential fluorescent contrast agent in cancer cells FL imaging studies. cells. After the addition of NaClO, the FL signal in the Ce6@Lum-AuNPs group was significantly higher than that in the normal cells, which may be due to the special microenvironment in the cancer cells, such as a certain amount of H2O2, enhancing the FL. The relative FL intensities of L02 and HepG2 cell cross-sections were shown in Figure 8B-D. Therefore, we believe that Ce6@Lum-AuNPs can be used as a potential fluorescent contrast agent in cancer cells FL imaging studies.

Conclusions
In summary, we have explored the possibility of designing and constructing fluorescent Ce6@Lum-AuNPs as the FL contrast agent for cancer cell recognition and imaging analysis. Firstly, luminol-gold nanoparticles (Lum-AuNPs) have been readily built, and then successfully loaded the fluorescent receptor Ce6 to prepare the FL nanoprobes (Ce6@Lum-AuNPs) with green synthesis, i.e., with biocompatible agents and mild temperature. The as-prepared fluorescent Ce6@Lum-AuNPs can efficiently and sensitively realize FL bioimaging of cancer cells. The relevant bio-sensing mechanism is pertaining to the presence of the relatively high concentration of hydrogen peroxide in cancer cells that can readily interact with luminol in collaboration with ClO − to produce bright chemiluminescence and thus activate the Ce6 component to emit NIR FL. Therefore, this novel strategy of self-luminescent nanoprobes is developed to achieve highly sensitive fluorescent bioimaging of cancer cells in situ. Using the microenvironment of cancer cells, NIR FL can be successfully emitted for the precise cancer cell image, laying a promising way for the accurate early diagnosis and treatment of cancers.

Conclusions
In summary, we have explored the possibility of designing and constructing fluorescent Ce6@Lum-AuNPs as the FL contrast agent for cancer cell recognition and imaging analysis. Firstly, luminol-gold nanoparticles (Lum-AuNPs) have been readily built, and then successfully loaded the fluorescent receptor Ce6 to prepare the FL nanoprobes (Ce6@Lum-AuNPs) with green synthesis, i.e., with biocompatible agents and mild temperature. The as-prepared fluorescent Ce6@Lum-AuNPs can efficiently and sensitively realize FL bioimaging of cancer cells. The relevant bio-sensing mechanism is pertaining to the presence of the relatively high concentration of hydrogen peroxide in cancer cells that can readily interact with luminol in collaboration with ClO − to produce bright chemiluminescence and thus activate the Ce6 component to emit NIR FL. Therefore, this novel strategy of self-luminescent nanoprobes is developed to achieve highly sensitive fluorescent bioimaging of cancer cells in situ. Using the microenvironment of cancer cells, NIR FL can be successfully emitted for the precise cancer cell image, laying a promising way for the accurate early diagnosis and treatment of cancers.