Characterization of probes. The ROX-labelled ATP-aptamer (Apt-ROX) can be facilely and selectively adsorbed onto the prepared TC nanosheets by hydrogen bond and metal chelate interaction between the aptamer and TC nanosheets, guaranteeing the proximity of ROX to TC nanosheets surface (Fig. 1a). As thus, the fluorescence of ROX is rapidly and efficiently quenched by the TC nanosheets due to long-range energy transfer from ROX to these two-dimensional TC nanosheets (Fig. 1a). The as-prepared TC/Apt complex could be utilized for in vivo imaging ATP in tumour tissues of these mouse models based on signal off/on switch mechanism (Figs. 1b & 1c). Typically, the TC/Apt can burst into live cells based on active endocytosis, and then finally distribute in cellular cytoplasm (Fig. 1d). Then the hybridization between intracellular ATP and aptamer occurs due to the strong affinity between target and aptamer. Such hybridization leads to release of ROX-tagged aptamer from quencher surface, thus recovering the fluorescence of ROX under the 545-nm excitation (Fig. 1d). In addition to the in vivo imaging analysis, the resultant TC/Apt probes have the ability to quantitatively and sensitively detect ATP content in real samples, such as mouse and human serum (Fig. 1e). Firstly, the surface topography of as-prepared TC/Apt is imaged by the transmission electron microscopy (TEM), high-resolution TEM (HRTEM), and scanning electron microscopy (SEM), respectively. Both TEM and SEM images (Figs. 2a & 2b) reveal that the resultant TC/Apt present sheet-like structure with obvious ~ 200–500 nm lateral dimensions, which is consistent with pristine TC sheets (Additional file 1: Figure S1a & S1b). Furthermore, these TC/Apt platforms are characterized by the photoluminescence (PL) spectroscopy, UV-Vis absorption spectroscopy, Raman spectroscopy, Fourier transform infrared (FTIR) spectroscopy, and Zeta potentials to validate successful conjugation of as-prepared TC nanosheets with ROX-tagged aptamer. Typically, as revealed in absorption spectra in Fig. 2c, pure Apt has a maximal peak at ~ 585 nm (yellow line) assigned to ROX moiety 25, and pure TC nanosheets have a strong absorption at ~ 785 nm attributed to the modified Al oxoanions 24. As a result, TC/Apt complex shows two typical peaks at ~ 585 nm and ~ 785 nm. Moreover, Fig. 2d provides Raman spectra of TC nanosheets, Apt, and TC/Apt. In particular, three characteristic Raman peaks at 867, 1137, and 1615 cm− 1 are observed in Apt, assigned to ν(C−C) ring-stretch of ROX 26, 27. These Raman bands can be observed in TC/Apt complex rather than in pure TC nanosheets.
In order to evaluate the quenching efficiency (QE) of TC nanosheets against ROX, graphene oxide (GO) with the same concentration of 200 µg/mL is selected for a comparison. As shown in PL spectra in Fig. 2e, 500 nM Apt presents a typical emission peak at 610 nm, which sharply decreases when it is absorbed by GO to form GO/Apt complex. Typically, the PL intensity of pure Apt at 610 nm is ~ 9.8-fold stronger than that of GO/Apt complex, and the corresponding QE is calculated to be ~ 90% 18. On the contrary, the stronger fluorescence quenching capacity of TC nanosheets against ROX is observed in Fig. 2f. The PL spectrum of TC/Apt complex is in line with pure TC nanosheets, and the corresponding QE is calculated to be ~ 97%. The superior QE of TC nanosheets is due to their higher mass extinction coefficients 24. Furthermore, fluorescence quenching ability of TC nanosheets would not be influenced by both different temperatures and pH values, suggesting the relatively stable structure of as-prepared TC/Apt platforms in various environments (Additional file 1: Figures S2-3). The surface groups of the as-prepared TC/Apt are characterized by FTIR (Additional file 1: Figure S4). With regard to Zeta potentials (Additional file 1: Figure S5), Zeta potential decreases from + 20.7 mV to + 10.1 mV when Apt links to TC.
Quantitative determination of ATP. The concentration ratio of TC nanosheets to Apt is a vital factor for fluorescence quenching effect of TC/Apt platforms. Figure 3a displays PL spectra of TC/Apt complex, produced in different concentration ratios of TC nanosheets to Apt (i.e., 1:0.625-1:160). Particularly, the weakest fluorescence intensity of TC/Apt is observed when the concentration ratio equals to or more than 1:2.5. As shown in the corresponding agarose gel electropherogram of Fig. 3b, no free aptamer bands can be observed when the concentration ratio of TC nanosheets to Apt is above 1:2.5, also confirming the ratio of 1: 2.5 is the optimal concentration ratio to prepare TC/Apt. Figures 3c and 3f respectively show schematic illustration of TC/Apt and GO/Apt for fluorescent detection of ATP. Figure 3d gives a series of PL spectra of TC/Apt under the 545-nm excitation when adding ATP solutions with different concentrations (e.g., 0.001-10.0 mM). Specifically, the fluorescence intensity of TC/Apt gradually grows with increase of ATP concentrations, revealing more Apt chains are released from TC sheets. The ATP concentration-dependent fluorescence change is further quantitatively explored in scatter curve of normalized fluorescence intensity ((F-F0)/F0) at the 610 nm versus ATP concentration (Fig. 3e). Herein, F or F0 represent the fluorescence intensity of TC/Apt treated with (F) or without ATP (F0), respectively. As revealed, the whole process from signal-off to signal-on state shows an ATP concentration-dependent manner. Notably, when the ATP concentration reaches 1.5 mM, the maximal (F-F0)/F0 value is achieved. While when ATP concentration further increases, no further significant enhancement is observed, indicating no more Apt is dissociated from TC nanosheets triggered by ATP. In addition, a good linearity is presented between the relative (F-F0)/F0 and ATP concentration in the range of 1 µΜ to 1.5 mM (Fig. 3e inset). The corresponding regression equation is Y = 14.53 X + 0.32 with a good correlation coefficient of r2 = 0.998, where, Y represents (F-F0)/F0 value and X means ATP concentration. The corresponding limit of detection (LOD) is calculated to be ~ 0.2 µΜ by setting the signal-to-noise ratio of 3:1 (Fig. 3e inset). Comparatively, the dynamic range of GO/Apt is from 0.5 mM to 1.5 mM under the same conditions, much narrower than that of TC/Apt (e.g., 1 µM-1.5 mM), as shown in Fig. 3g. Accordingly, the LOD of GO/Apt is down to 0.46 mM by setting the signal-to-noise ratio of 3:1 (Fig. 3h), which is two orders of magnitude higher than that of TC/Apt (e.g., 0.2 µΜ). The superior dynamic range and sensitivity of TC/Apt over GO/Apt is partially due to their higher quenching efficiency of the TC over GO (e.g., ~ 97% versus ~ 90%), as mentioned above (Additional file 1: Table S1). Afterwards, the selectivity of TC/Apt-based platform for detection of ATP molecules is evaluated against other similar molecules such as uridine triphosphate (UTP), cytidine triphosphate (CTP), guanosine triphosphate (GTP), and adenosine monophosphate (AMP) (Additional file 1: Figure S6), different ions (Additional file 1: Figure S7), as well as various amino acids (Additional file 1: Figure S8). As depicted in Additional file 1: Figures S6-S8, those interfering species display much weak fluorescence response, whereas the ATP sample exhibits relatively strong fluorescence response, verifying the good selectivity of prepared TC/Apt-based probes.
Detection of ATP in vitro. Next, we primarily evaluate the cytotoxicity of TC/Apt probes. The corresponding viabilities of treated cells are above ~ 85% (Additional file 1: Figure S9), suggesting low cytotoxicity of the resultant TC/Apt. Furthermore, the TC/Apt entering into cells is through the energy-consuming endocytosis 28, 29, as revealed in Additional file 1: Figures S10-13. Based on above results, the feasibility of these TC/Apt-based probes for sensing of intracellular ATP in cytoplasm is systematically evaluated. Experimentally, the unmodified TC (100 µg/mL) and TC/Apt probes (100 µg/mL) are respectively incubated with cancer cells (e.g., HeLa, MCF-7, and 4T1 cells) and normal cells (e.g., ARPE-19 cells) at 37 oC for 12 h, followed by the CLSM imaging. As shown in confocal images of the Figs. 4a-4d, no fluorescence can be measured in cells without any treatments (control groups, Ctrl). Similarly, TC-treated cells also show negligible fluorescence signals. On the contrary, distinct red fluorescence can be observed in TC/Apt-treated HeLa (Fig. 4a), MCF-7 (Fig. 4b), and 4T1 cells (Fig. 4c). Comparatively, the relatively weak red fluorescence can be detected in these TC/Apt-treated ARPE-19 cells (Fig. 4d). These experimental results are consistent with previously reported results that ATP contents in cancer cells are much higher than those in normal cells 5–7. For further quantitative comparison, the fluorescence intensity for each image is analyzed by using the commercial image software (Leica LAS AF Lite). The mean fluorescence intensity of Ctrl, TC, and TC/Apt groups is displayed in Fig. 4e. The mean fluorescence intensity of these TC/Apt-treated cancer cells is significantly higher than control groups (e.g., ~ 3.6–4.8 folds, p < 0.001) or TC groups (e.g., ~ 3.5–4.7 folds, p < 0.001), which is consistent with cellular imaging results mentioned above. Also, Fig. 4e further exhibits that the mean fluorescence intensities of cancer cells determined by TC/Apt probes are significantly higher than those of normal cells (e.g., ~ 1.7 fold of 4T1 cells (p < 0.05), ~ 1.9 fold of MCF-7 cells (p < 0.01), and ~ 2.0 fold of HeLa cells (p < 0.01)), which are in accordance with above cellular fluorescence images. These results demonstrate that TC/Apt probes are available for in situ monitoring of intracellular ATP in living cells. To demonstrate the capability of the TC/Apt to monitor ATP level changes inside living cells, the calcium ion (Ca2+) and etoposide are used to promote the ATP production of the cancer cells. In previous reports, Ca2+ and etoposide as apoptotic stimuli are widely employed for the elevation of cytosolic ATP level 18, 29. For both HeLa and MCF-7 cells, in comparison to relatively weak fluorescence of cells without incubation with Ca2+ or etoposide (untreated groups), relatively strong fluorescence is measured in cells incubated with Ca2+ or etoposide (Figs. 5a & 5c). For the quantitative evaluation, mean fluorescence intensities of untreated, Ca2+ and etoposide groups are given in Figs. 5b & 5d. Compared with untreated cells (control groups), the mean fluorescence intensity increases by ~ 18% in Ca2+-treated HeLa cells, by ~ 20% in etoposide-treated HeLa cells (Fig. 5b), by ~ 16% in Ca2+-treated MCF-7 cells and by ~ 21% in these etoposide-treated MCF-7 cells (Fig. 5d). The similar results are observed in 4T1 cells (Additional file 1: Figure S14). These data indicate that TC/Apt probes have the ability to monitor level changes of intracellular ATP.
Detection and imaging of ATP in body fluid. To evaluate the feasibility of the developed probes for imaging and sensing ATP in real complex samples, the fluorescent TC/Apt probes are utilized to detecting ATP with various concentrations in the different body fluid (Fig. 6a). In detail, serum and urine samples are extracted from mouse or human (Step 1). After that, series of concentrations of standard ATP covering from 0.001 to 5 mM are separately spiked into the 0.1% diluted different body fluid samples, respectively (Step 2) 10. Finally, the fluorescence intensity at the 610 nm can be detected and calculated from a series of PL spectra of ROX dyes after adding the 200 µg/mL TC/Apt probes into above body fluid samples containing ATP with various concentrations (Steps 3–4). As depicted in these Figs. 6b-d, when the concentration of adding ATP is 0 mM, the fluorescence intensity at 610 nm of TC/Apt probes can be detected as ~ 5×105, suggesting that the original body fluid samples contain a certain concentration of ATP molecules. With the increase of adding ATP concentration, fluorescence intensity at the 610 nm of TC/Apt probes in real samples gradually increases from ~ 5×105 to ~ 16×105, indicating that the prepared TC/Apt probes offer the ability to detect the ATP content in different body fluid samples. Together, these results demonstrate that the as-prepared TC/Apt probes can be used to detect the ATP content in real complex samples. Furthermore, the 4T1 or MCF-7 tumor-bearing mouse model is employed for verify the feasibility of TC/Apt probes for imaging ATP in vivo (Fig. 6e). In detail, Fig. 6f displays that the TC/Apt-based fluorescent probes can exhibit more obvious fluorescence signals on the right back tumor site of the 4T1 tumor-bearing mouse after intratumoral injection with 200 µg/mL TC/Apt than that of left normal sites. For quantitative comparisons, the mean fluorescence intensity detected in the 4T1 tumor tissue is significantly higher than that of normal skin tissues (p < 0.01) (Fig. 6g). Meanwhile, the MCF-7 tumor-bearing mouse treated by the TC/Apt shows the similar fluorescence imaging results to 4T1 tumor tissue (Figs. 6h & 6i). These data indicate that TC/Apt has the ability to image ATP in vivo.